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molecules Article Method Validation for Determination of Thallium by Inductively Coupled Plasma Mass Spectrometry and Monitoring of Various Foods in South Korea Yeon-hee Kim 1 , Wook-jin Ra 1 , Solyi Cho 2 , Shinai Choi 3 , Bokyung Soh 1 , Yongsung Joo 4 and Kwang-Won Lee 1, * 1 2 3 4 *



 Citation: Kim, Y.-h.; Ra, W.-j.; Cho, S.; Choi, S.; Soh, B.; Joo, Y.; Lee, K.-W. Method Validation for Determination of Thallium by Inductively Coupled Plasma Mass Spectrometry and Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Korea; helly27@korea.ac.kr (Y.-h.K.); ra228@korea.ac.kr (W.-j.R.); bokyung12345@korea.ac.kr (B.S.) Advanced Food Safety Research Group, School of Food Science and Technology, Chung-Ang University, Anseong-si 17546, Korea; syjo@knacon.co.kr KnA Consulting, Yongin-si 16942, Korea; shchoi@knacon.co.kr Department of Statistics, Dongguk University-Seoul, Seoul 04620, Korea; yongsungjoo@dongguk.edu Correspondence: kwangwon@korea.ac.kr; Tel.: +82-2-3290-3027 Abstract: Thallium (Tl) is a rare element and one of the most harmful metals. This study validated an analytical method for determining Tl in foods by inductively coupled plasma mass spectrometry (ICP-MS) based on food matrices and calories. For six representative foods, the method’s correlation coefficient (R2 ) was above 0.999, and the method limit of detection (MLOD) was 0.0070– 0.0498 µg kg−1 , with accuracy ranging from 82.06% to 119.81% and precision within 10%. We investigated 304 various foods in the South Korean market, including agricultural, fishery, livestock, and processed foods. Tl above the MLOD level was detected in 148 samples and was less than 10 µg kg−1 in 98% of the samples. Comparing the Tl concentrations among food groups revealed that fisheries and animal products had higher Tl contents than cereals and vegetables. Tl exposure via food intake did not exceed the health guidance level. Monitoring of Various Foods in South Korea. Molecules 2021, 26, 6729. Keywords: thallium; trace element; food contamination; risk assessment; ICP-MS https://doi.org/10.3390/ molecules26216729 Academic Editors: Željka Fiket and Martina Furdek Turk Received: 29 September 2021 Accepted: 3 November 2021 Published: 6 November 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 1. Introduction Since the industrial revolution, modern technological development and associated environmental pollution have increased the exposure to trace metals. With novel applications in microelectronics, chemotherapies, and other emerging technologies, the toxicological significance of some uncommon or infrequently used metals has risen [1]. In this regard, thallium (Tl) is rare but is one of the most harmful metals. William Crookes discovered it in 1861, and the metallic form was first obtained by Lamy in 1862 [2]. The name is derived from the Greek word “thallos,” which means young green twig [2]. Tl can be discovered in pure form or in combination with other elements. Pure Tl has no odor or taste. It is utilized in the production of optic lenses and glasses and the semiconductor and pharmaceutical industries [3]. Tl was used as a rodenticide and insecticide until 1972 but was prohibited because of its potential toxicity to humans [4]. Because the Tl ion charge and radius are similar to those of potassium ion, Tl can interfere with the biological functions of potassium [5]. Gastroenteritis, polyneuropathy, and alopecia are three significant symptoms of Tl poisoning in humans [6]. Alopecia is a specific symptom of Tl poisoning. Studies of Tl toxicity in humans include case studies, clinical reports, and medical investigations. Epidemiological studies associated with long-term exposure to Tl are restricted by small scales and insufficient information. The average lethal oral dose in adults has been estimated to be 10–15 mg kg−1 [7]. If not treated, it generally takes 10–12 days to die, but there are reports of death within 8–10 h [8]. 4.0/). Molecules 2021, 26, 6729. https://doi.org/10.3390/molecules26216729 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 6729 2 of 14 The regulation for Tl sets a maximum contaminant level (MCL) of 0.2 µg L−1 in drinking water in the United States [9], and 0.8 µg L−1 in fresh water and 1 mg kg−1 dry weight in soil in Canada [10,11]. In South Korea, food regulations for most common toxic metal(loid)s such as cadmium, lead, mercury, and arsenic are established and managed to ensure safety throughout food consumption [12]. In contrast, preventive research and analytical method verification are necessary to avoid food accidents and respond to possible hazards from excess Tl. Heavy metal analysis can be performed using such analytical techniques as inductively coupled plasma atomic emission spectrometry (ICP-AES), graphite furnace atomic absorption spectrometry (GF-AAS), and inductively coupled plasma mass spectrometry (ICP-MS). The latter is suitable for trace element analysis because of its high resolution and two to three times lower detection limit than ICP-AES [13]. In recent years, the number of studies on food analysis using ICP-MS has increased [14–18]. Given that there were no detailed studies on Tl content in various food products in the South Korean market, we validated a method for Tl determination by ICP-MS, monitored the commonly consumed foods in South Korea, and assessed the risk based on the results to provide foundational information. 2. Results and Discussion 2.1. Method Validation and Quality Control We validated the external standard calibration curve for Tl. Regression of the correlation (R2 ) showed good linearity: above 0.999. Trueness was represented by certified reference material (CRM) (BCR-679, white cabbage). The measured average concentration was 3.03 ± 0.36 µg kg−1, and the mean recovery was 101%. The relative standard deviation (SD) was 2.94%. Six representative foods were tested for accuracy and precision by adding standard solutions at low (0.1 µg kg−1 ), medium (0.5 µg kg−1 ), and high (1.0 µg kg−1 ) concentrations (Table S1). The intraday accuracy ranged from 82.06% to 119.81%, interday accuracy—from 92.05% to 110.44%; the intraday precision—from 0.88% to 9.08%, interday precision—from 1.09% to 9.79%. We observed a recovery reduction in the salted solid matrix representative food (sea salt) to less than 60% during the method validation. Salts (i.e., sodium, calcium, chloride, and potassium) cause interference in ICP-MS [19]. Higher matrix salt concentrations reduce the signal and lower the ionization potential, resulting in higher matrix effect [20]. Sample dilution can reduce the severity of the matrix effect and instrument drift [20,21]. It is recommended to keep the total dissolved solid level of samples less than 0.2% when using ICP-MS [21]. The original sample weight was 0.5 g, and the sample decomposition solution weight was 20 g. To reduce the salt interference effect, we adjusted the sample weight from 0.5 g to 0.1 g, and the decomposition solution was diluted to 80 g with distilled water. As a result, the accuracy of sea salt determination was restored from 92.05% to 101.42%. The traditional South Korean diet includes high amounts of sodium [22]. The salt interference effect can be reduced in evaluating foods with high salt content by diluting sample solutions to prevent low recovery. The method limit of detection (MLOD) for representative foods ranged from 0.0070 to 0.0498 µg kg−1 , and the method limit of quantification (MLOQ) ranged from 0.0222 to 0.1585 µg kg−1 (Table 1). We calculated the measurement uncertainty to a non-fatty matrix (rice) that spiked the Tl standard solution’s intermediate concentration (0.5 µg kg− 1 ). The expanded uncertainty for rice was 0.51 ± 0.03 µg kg−1 . The expanded uncertainty of the measurement is stated at an approximately 95% confidence level using coverage of k = 2. If the target uncertainty is not established in a regulation, an additional 20–30% tolerance can be considered to allow for variability in the uncertainty calculation process [23]. Molecules 2021, 26, 6729 3 of 14 Table 1. Selection of representative foods and validation parameters of Tl analysis. Classification Representative Food Regression Equation R2 MLOD a (µg kg−1 ) MLOQ b (µg kg−1 ) Non-fatty Solid Hydrated Solid Fatty Solid Salty Solid Non-Fatty Liquid Fatty Liquid Rice Apple Beef Sea Salt Orange Juice Sesame Oil y = 117366x − 208.52 y = 38252x + 774.86 y = 85346x − 6.9005 y = 67663x + 165.49 y = 88399x − 441.29 y = 111833x − 120.50 1 0.9999 0.9997 0.9999 1 1 0.0092 0.0498 0.0070 0.0207 0.0126 0.0225 0.0293 0.1585 0.0222 0.0661 0.0401 0.0715 a MLOD refers to the method limit of detection, S/σ = 3.14; b MLOQ refers to the method limit of quantification, S/σ = 10. 2.2. Concentration of Tl in Foods 2.2.1. Concentration of Tl in Agricultural, Fishery, and Livestock Products We measured the Tl concentration in agricultural, fishery, and livestock products commonly consumed in the South Korean market and summarized the results into 13 subgroups considering the Korea Food Code (Table 2, Tables S2–S4). Tl above the MLOD was detected in 120 out of 175 samples. The total number of cereal types tested was 32, with Tl exceeding MLOD in 16 of them. The mean value was 0.83 µg kg−1, and the highest value was 5.27 µg kg−1 for sorghum, whereas Tl in rice was reported to be 26.8 µg kg−1 in a Polish study [24]. Tl contents in rice, barley, and oats were below MLOD in the current study. Tl levels in tuber crops, such as potatoes and sweet potatoes, exceeded the MLOD. The mean Tl content in tuber crops was 6.67 µg kg−1 , and the highest of 13.21 µg kg−1 was detected in sweet potatoes. Tl content in potatoes, 4.04 µg kg−1 , was similar to that in an Italian study (6.90 µg kg−1 ) and higher than that in a South African one (1.03 µg g−1 ) [25,26]. In the bean category, three types of beans—white, black, and kidney beans—were examined. In kidney beans, Tl content was below the detection limit. The average Tl content in beans was 0.49 µg kg−1 , lower than the Italian study result (13.4 µg kg−1 ) [25]. Apple, mandarin, and pear were the three fruits examined. Tl levels in all mandarins were below MLOD, whereas Tl was detected in all pears. The mean concentration in the fruit group was 1.48 µg kg−1 (Table 2). In Italy, the contents in five fruits varied from 0.7 to 3.1 µg kg−1 [25], and we obtained similar values. Fifty vegetables were categorized as root, leafy, and other vegetables. In the case of root vegetables including carrot, white radish, garlic, onion, burdock root, and ginseng, Tl was detected in 14 out of 19 samples, with concentrations ranging from 0.02 to 7.11 µg kg−1 , and the highest detected content was in white radish. The mean content reduced in the following order: white radish > ginseng > garlic > carrot > burdock root > onion. Tl was found in 14 out of 16 leafy vegetable samples, ranging from 0.02 to 15.93 µg kg−1 , with most Tl detected in cabbage. The mean contents in lettuce and cabbage were relatively high in this group. A previous study discovered that the Brassicaceae family (cabbage, rape, turnip, and mustard) contains high levels of Tl [27]. Cabbage showed the highest concentration of Tl (4.13 ± 6.82 µg kg−1 ) in our category, although the SD across samples was large, indicating the differential Tl content in the soils where the samples were grown. Tl concentrations were below MLOD in zucchini and cucumber and were 0.60 and 0.49 µg kg−1 in one sample of red pepper and eggplant, respectively. Tl concentrations in native plants were approximately 0.05 mg kg−1 [28], and in Chinese crops, they ranged from 0.01 to 0.06 mg kg−1 [29]. In the case of livestock products, Tl in chicken, beef, and pork ranged from 0.51 to 5.72 µg kg−1 with the mean value of 2.46 µg kg−1 . The highest Tl level among animal products was in eggs, with the mean value of 2.46 µg kg−1 , more than that in a Belgian study [30]. Tl in eggs came endogenously from chickens, and the natural environment in which they grew up was conceivable to contribute [30,31]. The mean concentration in meats was 0.79 µg kg−1 , similar to that reported in a Swiss study [32]. Molecules 2021, 26, 6729 4 of 14 Table 2. Concentration (fresh weight) and detection frequency of Tl in agricultural, fishery, and livestock products. Category Agricultural Products Livestock Products Fishery Products Subcategory (n) a Cereals (32) Beans (10) Tuber crops (6) Vegetables (50) Mushrooms (6) Fruits (9) Eggs (3) Meats (9) Fishes (21) Cephalopoda (6) Shellfishes (10) Crustaceans (4) Sea Algae (9) Frequency of Detection b (%) Min–Max c (µg kg−1 ) Median (µg kg−1 ) Mean ± SD c (µg kg−1 ) 50 0.00–5.27 0.20 0.83 ± 1.47 50 0.00–2.31 0.00 0.49 ± 0.80 100 3.78–13.21 5.13 6.67 ± 3.70 66 0.02–15.93 0.34 1.59 ± 3.21 0 0.02–0.02 0.02 0.02 ± 0.00 56 0.02–7.00 0.34 1.48 ± 2.39 100 0.65–5.72 1.03 2.46 ± 2.31 100 0.51–1.63 1.00 0.79 ± 0.38 100 0.41–1.87 1.03 1.06 ± 0.37 100 1.04–1.22 1.07 1.10 ± 0.06 100 1.20–6.02 1.88 2.55 ± 1.46 100 1.07–1.79 1.14 1.28 ± 0.29 22 0.02–12.72 0.02 1.21 ± 1.40 a n is the number of samples; b frequency of detection is the percentage of samples above the method detection of limit (MLOD) in each category; c results < MLOD were set equal to 1/2 MLOD values. Fishery products were classified into fish, cephalopodan, shellfish, crustacean, and sea alga products. Tl content was higher than the detection limit in 44 out of the 50 samples tested in this category (Table 2). Tl contents in fishery products ranged from 0.02 to 12.72 µg kg−1 , with a total average concentration of 1.55 µg kg−1 . Tl levels in fishes varied from 0.41 to 1.87 µg kg−1 , with the mean value of 1.06 µg kg−1 . These values were higher than those in a Vietnamese study (0.012–0.050 µg g−1 ), but similar to those in the Canadian (0.01–19.26 ng g−1 ) and Iranian (0.02–0.30 µg kg−1 ) ones [33–35]. As for cephalopods, we analyzed squid and octopus, and their average Tl content was 1.10 µg kg−1 . Crustaceans examined were four types of crabs, with an average Tl concentration of 1.28 µg kg−1 . The average Tl content in shellfish was 2.55 µg kg−1 , with the highest concentration (6.02 µg kg−1 ) observed in the manila clam. Sea algae were investigated in three types: laver, sea mustard, and hijiki (Table S4). The hijiki contained the least quantity of Tl, with Tl concentrations of 5.21 and 12.72 µg kg−1 in one sample of laver and sea mustard samples, respectively. The SD of Tl levels among sea algae and shellfish was higher than in other species. Since each sample originated from a different origin site, such a large SD might represent habitat variations related to the marine environment. The Tl content in aquatic creatures is influenced by seawater along the coast they inhabit. The Tl concentration in seawater is about 0.2–20 µg kg−1 , affected by volcanic events, airborne particles, and deposit inflows from rivers and soil [36]. As a result, if polluted river water or sediment reaches a habitable area, it may affect the Tl concentration of aquatic species. The presence of Tl in sediment or soil deposits enables the metal to move into benthic species such as shellfish (zoobenthos) and sea algae (phytobenthos) rather than fish and cephalopods due to their habiting nature. In recent years, numerous and broad investigations on Tl contamination have been undertaken in China [37–41]. There are Tl deposits in India, Japan, and Uzbekistan in Asia, but China has the most Tl deposits (i.e., Lanmuchang, Molecules 2021, 26, 6729 5 of 14 Nanhua, Yunfu, and Chengmenshan deposits) [42]. The presence of Tl in crops is related to its level in the soil. Tl levels in the crust range from 0.1 to 1.7 mg kg−1 , with sulfide ores being the most common source [43]. Lee. et al. conducted a study on soil in South Korean locations and reported the estimated Tl content of 1.20–12.91 mg kg−1 in the soil near a cement industry facility, and 0.18–1.09 mg kg−1 near a smelter and mine [44]. In addition, 2.28 ± 1.39 mg kg−1 of Tl was found in rhizospheric soils near cement plants in China [45]. Although the Tl content of commonly consumed foods in South Korea is low, additional studies on areas with potential Tl contamination are required, considering the concentration of Tl in contamination-prone parts of the country. 2.2.2. Concentration of Tl in Processed Foods The processed foods were monitored on 129 samples of commercial products consumed by the general population (Table 3). Canned foods were excluded because of the possibility of heavy metal cross-contamination from packaging. Tl was detected in 28 out of 129 samples of beverages and other processed foods, with considerable supplementary data (Tables S5 and S6). Table 3. Concentration (fresh weight) and detection frequency of Tl in processed foods. Category Beverages Other Processed Foods Subcategory (n) a Infusion Teas (18) Other Beverages (21) Snacks (6) Salted Seafood processing Products (12) Pickled Foods (12) Sauces (18) Oil Products (27) Dairy Products (6) Cereal Products (3) Livestock Processing Products (3) Noodles (3) Frequency of Detection b (%) Min–Max c (µg kg −1 ) Median (µg kg −1 ) Mean ± SD c (µg kg −1 ) 39 0.01–1.99 0.52 0.61 ± 0.63 33 0.01–4.12 0.01 0.49 ± 0.97 100 0.01–0.01 1.10 0.01 ± 0.00 25 0.01–1.41 0.01 0.30 ± 0.52 8 0.01–19.46 0.01 1.63 ± 5.38 0 0.01–0.01 0.01 0.01 ± 0.00 0 0.01–0.01 0.01 0.01 ± 0.00 0 0.01–0.01 0.01 0.01 ± 0.00 0 0.00–0.00 0.00 0.00 ± 0.00 0 0.01–0.01 0.01 0.01 ± 0.00 0 0.01–0.01 0.01 0.01 ± 0.00 a n is the number of samples; b frequency of detection is the percentage of samples above the method detection of limit (MLOD) in each category; c results < MLOD were set equal to 1/2 MLOD values. Beverages were subdivided into infusion tea and other beverages (e.g., soda, coffee, and sports drinks). Other processed foods included various processed products, such as oils, snacks, and salted foods (e.g., sauces, pickled foods, salted seafood, and ham). In the beverage category, Tl was detected in 14 out of 39 samples. Tl concentration in tea infusions ranged from 0.01 to 1.99 µg kg−1 . Matte tea had the highest average concentration (1.50 µg kg−1 ), followed by Solomon-seal (Polygonatum odoratum var. pluriflorum) and black tea (Table S5). Our results were lower than the Tl concentrations in herbal tea infusions reported in Brazil [46]. Regarding other beverages, the average Tl concentration in red ginseng drink was 2.49 µg kg−1 , followed by fruit juice (0.68 µg kg−1 ) and soy milk (0.24 µg kg−1 ). Samples with mainly undetected Tl and limited sample size were classified as other processed foods (Table S6). Potato crisps and fruit jelly were categorized as snacks. The average Tl concentration in potato crisps was 3.49 µg kg−1 , much lower than in a New Zealand study (0.17 mg kg−1 ) [47]. The average Tl level in fruit jelly was 0.51 µg kg−1 . In the processed salty seafood category, Tl was only detected in fish cakes (1.19 µg kg−1 ). In the pickled food category, one of the three radish kimchi samples had a Tl content of Molecules 2021, 26, 6729 6 of 14 19.46 µg kg−1 , indicating that the Tl found in the sample might have come from the raw material. Tl was not detected in sauces, oil and dairy products, cereals, livestock processed foods, and noodles. In our study, processed foods showed a lower Tl detection frequency than raw materials. Processed foods have lower raw material contents and are supplemented with water and various food additives. Certain processed foods require the use of food extracts rather than raw foods. As a result, the metal concentration in the final product was somewhat diluted. Furthermore, cooking processes, such as blanching, boiling, and frying, can lower the amount of metal in food. Several previous studies have reported this trend [48,49]. For these reasons, heavy metal levels in processed foods are often low. Toxic metal(loid)s (including nickel, chromium, and manganese) can contaminate cooking utensils or packaging processes [50]. Contrastingly, Tl in foods can come from raw materials cultivated in a polluted environment (i.e., soil and river). 2.3. Comparing the Concentrations of Tl among Food Groups We divided the food groups based on biological similarities and compared the intragroup and intergroup differences in Tl concentration (Figure 1). The classification is as follows: cereals and beans (CB), fruits and vegetables (VF), livestock products (LP), and fishery products (FP). The processed foods were excluded from the comparison because Tl was not detected in most of their samples. When cereals and beans were compared, the median Tl value of beans was higher than that of cereals, but the difference was not statistically significant (Figure 1a). Legumes are the fruits of plants in the family Fabaceae, while cereals are fruits (caryopsis) with a seed coat (testa). The fact that there was no significant difference between the groups might be due to their comparable morphological forms. In the VF group, vegetables were subdivided into root vegetables, leafy vegetables, tuber crops, and other vegetables. The Tl concentration reduced in the following order: tuber crops > root vegetables > leafy vegetables > fruits > other vegetables (Figure 1b). A tuber is a nutrient storage organ that grows underground and belongs to the root type, but we separated it based on the classification standard of the Korea Food Code [12] and monitored potato (Solanum tuberosum L. ) and sweet potato (Ipomoea batatas) in the group (Table S2). The Tl concentration in sweet potato (9.29 µg kg−1 ) was higher than that of potato (4.04 µg kg−1 ), given that potato is a stem tuber crop and sweet potato is a root tuber crop. In a recent Chinese study, the bio-concentration factor (BCF) and transfer factor (TF) of Tl in sweet potato were more than one, indicating that the plant can accumulate metals [51]. The median Tl concentration in root vegetables was negligibly higher than that in leafy vegetables and fruits. In the other vegetable group, which included eggplant, red pepper, zucchini, and cucumber, Tl content was significantly (p < 0.05) lower than in the other VF groups. Based on the biological classification, fishery products were divided into fishes, cephalopods, shellfish, crustaceans, and sea algae. Tl levels in aquatic animals were significantly (p < 0.05) higher than those in aquatic plants among the FP groups (Figure 1c). While there was no significant difference between the aquatic animals, the shellfish group had the highest median Tl value. Livestock products were classified as eggs and meats. The median value of eggs was insignificantly higher than that of meats (Figure 1d). Examining the significant differences among the CB, VF, LP, and FP groups revealed that Tl content decreased in the following order: LP and FP > VF > CB (p < 0.05). Tl concentrations in animal foods (LP and FP) were significantly (p < 0.05) higher than those in the CB and VF groups (Figure 1e). Fisheries and livestock products belong to the upper trophic level of the food chain. Although sea algae are in the marine product group, Tl concentration in animals is apparently higher than that in plants. In other words, it seems that Tl accumulation can occur as it goes up the food chain. Toxic metal(loid)s accumulate in the food chain in natural environments [52,53]. Tl is enriched in the natural environment through anthropogenic sources (e.g., cement production and coal combination) and natural Molecules 2021, 26, x FOR PEER REVIEW 7 of 14 Molecules 2021, 26, 6729 7 of 14 Tl value. Livestock products were classified as eggs and meats. The median value of eggs was insignificantly higher than that of meats (Figure 1d). sources, such as volcanic activity and mineral ore mining [36]. Tl accumulates in plants and animals through the food chain, eventually causing harm to humans. (a) (b) (c) (d) (e) Figure 1. Box plots thallium (Tl) foodgroups groupsbybycategories. categories.(a) (a)cereals cerealsand andbeans, beans, (b) (b) fruits, fruits, vegetables, and tubers, Figure 1. Box plots of of thallium (Tl) inin food tubers, livestock products, fishery products, cereals beans; vegetable fruits; livestock products; (c) (c) livestock products, (d)(d) fishery products, (e) (e) CB,CB, cereals andand beans; VF,VF, vegetable andand fruits; LP,LP, livestock products; FP, FP, fishery products (line = median, box = 25th and 75th percentiles, whiskers = minimum and maximum observations fishery products (line = median, box = 25th and 75th percentiles, whiskers = minimum and maximum observations below below upper fence); IQR (Interquartile Range) 75th–25th percentile. Means within box plots with different letters are upper fence); IQR (Interquartile Range) 75th–25th percentile. Means within box plots with different letters are significantly different at p < 0.05 by testing between mean using t-test or one-way ANOVA with Tukey’s multiple comparison. Molecules 2021, 26, 6729 8 of 14 2.4. Health Risk Assessment for Thallium through Food Consumption We evaluated Tl exposure through food intake based on the monitoring results. Food intake and the average weight by age were calculated from the National Health and Nutrition Survey in Korea (KNHANES) data [54]. The information on chronic exposure to Tl in the human impact case study is limited because it is mainly associated with unintentional intake or suicide attempts. A recent study by Andrew et al. used a healthbased guidance value (HBGV) approach based on the tolerable daily intake (TDI) of the Netherlands National Institute for Public Health and the Environment (RIVM) while considering human toxicological uncertainty [47,55]. In the present study, the same method was applied to evaluate the dietary exposure and risk assessment for Tl. Table 4 displays the exposure and risk assessment results based on the Tl concentrations in foods. The exposure through food consumed was very low, below 0.0032 µg kg−1 bw day−1 for all food groups. Among them, the most exposed food group was vegetables (0.0000–0.0032 µg kg−1 bw d−1 ), followed by tubers (0.0012–0.0029 µg kg−1 bw d−1 ), eggs (0.0003–0.0028 µg kg−1 bw d−1 ), and fruits (0.0000–0.0011 µg kg−1 bw d−1 ). We estimated the dietary exposure level specifically by age in both lower bound (LB) and upper bound (UB) (Tables S7 and S8). At 0–2 years, Tl exposure through vegetables was the highest, with 0.0000–0.0055 µg kg−1 bw d−1 . The exposure through eggs reached a maximum level at 3–6 years (0.0010–0.0086 µg kg−1 bw d−1 ), 7–12 years (0.0006–0.0052 µg kg−1 bw d−1 ), and 13–19 years (0.0003–0.0028 µg kg−1 bw d−1 ), respectively. In adults aged 20–64 years, exposure through vegetables ranged from 0.0000 to 0.0031 µg kg−1 bw d−1 , and in the people aged 65 years and older, tubers were the highest at 0.0009–0.0043 µg kg−1 bw d−1 . Because our detection limits and Tl concentrations were low, the difference between LB and UB was minimal. Thus, we reported the exposure dose and % HBGV based on the UB (Table 4). Table 4. Exposure assessment and risk characterization for Tl. Food Group Agricultural Products Livestock Products Fishery Products Processed Products Cereals Fruits Beans Mushrooms Tuber Crops Vegetables Eggs Meats Crustaceans Fishes Cephalopoda Shellfishes Seaweeds Beverages Other Processes Products Concentration (µg kg−1 ) Exposure Dose (µg kg−1 bw day−1 ) % HBGV 0.01–5.27 0.01–7.00 0.01–2.32 0.01 3.78–13.21 0.01–15.93 0.65–5.72 0.51–1.63 1.07–1.79 0.41–1.87 1.04–1.22 1.20–6.02 0.01–12.72 0.01–4.12 0.0000–0.0001 0.0000–0.0011 0.0000–0.0001 0.0000–0.0000 0.0012–0.0029 0.0000–0.0032 0.0003–0.0028 0.0001–0.0009 0.0000–0.0000 0.0000–0.0001 0.0000–0.0001 0.0000–0.0001 0.0000–0.0002 0.0000–0.0003 0.0000–0.0345 0.0000–0.5625 0.0000–0.0549 0.0001–0.0002 0.6050–1.4682 0.0001–1.5807 0.1604-1.4109 0.0502-0.4326 0.0130-0.0216 0.0027-0.0440 0.0143-0.0445 0.0106-0.0606 0.0000-0.0900 0.0001-0.1549 0.00–19.46 0.0000–0.0014 0.0000-0.6881 HBGV is the health-based guidance value: % HBGV = (estimated daily intake/HBGV) × 100% HBGV was calculated based on HBGV of Tl = 0.2 µg kg−1 bw day−1 . In all age and dietary groups, no exposure estimation surpassed the health-based guidance value. Our results indicate that the risk of exposure through Tl-contaminated food ingestion is at a minimum level. Molecules 2021, 26, 6729 9 of 14 3. Materials and Methods 3.1. Instrumentation Tl was determined using an Agilent 7700x (Agilent Technologies, Santa Clara, CA, USA) ICP-MS with ion monitoring at mass-to-charge ratios (m/z) 203 and 205 to gather the data. The specific instrument parameters and analytical conditions are described in Table S9. 3.2. Reagents and Solutions The reagents used for this study were of analytical grade, and the water was deion-ized and purified below 18.2 MΩ using a YL WPS System (Young Lin Instrument Co., Anyang-si, Kyunggi-do, Korea). As a standard solution for Tl, the periodic table mix 1 for the ICP solution (Sigma-Aldrich, St. Louis, MO, USA) was diluted with 2.5% nitric acid to 100 µg kg−1 of stock solution. Analytical grade nitric acid (Chemitop, Jin-cheon, Cheongbuk-do, Korea) and hydrogen peroxide (Chemitop, Jincheon, Cheongbuk-do, Korea) were used. BCR-679 (white cabbage) certified reference materials (CRM) for method validation and quality assurance were purchased from the European Commission (European Com-mission, Joint Research Centre, Institute for Reference Materials and Measurements, Geel, Belgium). 3.3. Sample Collection and Processing According to KNHANES, monitored samples were chosen to represent commonly consumed foods. Additionally, we referred to a report on the reassessment of toxic metal(loid)s criteria in South Korean food published by the Korea Ministry of Food and Drug Safety (MFDS) [54,56]. For representativeness, agricultural (113 samples), animal (12 samples), and fishery products (50 samples) were purchased from more than three distinct locations. For processed foods (129 samples), more than three products were purchased per manufacturer. Before homogenizing, the samples were washed with deionized water, and the surface moisture and non-edible components (e.g., peels, seeds, and bones) were removed. Fruits and vegetables were dried at 65 ◦ C for 18 h before homogenization with a lab blender for 60 s and sealed at −18 ◦ C. For infusion teas, 1.2 g of tea sachet was immersed in 100 mL of distilled water at 80 ◦ C for 2 min. We used the infused water as the samples. 3.4. Sample Preparation The current study’s sample preparation procedure was based on the elemental analysis manual of the US Food and Drug Administration (US FDA) [57] and guidelines for the analysis of toxic metal(loid)s in food by MFDS [58]. We classified foods into six categories based on their energy (i.e., calories) and matrix. The calorific value of each food was determined using the MFDS food nutrient database [59]. The classification included nonfatty solids, hydrated solids, fatty solids, salty solids, non-fatty liquids, and fatty liquids. Table 5 lists the sample weights for various sample classifications. The samples were pre-decomposed on a hot plate with 4 mL of nitric acid (70 vol%) and 1 mL of hydrogen peroxide (30 vol%) in a polytetrafluoroethylene (PTFE) vessel. After cooling at 25 ◦ C, 3 mL of nitric acid (70 vol%) was added. PTFE vessels were sealed and placed in a microwave digestion system (ETHOS Easy, Milestone, Italy). After digestion, the samples were cooled at 25 ◦ C and diluted to 20.0 g (in the case of salty foods, they were diluted to 80.0 g) with distilled water. All samples were analyzed in triplicates. The hydrated solid foods were converted to wet-based concentrations by considering the moisture content before drying. Molecules 2021, 26, 6729 10 of 14 Table 5. Classification into food matrix groups and sample weights for preparation. Classification Sample Weight (g) Food Group Non-Fatty Solid Hydrated Solid Fatty Solid Salty Solid Non-Fatty Liquid Fatty Liquid 0.5 0.5 0.5 0.1 0.5 0.3 Cereals, Tubers, Beans, Snacks, Cereal Products Fruits, Vegetables, Mushrooms, Sea Algae Meats, Eggs, Fishes, Cephalopods, Shellfishes, Crustaceans Sauces, Pickled Foods, Salt-Added Products, Noodles Infusion Teas, Other Beverages Oil Products, Dairy Products 3.5. Single-Lab Validation and Quality Control Food comprises various organic components. In the method validation for determining toxic metal(loid)s, the matrix effect is one of the critical factors in ICP-MS analysis. We selected six representative foods for each category, taking into account the effects of matrices, such as salts and fats, and validated the method for determining Tl. We confirmed the factors for method validation in terms of linearity, trueness, accuracy, and precision. The linearity for external standard calibration curves was measured in stock solution diluted to 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, and 2.0 µg kg−1 . The trueness of Tl determination was verified using CRM Tl mass fractions. The measured values were compared with the certification values seven times. The accuracy and precision were calculated intraday and interday. The standard solution was spiked into each representative sample, resulting in the final concentrations of 0.1 µg kg−1 (low concentration), 0.5 µg kg−1 (medium concentration), and 1.0 µg kg−1 (high concentration) in seven replicates, respectively. The measurement uncertainty was calculated for the rice matrix spiked with a medium concentration of Tl standard solution. The sources of measurement uncertainty were expressed in the form of a fishbone diagram (Figure S1). The MLOD and MLOQ were determined using the standard addition method, which involved adding five different Tl concentrations (0.05, 0.1, 0.25, 0.5, and 1.0 µg kg−1 ) to representative foods for calibration curve with seven repetitions. The equations are as follows: MLOD = 3.14× S/σ and MLOQ = 10 × S/σ (1) where 3.14 = student t factor for seven replicates, S = slope mean, and σ = standard deviation of intercept. For quality control, CRM was analyzed for every batch of samples, and recovery was checked within 80–120%. 3.6. Statistical Analysis The analysis data were processed using SPSS 21 (IBM Corporation, New York, NY, USA) to compare the levels among the food groups. Significant differences were defined using t-tests or one-way ANOVA. Statistical significance was set at p < 0.05. For statistical calculation, values below MLOD were replaced with half the MLOD values, and its natural logarithm was used. 3.7. Risk Assessment and Exposure to Thallium Risk assessment was conducted based on monitoring data, daily consumption value, and the average body weight derived from KNHANES. To evaluate the risk of potential exposure, we used the HBGV approach. In the present study, % HBGV was calculated using 0.2 µg kg−1 bw day−1 as the HBGV of Tl [46]. Estimated daily intake (EDI) and health risk (% HBGV) were determined using the following equations: EDI (µg/kg bw/day) = Daily consumption (g/day) × Tl concentration (µg/kg) Average body weight (kg) (2) Molecules 2021, 26, 6729 11 of 14 where average body weights (kg) by age are: 0 to 2 years (11.52 kg), 3 to 6 years (18.75 kg), 7 to 12 years (35.76 kg), 13 to 19 years (59.72 kg), 20 to 64 years (64.93 kg), over 65 years (60.20 kg). EDI % HBGV = × 100 (3) HBGV 4. Conclusions ICP-MS was used to validate analytical methods for six typical foods selected based on food matrices and calories. Tl levels were monitored in 304 commonly consumed food items. As a result, Tl was detected in 148 samples, with 97% of the measured samples containing less than 10 µg kg−1 Tl. We examined Tl levels in agricultural, fishery, and livestock products and found that Tl concentrations in animal foods are higher than in plant foods. The results confirmed that the level of Tl exposure through food intake was within the health-based guidance value. Further research and regular monitoring of Tl in foods are necessary to protect human health and avoid food-borne contamination. Supplementary Materials: The following are available online: Table S1: Evaluation of intraday and interday accuracy and precision at low, medium, and high concentrations of thallium (Tl) in representative foods; Table S2: Tl content (fresh weight) in specific items of agricultural products; Table S3: Tl content (fresh weight) in specific items of livestock products; Table S4: Tl content (fresh weight) in specific items of fishery products; Table S5: Tl content (fresh weight) in specific items of beverages; Table S6: Tl content (fresh weight) in specific items of other processed foods; Table S7: Estimated dietary exposure to Tl by age (Lower Bound); Table S8: Estimated dietary exposure to Tl by age (Upper Bound); Table S9: Instrumental conditions of ICP-MS for the determination of Tl; Figure S1: Fishbone diagram for Tl analysis uncertainty for rice. Author Contributions: Conceptualization, methodology, data curation, software, formal analysis, and writing—original draft preparation, Y.-h.K. and K.-W.L.; validation, Y.-h.K. and W.-j.R.; formal analysis, Y.-h.K., W.-j.R., and B.S.; risk assessment, S.C.(Solyi Cho) and S.C.(Shinai Choi); data curation and supervision, Y.J.; supervision, writing—review and editing, K.-W.L.; project administration and funding acquisition, K.-W.L. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Ministry of Food and Drug Safety, Republic of Korea (grant number 20162MFDS037) in 2020. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: This work was supported by the School of Life Sciences and Biotechnology for BK21 PLUS, Korea University. 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Клинический случай І Case report DOI: 10.33925/1683-3031-2023-642 Эстетическая и функциональная реабилитация детей раннего возраста с использованием искусственных коронок из диоксида циркония С.В. Каменева, Д.М. Омехина, Н.В. Кущенко, А.В. Голоднюк Первый Санкт-Петербургский государственный медицинский университет имени академика И.П. Павлова, Санкт-Петербург, Российская Федерация АННОТАЦИЯ Актуальность. Необходимость в функциональной реабилитации детей раннего возраста – достаточно распространенная проблема в современной детской стоматологии. Это связано с тем, что ребенок в возрасте до трех лет имеет поведенческие особенности, которые затрудняют оказание стоматологической помощи. Относительно недавно появились стандартные детские коронки из диоксида циркония, обладающие высокими эстетическими и механическими характеристиками Цель исследования. Проанализировать на примере клинических случаев применение коронок из диоксида циркония для функциональной реабилитации и профилактики заболеваний пародонта у детей раннего возраста. Описание клинического случая. В исследование представлен клинический случай функциональной и эстетической реабилитации детей в возрасте до трех лет при помощи искусственных коронок из диоксида циркония. Заключение. Коронки из диоксида циркония являются перспективной альтернативой другим способам восстановления центральных зубов у детей раннего возраста. На основании клинического случая показано, что циркониевые коронки выглядят естественно, в полной мере восполняют дефект, восстанавливают нарушенную функцию без отдаленных осложнений, а также не оказывают отрицательного воздействия на состояние пародонта. Ключевые слова: детская стоматология, острый детский кариес, коронки из диоксида циркония, профилактика заболеваний пародонта у детей. Для цитирования: Каменева СВ, Омехина ДМ, Кущенко НВ, Голоднюк АВ. Эстетическая и функциональная реабилитация детей раннего возраста с использованием искусственных коронок из диоксида циркония. Стоматология детского возраста и профилактика. 2023;23(2):184-190. DOI: 10.33925/1683-3031-2023-642. Aesthetic and functional rehabilitation of young children using zirconia crowns S.V. Kameneva, D.M. Omekhina, N.V. Kushchenko, A.V. Golodniuk Pavlov First Saint Petersburg State Medical University, Saint Petersburg, Russian Federation ABSTRACT Relevance. The need for functional restoration in young children is a sufficiently common problem in modern pediatric dentistry due to the behaviour characteristics of a child under three years old, which hinder dental treatment. Prefabricated zirconia crowns for children appeared relatively recently and have high aesthetic and functional properties. Purpose. To analyze the application of zirconia crowns for functional rehabilitation and periodontal disease prevention in young children, exemplified by clinical cases. Clinical case description. The study presents a clinical case of functional and aesthetic restoration with zirconia crowns in a child under three years old. Conclusion. Zirconia crowns are a promising alternative to other methods of restoring anterior teeth in a toddler. The clinical case shows that zirconia crowns look natural, fully compensate for the defect, restore the lost function without long-term complications and do not adversely affect the periodontium. Key words: pediatric dentistry, severe early childhood caries, zirconia crowns, prevention of periodontal disease in children. For citation: Kameneva SV, Omekhina DM, Kushchenko NV, Golodniuk AV. Aesthetic and functional rehabilitation of young children using zirconia crowns. Pediatric dentistry and dental prophylaxis. 2023;23(1):184-190. (In Russ.). DOI: 10.33925/1683-3031-2023-642. 184 2023; 23(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis Клинический случай І Case report АКТУАЛЬНОСТЬ В современной детской стоматологии наблюдается тенденция снижения возраста пациентов, обращающихся к стоматологу для лечения. Наиболее распространенным заболеванием зубов в детском возрасте является кариес, 60-90% детей имеют хотя бы одну кариозную полость. Наиболее агрессивным видом кариеса считается острейший кариес временных зубов, который встречается у детей в возрасте до трех лет [1]. Он имеет свои особенности, среди которых можно выделить: типичную локализацию (резцы верхней челюсти), наличие трех-четырех кариозных полостей на одном зубе, плоскостное и циркулярное распространение кариеса, симметричность поражения, высокий риск вовлечения пульпы и периодонта с последующим отколом коронки. Все это в совокупности часто влечет за собой эстетические и функциональные нарушения. В раннем детском возрасте также высока вероятность травмы зубов, обусловленная пиком любознательности и развития детей при отсутствии «охранительного» рефлекса. Исходя из клинической практики и проведенных исследований, вывихи и переломы коронок верхних центральных резцов встречаются наиболее часто [2]. Обширное разрушение передней группы зубов вследствие различных причин ведет как к функциональному, так и к эстетическому нарушению. С точки зрения функционального нарушения важно рассмотреть два аспекта. Во-первых, при разрушении передней группы зубов происходит нарушение функции откусывания и отрывания пищи, что влечет за собой перекладывание данной функции на боковой отдел зубов, который не приспособлен к ней за счет отсутствия режущих краев. Во-вторых, передние зубы играют важную роль в звукообразовании. Так, при разрушении или утере резцов формируется передний «открытый» прикус, который характеризуется наличием вертикальной щели в переднем отделе зубного ряда, которая, в свою очередь, влияет на ухудшение произношения таких звуков, как Л, Т, Д, Н, а также ведет к межзубному произношению свистящих и шипящих звуков [3]. Проблема эстетической неполноценности вследствие разрушения зубов носит очень серьезный характер. Это обосновано тем, что внешний вид ребенка влияет на его социализацию. Как правило, путь социализации ребенка начинается с двух лет в дошкольном учреждении. К этому возрасту дети способны сравнивать предметы, а также проявлять интерес к собственному отражению в зеркале. Безусловно, в раннем возрасте дети не будут подвержены насмешкам со стороны сверстников, но видеть отличие своих разрушенных зубов от здоровых они будут. При восстановлении разрушенных зубов необходимо помнить и о риске возникновения локальных заболеваний пародонта, обусловленных неправильно восстановленным контактным пунктом [4]. Необходимость в функциональной реабилитации детей раннего возраста – достаточно распространенная проблема в современной детской стоматологии. Это связано с тем, что ребенок в возрасте до трех лет имеет поведенческие особенности, которые затрудняют оказание стоматологической помощи. Применение стандартных коронок из нержавеющей стали у детей является рекомендованным методом протезирования детей при поражении кариесом двух и более поверхностей зуба. Этот клинически эффективный и безопасный вариант восстановления зубов часто отклоняется родителями по причине неудовлетворительной эстетики. Родители предпочитают обычные реставрации с использованием эстетических материалов – композитов или СИЦ. На сегодняшний день все большее распространение для восстановления зубов в детской стоматологии получают стандартные коронки из диоксида циркония. Высокий спрос на использование искусственных коронок из диоксида циркония объясняется их более высокими эстетическими характеристиками. Стоит также отметить, что циркониевые коронки не уступают по механическим характеристикам коронкам из нержавеющей стали и могут применяться у детей любого возраста как в переднем отделе, так и в боковом. Но несмотря на повышенный спрос, искусственные коронки из диоксида циркония не применяют повсеместно, причина тому – отсутствие литературы с доказательствами эффективности применения данного вида коронок у детей раннего возраста. Поэму целью данного исследования стал анализ клинического случая применения коронок из диоксида циркония у детей раннего возраста. ОПИСАНИЕ КЛИНИЧЕСКОГО СЛУЧАЯ Пациент А. Пол: женский. Дата рождения: 10.10.2021 г. (на момент обращения возраст 1 год 7 месяцев). Жалобы: дефект резцов верхней челюсти. Нарушение приема пищи, отказ ребенка от кислой и сладкой пищи. Анамнез жизни: 1. Перенесенные заболевания: аллергоанамнез не отягощен, заболевания со стороны сердечно-сосудистой системы, эндокринной системы и ЖКТ отсутствуют. 2. Инфекционные заболевания родители отрицают. 3. Непереносимость лекарственных препаратов: отсутствует. 4. Наследственность: не отягощена. 5. Беременность у матери первая, токсикоз I триместра беременности, родоразрешение путем кесарева сечения, гипоксия плода. Искусственное вскармливание с 1,5 месяцев. 6. Вредные привычки: ночное кормление молочными продуктами из бутылочки. 7. Гигиена полости рта: нерегулярная чистка зубов один раз в день при помощи силиконовой зубной щетки и зубной пасты «Дракоша». Развитие настоящего заболевания: родители обратились в клинику с целью реставрации разрушенных центральных резцов верхней челюсти. 2023; 23(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis 185 Клинический случай І Case report Внешний осмотр: общее состояние удовлетворительное, сознание ясное, положение активное, тип телосложения нормостенический, рост 83 см, вес 11 кг. Температура тела 36.6, кожа нормального цвета без высыпных элементов, конфигурация лица не изменена, лимфатические узлы овальные, пальпация безболезненна, симметричная. Осмотр полости рта: слизистая оболочка бледнорозовая, умеренно увлажнена, высыпных элементов нет. В полости рта присутствует 10 зубов (5.4, 5.2, 5.1, 6.1, 6.2, 6.4, 7.2, 7.1, 8.1, 8.2). На вестибулярной поверхности 5.2, 5.1, 6.1, 6.2 отмечаются симметричные пятна. При зондировании на 5.1 и 6.1 зубе под слоем пораженной эмали обнаружен размягченный дентин. 5.2 и 6.2 зуб разрушены, пульпарная камера вскрыта. Индекс Loe & Silness: 5.2, 5.1, 6.1, 6.2 – 2 балла (умеренное воспаление – покраснение, отечность, кровоточивость при зондировании); 5.4, 6.4, 7.2, 7.1, 8.1, 8.2 – 1 балл (незначительное воспаление – легкая отечность, отсутствие кровоточивости при зондировании); общая оценка: 1.4 балл (гингивит средней тяжести). Основной диагноз: К00.40 Гипоплазия эмали 5.1, 5.2, 6.1, 6.2 зубов. Осложнение: К.02.1 Кариес дентина 5.1, 6.1 зубов. К.04.0 Обратимый пульпит 5.2, 6.2 зубов. Ход лечения Лечение проведено в условиях общего обезболивания (ингаляционный газ Севоран). В условиях изоляции при помощи системы коффердам проведена механическая и медикаментозная обработка кариозных полостей 5.1 и 6.1 зуба. 5.2 и 6.2 зуб – пульпотомия с применением препарата «Биодентин», изолирующая подкладка IRM. Произведена фиксация искусственных коронок из диоксида циркония на 5.1, 5.2, 6.1, 6.2 зубы при помощи цемента «Фуджи 1». Рекомендации: 1. Посещение стоматолога 1 раз в 3 месяца. 2. Гигиена полости рта 2 раза в день под контролем родителей при помощи мягкой зубной щетки и пастой с содержанием фтора 1000 ppm. ОБСУЖДЕНИЕ Восстановление разрушенных передних молочных зубов у детей требуется для профилактики нарушения прикуса, нормализации функции откусывания пищи и звукообразования, предотвращения развития заболеваний пародонта. Формирование нейтрального прикуса зависит от соотношения челюстей и положения зубов. В свою очередь, челюсти Рис. 1. Клиническая картина до начала лечения. Гипоплазия эмали 5.1, 5.2, 6.1, 6.2 зубов, осложненная кариозным процессом: а) вестибулярная поверхность; а/a б) небная поверхность Fig. 1. Clinical pictures before the treatment. Enamel hypoplasia of teeth 5.1, 5.2, 6.1, 6.2 complicated by caries: a) labial surface b) palatal surface б/b а/a б/b 186 Рис. 3. Клиническая картина после окончания лечения. Искусственные коронки из диоксида циркония на 5.1, 5.2: а) вестибулярная поверхность; б) небная поверхность Fig. 3. Clinical pictures before the treatment. Enamel hypoplasia of teeth 5.1, 5.2, 6.1, 6.2 complicated by caries: a) labial surface b) palatal surface Рис. 2. Прицельная рентгенограмма 5.1, 5.2, 6.1, 6.2 зубов Fig. 2. Periapical X-rays of teeth 5.1, 5.2, 6.1, 6.2 Рис. 4. Контрольная прицельная рентгенограмма после фиксации коронок на 5.1, 5.2, 6.1, 6.2 зубы Fig. 4. Control X-ray after crown cementation to teeth 5.1, 5.2, 6.1, 6.2 2023; 23(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis Клинический случай І Case report развиваются и растут за счет жевательной нагрузки как на кость, так и на зубы во время жевания. Выпадение из окклюзии хотя бы одного зуба ведет к перераспределению жевательного давления и, как следствие, к нарушению роста челюстей и формированию патологического прикуса [5]. Кроме скелетной формы патологического прикуса, вызванной неправильным ростом челюстей, существует зубная форма, обусловленная неправильным положением зубов и их ретенцией. В большинстве случаев (61,8%) ретенция постоянных резцов обусловлена ранней потерей и разрушением временных резцов [6]. В свою очередь, неправильно расположенные центральные резцы влияют на формирование постоянного прикуса. Торк центральных резцов верхней и нижней челюсти имеет важное значение в становлении прикуса у ребенка. Во-первых, торк влияет на глубину резцового перекрытия – может сформироваться открытый, глубокий или прямой прикус во фронтальной плоскости. Во-вторых, значение угла наклона центральных резцов влияет на положение больших и малых коренных зубов. В свою очередь, положение жевательной группы зубов влияет на межальвеолярную высоту и формирование нейтрального прикуса. Особую роль играет оральная поверхность верхних центральных резцов, так как она является направляющей при движении нижней челюсти вперед. При нарушении длины и угла наклона верхних центральных резцов происходит изменение суставной и зубной траектории нижней челюсти [7]. Применение стандартных коронок из нержавеющей стали (SSC) у детей является рекомендованным методом протезирования детей при поражении кариесом двух и более поверхностей зуба. Этот клинически эффективный и безопасный вариант восстановления зубов часто отклоняется родителями по причине неудовлетворительной эстетики. Прямая реставрация молочного зуба композитом – одна из наиболее классических методик восстановления разрушенного зуба. К ее преимуществам следует отнести: эстетичность (возможность подбора цвета в стоматологическом кресле), доступность и дешевизну. Среди недостатков прямых реставраций композитным материалом следует выделить: прямую зависимость результата реставрации от мануальных навыков врача, большое количество времени, необходимое для восстановления анатомической формы зуба, низкую износостойкость, высокую степень усадки, необходимость полной изоляции зуба от ротовой жидкости, высокий риск сколов при объемном восстановлении, небольшой срок службы, нарушение краевого прилегания пломб (и, как следствие, рецидив кариозного процесса), нуждаемость в регулярной полировке пломбировочного материала с целью профилактики возникновения ретенционных пунктов зубного налета, способных привести к воспалительным заболеваниям пародонта (например, локальному гингивиту). Прямые реставрации молочных зубов можно выполнить также стеклоиономерными цементами и их производными – компомерами. Главным преимуществом данных пломбировочных материалов является выделение в окружающую среду ионов фтора, за счет которого на границе твердых тканей зубов образуются фторапатиты, что обеспечивает дополнительную профилактику кариеса [8]. Однако они обладают весомым недостатком в виде высокого водопоглощения, что ведет к гигроскопическому расширению материала. Стоит отметить, что эстетичность и цветостойкость у них ниже, чем у композиционных материалов. Альтернативной методикой восстановления разрушенных молочных зубов являются искусственные коронки. В современной детской стоматологии для восстановления передних молочных зубов применяются металлические коронки с композитной облицовкой, стрип-коронки и циркониевые коронки. Металлические коронки с композитной облицовкой – это детские стандартные штампованные коронки, покрытые слоем композиционного материала, имеющие различную форму и размер. К преимуществам коронок данного вида стоит отнести: эстетичность, отсутствие рецидива кариеса, щадящее препарирование зуба без создания уступа, низкую стоимость. Однако данные коронки имеют большое количество недостатков. Среди них можно выделить: низкую цветостойкость и износостойкость, слабую адгезию композита с металлической основой, неудовлетворительную прочность композита на сжатие (ведет к появлению сколов), высокий риск расцементировки. Данный вид искусственных коронок является приоритетным при восстановлении зубов с гипоминерализованной эмалью [9]. Альтернативным эстетическим вариантом для реставрации зубов переднего отдела являются стрипкоронки. Они представляют собой целлулоидные колпачки, которые имеют различную форму. Колпачки после припасовки заполняются композиционным материалом (преимущественно микронаполненным или гибридным) [10]. Данная методика имеет ряд преимуществ: эстетичность, щадящее препарирование, простота методики, низкая стоимость и времязатратность. Несмотря на доступность данной методики, повсеместное ее применение ограничено из-за ряда негативных свойств: необходимость полной изоляции, высокая степень усадки из-за объема композиционного материала, недостаточная прочность материала на сжатие, низкая износостойкость, образование ретенционных пунктов из-за несовершенства полировки, высокий риск осложнений со стороны пародонта [11]. Согласно данным литературы искусственные коронки из диоксида циркония на передние зубы – это один из способов реабилитации детей раннего возраста. 2023; 23(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis 187 Клинический случай І Case report Стандартные коронки из диоксида циркония получают все большее распространение для восстановления фронтальной группы зубов у детей. Среди положительных сторон данных коронок, кроме эстетики, можно выделить: биосовместимость, предупреждение рецидива кариеса, высокую прочность на изгиб, стабильность цвета, низкую степень износа. Кроме этого, при сравнении уровня гигиены у детей спустя год после протезирования было выявлено, что у детей, зубы которых покрыты коронками из диоксида циркония, зубной налет скапливается в меньшей степени, чем у детей, чьи зубы покрыты иными видами искусственных коронок. В результате снижается риск возникновения заболеваний пародонта (в частности локального гингивита), отсутствует негативное влияние на пародонт зуба. При протезировании зубов коронками из диоксида циркония кровоточивость отмечается в меньшем количестве случаев, чем при протезировании композитными коронками и коронками из нержавеющей стали [11]. К недостаткам циркониевых коронок можно отнести большую редукцию твердых тканей по сравнению с коронками из нержавеющей стали, а также сложность регулировки для механической ретенции [12]. При фиксации искусственных коронок из диоксида циркония лишь на одну из челюстей происходит стирание твердых тканей зубов-антагонистов. Было проведено исследование, во время которого отслеживали степень износа зубов-антагонистов по индексу Смита и Найта спустя 3, 6, и 12 месяцев после фиксации циркониевых коронок. Спустя 3 и 6 месяцев состояние эмали не изменилось. Спустя 12 месяцев у 11,7% исследуемых была отмечена минимальная потеря контуров и минимальные изменения поверхности эмали (р < 0,02). Данная динамика свидетельствует о том, что со временем стираемость противоположных зубов увеличивается и данные коронки лучше не использовать у пациентов с низкой степенью минерализации эмали, либо необходимо фиксировать искусственные коронки и на зубы-антагонисты с целью профилактики износа [11]. В то же время Murad Alrashdi и соавторы (2022) на основании обзора литературы утверждают, что достоверно значимого стирания зубов-антагонистов не было выявлено. Фиксация края искусственной коронки в области биологической ширины зуба при тонкой альвеолярной кости может привести к атрофии костной ткани и рецессии десны. Расположение края искусственной коронки глубже биологической ширины зуба может повлечь за собой рецидивирующий гингивит, при этом уровень костной ткани остается неизменным [14]. Стоит также отметить, что в качестве осложнений встречаются редкие случаи расцементировки искусственной коронки, которая происходит за счет того, что несоответствие формы культи зуба и стандартной коронки компенсируется большим количеством композиционного цемента. Циркониевые коронки, как и другие конструкции, имеют свои показания, к ним следует отнести: мультиповерхностный кариес, кариес с вовлечением режущего края, травму зуба, покрытие зубов после проведенного эндодонтического лечения по поводу пульпита и периодонтита, дисколорит передних зубов, «бутылочный» кариес. Однако коронки из диоксида циркония имеют относительные противопоказания: повышенная стираемость зубов, скученное положение зубов, воспаление пародонта [14]. ЗАКЛЮЧЕНИЕ Коронки из диоксида циркония являются перспективной альтернативой другим способам восстановления центральных зубов у детей раннего возраста. Эстетико-функциональная реабилитация детей раннего возраста с использованием искусственных коронок из диоксида циркония может быть предпочтительным вариантом лечения маленьких детей с зубами, имеющими обширные (более 50%) разрушения коронковой части зуба. Данный вид конструкций не только восполняет эстетический и функциональный дефекты и нарушенную функцию, но и препятствует дальнейшему разрушению зубного ряда, способствует правильному формированию прикуса и полноценному звукообразованию. Преимуществом циркониевых коронок является минимизация рисков возможных осложнений, что важно при лечении детей до трех лет. Лечение детей раннего детского возраста чаще всего проходит в условиях общего обезболивания, что осложняет возможности проведения повторного лечения для устранения осложнений. На основании клинических случаев показано, что циркониевые коронки выглядят естественно, в полной мере восполняют дефект, восстанавливают нарушенную функцию без отдаленных осложнений, а также не оказывают отрицательного воздействия на состояние пародонта. СПИСОК ЛИТЕРАТУРЫ 1. Frencken JE, Sharma P, Stenhouse L, Green D, Laverty D, Dietrich T. Global epidemiology of dental caries and severe periodontitis - a comprehensive review. J Clin Periodontol. 2017;44 Suppl 18:S94-S105. doi:10.1111/jcpe.12677 2. Муртазаев С, Махмудова Д. Современные мето- 188 ды лечения острых травм зубов у детей. Stomatologiya. 2021; 2(83):83–86. doi: 10.34920/2091-5845-2021-57 3. Фоменко ИВ. Влияние открытого и мезиального прикуса на качество звукопроизношения у детей при смене зубов. Современные методы диагностики, 2023; 23(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis Клинический случай І Case report лечения, и профилактики стоматологических заболеваний. 2018:86-88. Режим доступа: https://www.elibrary.ru/item.asp?id=36090440&pff=1 4. Орехова ЛЮ, Прохорова ОВ, Шефов ВЮ. Качественное анатомическое восстановление контактного пункта зубов – профилактика возникновения локализованных форм заболеваний пародонта. Пародонтология. 2020;25(1):10-15. doi: 10.33925/1683-3759-2020-25-1-10-15 5. Паршукова АИ, Шестакова ВИ. Методы ортопедического лечения детей при ранней потере молочных зубов. Приоритетные направления развития науки и образования. 2021:252-254. Режим доступа: https://www.elibrary.ru/item.asp?id=44578062 6. Бимбас ЕС, Шишмарева АС, Мельникова МА, Кайем ВМ, Шишмарева ЮС. Сочетание факторов – ранняя потеря верхних временных резцов и ротовое дыхание – в формировании ретенции постоянных резцов. Клинический случай. Проблемы стоматологии. 2018;14(3):73-78 doi: 10.18481/2077-7566-2018-14-3-73-78 7. Давыдов БН, Дмитриенко СВ, Доменюк ДА, Самедов ФВ, Иванюта ИВ, Иванюта СО. Современные концепции в подходах к определению индивидуальной позиции медиальных резцов у людей с физиологическими видами прикуса. Медицинский алфавит. 2019;4(34):16-22. doi: 10.33667/2078-5631-2019-4-34(409)-16-22 8. Луцкая ИК. Опыт использования компомера для пломбирования временных зубов. Современная стоматология. 2018;(3):43-47. Режим доступа: https://cyberleninka.ru/article/n/opyt-ispolzovaniyakompomera-dlya-plombirovaniya-vremennyh-zubov/viewer 9. Галонский ВГ, Сурдо ЭС, Макарчук МЮ, Бриль ЕА, Журавлева ТБ. Восстановление дефектов твердых тканей зубов искусственными коронками в клинической практике стоматологии детского возраста (обзор литературы, часть 2). Актуальные проблемы и перспективы развития стоматологии в условиях Севера. Сборник статей Межрегиональной научнопрактической конференции, посвященной 65-летию Медицинского института ФГАОУ ВО «Северо-Восточный федеральный университет имени М.К. Аммосова». 2022:339-355. Режим доступа: https://www.elibrary.ru/item.asp?edn=fqotrc 10. Yang JN, Mani G. Crowns for Primary Anterior Teeth. Int J Pedod Rehabil. 2017;1:75-78. doi: 10.4103/2468-8932.196491 11. Alaki SM, Abdulhadi BS, AbdElBaki MA, Alamoudi NM. Comparing zirconia to anterior strip crowns in primary anterior teeth in children: a randomized clinical trial. BMC Oral Health. 2020;20(1):313. doi: 10.1186/s12903-020-01305-1 12. Alzanbaqi SD, Alogaiel RM, Alasmari MA, Al Essa AM, Khogeer LN, Alanazi BS, et al. Zirconia Crowns for Primary Teeth: A Systematic Review and Meta-Analyses. Int J Environ Res Public Health. 2022;19(5):2838. doi: 10.3390/ijerph19052838 13. Татаринова ЕН, Соболева НН, Сбитнева ОВ. Вариант реабилитации детей с травмы зубов. Актуальные проблемы стоматологии детского возраста. Материалы VII Всероссийской научно-практической конференции. 2021:276-283. Режим доступа: https://www.elibrary.ru/item.asp?id=47370828&pff=1 14. Ninawe N, Suyash J,Badhe H, Honaje N, Bhaje P, Barjatya K. Zirconia Crowns In Pediatric Dentistry: A Review. Journal of Positive School Psychology. 2022;6(8):1718-1724. Режим доступа: https://journalppw.com/index.php/jpsp/article/ view/10035 REFERENCES 1. Frencken JE, Sharma P, Stenhouse L, Green D, Laverty D, Dietrich T. Global epidemiology of dental caries and severe periodontitis – a comprehensive review. J Clin Periodontol. 2017;44 Suppl 18:S94-S105. doi: 10.1111/jcpe.12677 2. Murtazaev S, Makhmudova D. Modern methods of treatment of acute dental injuries in children. Stomatologiya. 2021; 2(83):83–86 (In Russ.). doi: 10.34920/2091-5845-2021-57 3. Fomenko IV. Influence of open and mesial bite on the quality of sound production in children during tooth replacement.Modern methods of diagnosis, treatment, and prevention of dental diseases. 2018:86-88 (In Russ.). Available from: https://www.elibrary.ru/item.asp?id=36090440&pff=1 4. Orekhova LYu, Prokhorova OV, Shefov VYu. Qualitative anatomical restoration of the contact point of teeth – prevention of localized forms of periodontal diseases. Parodontologiya. 2020;25(1):10-15 (In Russ.). doi: 10.33925/1683-3759-2020-25-1-10-15 5. Parshukova AI, Shestakova VI. Methods of orthopedic treatment of children with early loss of deciduous teeth. Priority directions of development of science and education. 2021:252-254 (In Russ.). Available from: https://www.elibrary.ru/item.asp?id=44578062 6. Bimbas ES, Shishmareva AS, Mel’nikova MA, Kajem VM, Shishmareva US. The combination of factors – early loss of upper temporal incisors and mouth breathing – in the formation of retention of permanent incisors. Clinical case. Actual problems in dentistry. 2018;14(3):73-78 (In Russ.). doi: 10.18481/2077-7566-2018-14-3-73-78 7. Davydov BN, Dmitrienko SV, Domenyuk DA, Samedov FV, Ivanyuta IV, Ivanyuta SO. Modern concepts in approaches to determination of individual position of medial incisors in people with physiological types of bite. Medical Alphabet. 2019;4(34):16-22 (In Russ.). doi: 10.33667/2078-5631-2019-4-34(409)-16-22 8. Lutskaya IK. Experience in the use of compomer for the filling of temporary teeth. Sovremennaya stoma- 2023; 23(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis 189 Клинический случай І Case report tologiya. 2018;(3):43-47 (In Russ.). Available from: https://cyberleninka.ru/article/n/opyt-ispolzovaniyakompomera-dlya-plombirovaniya-vremennyh-zubov/viewer 9. Galonsky VG, Surdo ES, Makarchuk MU, Bril EA, Zhuravleva TB.Restoration of defects in the hard tissues of teeth with artificial crowns in the clinical practice of pediatric dentistry (literature review, part 2). Actual problems and prospects of dentistry development in the conditions of the North. Collection of articles of the Interregional scientific and practical conference dedicated to the 65th anniversary of the Medical Institute of the Northeastern Federal University named after M.K. Ammosov. 2022:339-355 (In Russ.). Available from: https://www.elibrary.ru/item.asp?edn=fqotrc 10. Yang JN, Mani G. Crowns for Primary Anterior Teeth. Int J Pedod Rehabil. 2017.1:75-78. doi: 10.4103/2468-8932.196491 11. Alaki SM, Abdulhadi BS, AbdElBaki MA, Alamoudi NM. Comparing zirconia to anterior strip crowns in primary anterior teeth in children: a randomized clinical trial. BMC Oral Health. 2020;20(1):313. doi: 10.1186/s12903-020-01305-1 12. Alzanbaqi SD, Alogaiel RM, Alasmari MA, Al Essa AM, Khogeer LN, Alanazi BS, et al. Zirconia Crowns for Primary Teeth: A Systematic Review and Meta-Analyses. Int J Environ Res Public Health. 2022;19(5):2838. doi:10.3390/ijerph19052838 13. Tatarinova EN, Soboleva NN, Sbitneva OV. Rehabilitation option for children with dental injuries. Actual problems of pediatric dentistry. Materials of the VII All-Russian Scientific and Practical Conference. 2021:276-283 (In Russ.). Available from: https://www.elibrary.ru/item.asp?id=47370828&pff=1 14. Ninawe N, Suyash J,Badhe H, Honaje N, Bhaje P, Barjatya K. Zirconia Crowns In Pediatric Dentistry: A Review. Journal of Positive School Psychology. 2022;6(8):1718-1724. Available from: https://journalppw.com/index.php/jpsp/article/ view/10035 СВЕДЕНИЯ ОБ АВТОРАХ Каменева Саида Вячеславовна, кандидат медицинских наук, доцент кафедры стоматологии терапевтической и пародонтологии Первого Санкт-Петербургского государственного медицинского университета имени академика И. П. Павлова, Санкт-Петербург, Российская Федерация Для переписки: sannysvk87@gmail.com ORCID: https://orcid.org/0000-0003-2929-6565 Автор, ответственный за связь с редакцией: Кущенко Николай Викторович, кандидат медицинских наук, доцент кафедры стоматологии терапевтической и пародонтологии Первого Санкт-Петербургского государственного медицин- ского университета имени академика И. П. Павлова, Санкт-Петербург, Российская Федерация Для переписки: nikolaiknv@mail.ru ORCID: https://orcid.org/0000-0003-1154-249X?lang=ru Омехина Дарья Михайловна, врач-стоматолог, Санкт-Петербург, Российская Федерация Для переписки: d.kuratch52@gmail.com ORCID: https://orcid.org/0000-0001-7937-0212 Голоднюк Анна Вячеславовна, врач-стоматолог детский, Санкт-Петербург, Российская Федерация Для переписки: d.kuratch52@gmail.com ORCID: https://orcid.org/0009-0009-5991-1603 INFORMATION ABOUT THE AUTHORS Saida V. Kameneva, DMD, PhD, Associate Professor, Department of Restorative Dentistry and Periodontology, Pavlov First Saint Petersburg State Medical University, Saint Petersburg, Russian Federation For correspondence: sannysvk87@gmail.com ORCID: https://orcid.org/0000-0003-2929-6565 Anna V. Golodniuk, DMD, Pediatric Dentist, Saint Petersburg, Russian Federation For correspondence: d.kuratch52@gmail.com ORCID: https://orcid.org/0009-0009-5991-1603 Corresponding author: Nikolai V. Kushchenko, DMD, PhD, Associate Professor, Department of Restorative Dentistry and Periodontology, Pavlov First Saint Petersburg State Medical University, Saint Petersburg, Russian Federation For correspondence: nikolaiknv@mail.ru ORCID: https://orcid.org/0000-0003-1154-249X?lang=ru Конфликт интересов: Авторы декларируют отсутствие конфликта интересов/ Сonflict of interests: The authors declare no conflict of interests Поступила / Article received 06.05.2023 Поступила после рецензирования / Revised 25.05.2023 Принята к публикации / Accepted 29.05.2023 Daria M. Omekhina, DMD, Saint Petersburg, Russian Federation For correspondence: d.kuratch52@gmail.com ORCID: https://orcid.org/0000-0001-7937-0212 190 2023; 23(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis 

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Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.15, 2023 www.iiste.org Effectiveness of the Matheladder as a Review Material on the Achievement and Motivation Levels of Grade 9 Learners in Trigonometry Ferrylou L. Guadiario Mindanao State University – Iligan Institute of Technology, Andres Bonifaco Ave., Tibanga, Iligan City ferrylou.guadiario@g.msuiit.edu.ph Douglas A. Salazar Mindanao State University – Iligan Institute of Technology, Andres Bonifaco Ave., Tibanga, Iligan City douglas.salazar@g.msuiit.edu.ph Abstract This study aimed to look into the effectiveness of the Matheladder as a review material for grade 9 learners’ achievement and motivation level in learning trigonometry. The study was administered to grade 9 learners on the fourth quarter from Acelo C. Badelles Sr. Memorial High School who were selected through purposive sampling. This study employed mixed method of research which used quasi-experimental design as the quantitative method. Quantitative and qualitative data was utilized from the achievement test, motivation questionnaire and interview guide questions. Findings revealed that the experimental groups have significantly higher achievement level compared to the control group. It also reveals that all experimental groups have significantly different motivation levels than the control group. The experimental groups, in particular, had a higher level of motivation than the control group. The drawn insights of the learners revealed that some learners demonstrated an interest in trigonometry and appreciated the game’s value in helping them comprehend the teachings in an enjoyable way. Furthermore, the game’s Question-and-Answer component, as well as the concept of winning the game, is one of the most captivating parts of the game. The participants had difficulty in answering the test due to the lack of sufficient knowledge, of how hard it is to understand the topics, unfamiliarity with the problem-solving process, and the negative notion of trigonometry. Following are some recommendations based on the foregoing conclusions: (1) Conduct the study in a longer span of time and with larger sample. (2) Encourage the use of the Matheladder game as a formative assessment instead of using it as a review material. (3) Contextualize and incorporate Matheladder in mathematics activities. (4) Replicate the study in the Grade 9 level in order to know the reasons behind the low achievement level in trigonometry. (5) Students and teachers need to focus on conceptual learning rather than rote memorization for meaningful understanding in trigonometry. Keywords: achievement level, educational board-game, Matheladder, motivation level, trigonometry DOI: 10.7176/JEP/14-15-05 Publication date:May 31st 2023 1. Introduction Mathematics is an integral part of education and is a required subject across all levels of the basic education curriculum. At the secondary level, Mathematics requires more than just performing the four fundamental operations in mathematics or using a formula to find an answer to a given problem. One of its main goals is to make students become effective problem solvers (SEI-DOST & MATHTED, 2011). Mathematics is an essential part of the educational curriculum, as it is used in both daily life and the study of other disciplines. However, it is commonly believed that the majority of students despise mathematics due to a variety of factors including instruction, learners' cognitive, emotional, and psychomotor characteristics, subject matter, and learning environment (Gafoor & Kurukkan, 2015). Moreover, trigonometry is one branch of mathematics that high school students still find difficult, despite the fact that it has been taught since elementary school (Gur, 2009). Students frequently make mistakes when solving trigonometric questions, such as using incorrect equations, sequences of operations, inappropriate use of sines and cosines, misinterpretations of languages, illogical conclusions, distorted definitions, and technical error computations (Tuna, 2013). The COVID-19 pandemic has wreaked havoc on education systems around the world, affecting almost 1.6 billion students across 190 countries and continents. The distance learning method is thought to be the most applicable to the COVID-19 pandemic condition. It is surely not simple to implement because it involves the preparation of facilities that support instructors, institutions, and students (Cortez, 2020). This 40 Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.15, 2023 www.iiste.org crisis, on the other hand, has sparked innovation in the education sector. We've seen some innovative methods to ensure the continuity of education and training (UNESCO, 2020). Remote learning at home is becoming more than a trend; it is becoming a necessity to ensure that education continues. According to recent research conducted by Mak (2021), the pandemic has affected children's motivation to learn. It is as frustrating as it is for teachers who are working incredibly hard to engage their students via new platforms in this new normal. For many students, playing games and discussing about games was clearly more essential than remotely completing homework (Miller, 2021). As a result, every concerned educator is looking for solutions to the problem. One method is to improve the teaching process by including instructional aids just like mathematical games. Mathematical games have been used to teach mathematics and science in many countries, including here in the Philippines. This is a result of the usefulness of games in the educational process. A study conducted by Veray (2016), which aimed to determine the effectiveness of engaging students through board games, revealed that the experimental group who was exposed to the board games has higher academic performance. The study also recommends engaging students in board games as part of improving their academic performance. Games can facilitate the mathematical environment as they release boredom, tension and establish a friendly atmosphere which allows for the growth of skills and knowledge. It has long been known that board games can be used as instructional materials for math-related disciplines. This study is a continuation of the undergraduate thesis conducted by Guadiario & Dela Cruz (2019) entitled “Development and Validation of Matheladder as an Enrichment Activity on Basics of Trigonometry”. Matheladder is a developed and validated board game that was patterned from the Snakes and Ladders game but with the infusion of Trigonometry on it. Furthermore, the board game was tried out on Grade 9 learners in Mindanao State University - Iligan Institute of Technology Integrated Developmental School (MSU-IIT IDS) on the fourth quarter of S.Y. 2018-2019. Results showed that Matheladder is creatively made and is well designed. It has no construction errors and sharp edges that can cause injury and it is very easy to use. The rules are also understandable and clear enough that all could easily participate. However, its effectiveness has not been explored. One of the recommendations of the study is to implement the board game. Hence, in this present study, the researcher looked into the effectiveness of the Matheladder as a review material on the learners’ achievement level and motivational level. 2. Methods 2.1 Research Design The researcher used the Mixed method of research which is an intellectual and practical synthesis based on the combination of quantitative and qualitative research. The experimental design was used under the quantitative research design. The quasi-experimental research approach was used for the quantitative data. Specifically, the researcher used the posttest-only group design where the learners are grouped into the experimental group and the control group. The control group had their traditional review session while the experimental groups received the intervention by utilizing the Matheladder board game as a review material through a series of trials. The achievement test was then given to both groups that shows the difference between the achievement levels of the students from the control and experimental groups. Furthermore, the qualitative data was obtained from the insights of the learners through the interview guide questions. 2.2 Research Setting and Participants This study was conducted at Acelo C. Badelles, Sr. Memorial High School. It is a government owned school of the Department of Education that provides junior and senior high school education. It is located at Purok 6, Tipanoy, Iligan City. 2.3 Participants of the Study The participants of the study are the Grade 9 learners in Acelo C. Badelles, Sr. Memorial High School which will be selected through purposive sampling. The selection for the control and experimental group was also done purposively. The chosen section in Grade 9 was divided into two groups as per direction of the school and as one of the protocols in implementing the limited face-to-face classes. There are 42 learners in the section but only 40 students were considered. The mean grades of the learners were used to show the comparability of the participants. 2.4 Research Instruments The study will use the following research instruments: 2.4.1 Achievement Test This is a 40-item multiple choice test questionnaire made by the researcher following the Most Essential Learning Competencies stipulated by Deped, and Table of Specification. The coverage of the test includes all the 41 Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.15, 2023 www.iiste.org topics discussed for the whole duration of the study which are: illustrates the six trigonometric ratios, finds the trigonometric ratios of special angles, illustrates angles of elevation and angles of depression, uses trigonometric ratios to solve real-life problems involving right triangles, illustrates laws of sines and cosines, and solves problems involving oblique triangles. The achievement test was given to both experimental and control groups. The test questionnaire was pre-evaluated by two in-service teachers and the section adviser/resource teacher. A dry-run was conducted with 30 learners in the Grade 10 level at Iligan Capitol College due to the availability and considering the online modality of the school. In addition, an item analysis was done where eleven of the questions out of forty items were revised and rest is to be retained. The researcher used the suggestions received during the pre-evaluation to finalize the instrument and make it ready for use. 2.4.2 Motivation Questionnaire The researcher adapted the motivation questionnaire which was developed by Fiorella et al., (2021) to assess learners' motivation toward trigonometry in both the control and experimental groups. It is modified accordingly for it to be suited to the study. This questionnaire contains several statements aim at investigating the level of learners’ motivation towards Trigonometry. This is composed of 19 Likert-scale questions that are indicated into five latent factors: intrinsic value, self-regulation, self-efficacy, utility value, and test anxiety. Intrinsic Value pertains to the student’s feeling that learning trigonometry is personally valuable. Self-regulation refers to a student’s tendency to engage in behavioral learning strategies. Self-efficacy is a student’s belief that they have the necessary competence and capability to perform well in trigonometry. Utility value is a student’s perception that trigonometry is personally relevant to their lives or future careers. Lastly, test anxiety refers to a student’s feeling of anxiety toward being assessed in trigonometry or compared with others. All items were on a fivepoint Likert-type instrument where students will assess their level of agreement in every statement. A dry-run was conducted to the students of Iligan Capitol College in Roxas Avenue, Iligan City. To identify the internal consistency of the motivation questionnaire, the Cronbach’s alpha was computed. It resulted with an overall Cronbach’s alpha of 0.808 which implies that the internal consistency of the motivation questionnaire exceeds the acceptable reliability. Therefore, the instrument is reliably good. 2.4.3 Interview Guide Questions The interview guide questionnaire is made by the researcher that consists of open-ended questions relevant to the study. This interview questionnaire serves as a reference for assessing the learners' experiences with Matheladder and to get their insights after playing the Matheladder board game. These guide questions were evaluated by two in-service teachers and the resource teacher. 2.5 Data Gathering Procedure The following procedure was undertaken by the researcher to conduct the study. a.) Asking for Permission To guarantee the credibility of the research process before collecting data, a letter had been prepared addressed to the Department of Education asking for permission to conduct the study in the chosen school. A formal letter addressed to the School Principal of Acelo C. Badelles, Sr. Memorial High School was also presented along with the letter from the DepEd in order to gain permission in entering and utilizing some school facilities during the conduct of the study. Consent letters addressed to the parents of the learners will also be given. Upon the approval of the school, a section from the Grade 9 level was selected as the participants of the study. The learners were grouped accordingly so as to form the desired groups. b.) Orientation The researcher introduced herself and explained the purpose of the visit. All participants were asked to join the orientation to be held at the school, bringing with them the consent letter signed by their parents or guardian. The concept of the Matheladder board game was introduced only to the experimental group to make the learners aware of what the game is all about, thus preparing them for the actual game. Prior to the conduct of the orientation and/or the study, certain protocols are observed including protective measures, hygiene practices, safety procedures, and necessary documents for the school and other stakeholders. c.) Implementation The experimental groups were able to utilize the Matheladder board game as review material before taking the achievement test while the control group was given the traditional way of review. This was conducted by batch due to the current pandemic situation and a series of trials was made. Groups 1, 2, and 3 was considered the trials 1, 2, and 3 respectively. This is to see if there is a consistency of the results from all the trials conducted. Upon completion of the activity, the achievement test and the motivation questionnaire were given which concludes the gathering of quantitative data. For the qualitative data, the researcher conducted an interview based on an interview guide question and provided a feedback slip for the insights of the learners. d.) Intervention Given the pandemic situation, the resource school has implemented a progressive limited face-to-face class. Following the Operational Guidelines on the Implementation of Face-to-Face Learning Modality, the chosen 42 Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.15, 2023 www.iiste.org section in Grade 9 was divided into two. The researcher is required to follow the guidelines so as the original schedule of the Grade 9 learners that is, the other half is following the MWF schedule while the other half is following the TTh schedule. The selected section has 42 learners but only 40 of them was considered as the participants of the study due to some absences. There are 20 learners for the MWF schedule and another 20 learners for the TTh schedule, both utilizing the 7:30am-9:30am classes. d.) Conducting the Posttest Upon completion of the review, the achievement test and the motivation questionnaire were given to both the experimental and control groups. The conduct of the posttest was done on Friday for the learners following the MWF schedule. On the other hand, the conduct of the posttest for the learners following the TTh schedule was on Friday afternoon, this is with regards to the special agreement between the researcher, resource teacher and the assistant principal. 2.6 Data Analysis The study's participants' achievement and motivation levels were determined using both quantitative and qualitative methods. The achievement test and Motivation Questionnaire provided quantitative data, while interview results provided qualitative data. The tables below are used for the interpretation of data. These serve as basis to classify and determine the description of the intervals they belong to. Table 2 Achievement Level Classification Score Range Rating Classification 36-40 90% above Outstanding 34-35 85% - 89% Proficient 32-33 80% - 84% Approaching Proficient 30-31 75% - 79% Developing 29-0 Below 74% Beginning Note: From https://www.ciit.edu.ph/k-to-12-grading-system/ With a passing average of 75 percent, the table indicates the achievement test score ranges, percentages, and achievement level classification. This is based on the K-12 curriculum grading system approved by the Department of Education. The score ranges are set based on the number of items on the achievement test given with the lowest interval 0-29 classified as ‘Beginning’ and the highest interval of 36-40 classified as ‘Outstanding’. This table is used to determine the classification of the learners ‘achievement level from the result on their achievement test scores. Table 3 Description and Interpretation of the Different Levels of Motivation Score Interpretation Description 4.51 – 5.0 Very Highly Motivated Always feel motivated towards trigonometry. 3.51 – 4.50 Highly Motivated Often feel motivated towards trigonometry. 2.51 – 3.50 Moderately Motivated Sometimes feel motivated towards trigonometry. 1.51 – 2.50 Lowly Motivated Rarely feel motivated towards trigonometry. 1.00 – 1.50 Very Lowly Motivated Never feel motivated towards trigonometry. Note: Adapted from the study “Online Learning and Students’ Mathematics Motivation, Self-Efficacy, and Anxiety in the “New Normal” by L. Mamolo, 2022. From https://doi.org/10.1155/2022/949634 Table 3 shows the description and interpretation of the different levels of motivation from the study of Mamolo (2021). The researcher made modifications that suited the present study. This is used to determine the classification of the learner’s motivation level based from their responses to the motivation questionnaire. 2.7 Statistical Tools The researcher will use the following statistical tools to analyze the gathered data. a. Mean and Standard Deviation This is used to measure the average achievement level of the respondents and to determine the index of variability or the dispersion of the students’ scores in the motivation questionnaire and achievement test. b. ANOVA This is used to compare the collected data from the experimental group and the control group and to determine if there is a difference between the two. It was computed using a licensed SPSS and tested at 0.05 level of significance. c. Thematic Analysis Thematic analysis was used in the analysis of qualitative data which came from the interview and insights of the students after the implementation of the study. 43 Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.15, 2023 www.iiste.org 2.8 Coding of Data The data gathered during the implementation of the study was coded accordingly. Since the study is comprised with two groups namely, experimental group and control group, it was coded as EG and CG respectively. Following the coding of each group, “EG1” was used to represent ‘Experimental Group 1’, and “CG1” for ‘Control Group learner 1’. 3. Results and Discussions Objective 1: Determine the achievement level of the Grade 9 learners in trigonometry. Table 4 Learner’s Achievement Level in Trigonometry (n=40) Group Mean Std. Dev. Interpretation 1 (Experimental Group 1) 29.6 (~30) 1.26 Developing 2 (Experimental Group 2) 29.9 (~30) 1.79 Developing 3 (Experimental Group 3) 29.4 1.84 Beginning 4 (Control Group) 19.1 2.77 Beginning Total 27.0 5.00 Beginning Table 4 presents the achievement level of the Grade 9 learners in trigonometry. Based on the calculated mean score of 19.1 for control group, it shows that the learners under this group exhibit “beginning” level of achievement in trigonometry. This is much lower when compared to the learners under the experimental groups. Two out of three in the experimental group obtained an average rating of 75%-79% with a mean score of approximately 29.6 and 29.9 which is interpreted as “Developing”. Moreover, the third group obtained a mean score of 29.4 which has the interpretation as “Beginning” but it doesn’t show much difference on the other experimental groups. It can be observed that the calculated mean of the experimental groups is on the range of 29.4 – 29.6 which shows a consistent result among the three trials conducted that is higher than the control group. On average, learners regardless of the intervention, still has low achievement level since it is only in the developing level. This indicates that the groups don’t have much difference in terms of their learning ability. However, it is still worthy to note that though they do have generally low achievement levels, it still shows that the experimental groups have higher achievement levels than the control group. As such, there was an improvement from the control and experimental group. It is clear enough that the learners in the experimental group have higher achievement levels compared to the control group. With this, it appears that the matheladder is an effective review material in learning trigonometry for the Grade 9 learners. Objective 2: Determine the learners’ motivational level towards Trigonometry. The researcher adapted a motivation questionnaire that was given to the participants as part of the posttest after playing Matheladder. Table 5 Learners’ Motivational Level toward Trigonometry (n=40) Group Mean Std. Dev. Description Interpretation 1 (Experimental Group 1) 3.71 0.24 Often feel motivated towards trigonometry. Highly Motivated 2 (Experimental Group 2) 3.90 0.15 Often feel motivated towards trigonometry. Highly Motivated 3 (Experimental Group 3) 4.08 0.21 Often feel motivated towards trigonometry. Highly Motivated 3.15 0.15 Sometimes feel motivated towards Moderately 4 (Control Group) trigonometry. Motivated Total 3.71 0.40 Table 5 presents the motivational level of the Grade 9 learners toward trigonometry for both control and experimental groups. The three experimental groups got a “High” remark while the control group has “Moderate” remark. Again, it can be observed that the control group obtain a lower mean score level of motivation when compared to the experimental groups. Clearly, Grade 9 learners who experienced the matheladder exhibit much higher motivational level compared to those learners who are not exposed to matheladder. That is, learners under experimental groups often feel motivated toward trigonometry while those learners from control group only sometimes feel motivated towards trigonometry. It shows that the matheladder is an effective review material for improving the motivational level of the Grade 9 learners towards trigonometry. 44 Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.15, 2023 www.iiste.org Objective 3: Compare the mean scores in the achievement level of the learners between the experimental and control group. Table 6 ANOVA Table on the Mean Score Difference in Learner’s Achievement Level Source SS df MS F Sig. (P-value) Between Groups 833.4 3 277.8 70.13 0.0001* Error Variance 142.6 36 3.96 Total 976 39 25.03 Table 6 presents the result of one-way analysis of variance (ANOVA) in testing the significant mean score difference of learner’s achievement level among groups. As observe, the computed p-value is lesser than the alpha level (0.0001<0.05). Therefore, there is sufficient evidence to believe that the mean score of the learner’s achievement level is significantly different among groups. This means that the achievement level of the learners under for experimental groups is higher compared to control group. This shows that the matheladder is an effective review material for improving the achievement level of the Grade 9 learners toward trigonometry. Table 7 Post Hoc Analysis: Bonferroni Comparison Test Groups Mean Mean Dif. (Control-Trial) Sig. 1(Experimental Group 1) 29.6 -10.5 0.0001* 2(Experimental Group 2) 29.9 -10.8 0.0001* 3(Experimental Group 3) 29.4 -10.3 0.0001* 4 (Control Group) 19.1 * Significant at 5% level (if p<0.05) Now, to see which among the groups differ significantly, the Bonferroni comparison test is used. Based on the result of Bonferroni multiple comparison test, it reveals that all experimental groups have significantly higher achievement level compared to the control group. Objective 4: Compare the mean scores in the motivational level of the learners between the two groups. Table 8 ANOVA Table on the Mean Score Difference in Learner’s Motivation Level Source SS df MS F Sig. (P-value) Between Groups 4.87 3 1.62 44.99 0.0001* Error Variance 1.30 36 0.04 Total 6.17 39 0.16 Table 8 presents the result of one-way analysis of variance (ANOVA) in testing the significant mean score difference of learner’s motivation level among groups. As presented in the table above, there is enough data to conclude that the mean score of motivation level for both control and experimental groups varies significantly. This means that matheladder is also an effective review material in improving the motivation of the Grade 9 learners toward trigonometry. Table 9 Post Hoc Analysis: Bonferroni Comparison Test Groups Mean Mean Dif. (Control-Trial) Sig. 1(Experimental Grp1) 4.21 -0.978 0.0001* 2(Experimental Grp2) 4.24 -1.002 0.0001* 3 (Experimental Grp 3) 4.30 -1.062 0.0001* 4 (Control Group) 3.24 * Significant at 5% level (if p<0.05) Moreover, the result of Bonferroni comparison test shows that all groups under experimental have significantly different motivation level compared to control group. Specifically, the experimental groups have statistically higher motivation level than the control group. Objective 5: Draw insights from the learner’s experience after playing Matheladder. After the conduct of the activity, a semi-structured interview was conducted to the learners under the experimental group. Here are some of the responses of the learners on the given questions. 1.) The use of Matheladder affected the participants’ feelings towards trigonometry. From the responses of the participants, seven out of thirty participants find Matheladder as fun and exciting. Positive feelings are also evident as it allows them to be comfortable with the lessons and have relived their boredom and anxiety. Furthermore, five participants responded that Matheladder helps in understanding the lessons in trigonometry more and in recalling the basics of trigonometric values. Though one participant is not much affected, there are some participants who showed some 45 Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.15, 2023 www.iiste.org liking towards trigonometry. 2.) Performance on the Matheladder game. The performance of the participants in playing the Matheladder turned out to be good. Nine participants claimed to have done better, some had fun and find it an exciting and amazing experience amidst the nervousness. Some were also delighted for winning the game. Moreover, EG18 also find it nice and worthy to be shared to others if given a chance. 3.) The participants’ experiences with their classmates when playing Matheladder. Ten out of thirty participants described their experience while playing the Matheladder with their classmates as happy and enjoyable. Twelve participants also find their experience fun and exciting. It showed how they enjoyed their time playing the Matheladder board game and also how challenging it was. Many have responded about how they had fun and learn at the same time. 4.) Comparing their enjoyment of trigonometry before the use of matheladder? Most of the participants have responded that they find trigonometry challenging before, but with Matheladder, they have realized that trigonometry can be fun and interesting while still challenging. In addition, four participants have mentioned again the helpfulness of the game in understanding the lessons in a fun way. 5.) The participants’ favorite aspect of the game. The Question-and-Answer portion of the game is one of the most likeable parts based on the responses of the participants. They find it more challenging on their end since the player can’t move to his/her next move if the question will be answered correctly. EG7 also pointed it out to be a good thing so as to know if he/she really learned something. Additionally, EG20 also mentioned that even though he/she didn’t get the correct answer, he/she still learned from it also because of the answers of the other players. It shows that sharing of ideas is present even if this game is not necessarily a group effort type of game. Three participants also like the part of winning which is a vital result of the game. Throwing and rolling the dice also sparked an interest in the other five participants. The overall game experience is also of the liking of the other six participants which highlighted their enjoyment in playing and meaningful company with their classmates. 4. Conclusions The main purpose of this study is to delve into the effectiveness of the Matheladder as a review material for Grade 9 learners in trigonometry. Findings reveal that the Matheladder is an effective review material. The learners in the control group are said to have a “beginning” level of achievement in trigonometry. This is considerably lower than the "developing” achievement level of the experimental group. There is enough information to conclude that the mean score of the learners’ achievement level differs significantly between groups. This suggests that the experimental groups’ learners’ achievement levels are higher than the control group. It also reveals that all experimental groups have significantly higher achievement level compared to the control group. Furthermore, the experimental group’s participants provided more positive answers to the motivation questionnaire. As can be seen, the control group had a lower mean motivation score than the experimental group. The learners under the control group only sometimes feel motivated towards trigonometry, whereas learners in the experimental groups often feel motivated towards trigonometry. There is enough evidence to establish that the mean motivation level score for both the control and experimental groups varies significantly. It also reveals that all experimental groups have significantly different motivation levels than the control group. The experimental groups, in particular, had a statistically higher level of motivation than the control group. Lastly, the drawn insights of the learners revealed that the Matheladder is fun, enjoyable, exciting, challenging, as well as helping in a better understanding of the lessons and recalling trigonometric values. Some learners also demonstrated an interest in trigonometry and appreciated the game’s value in helping them comprehend the teachings in an enjoyable way. The majority of the participants also recognized that with Matheladder, trigonometry can be both fun and challenging. Additionally, the game’s Question-and-Answer component, as well as the concept of winning the game, is one of the most captivating parts of the game. Furthermore, the participants had difficulty in answering the test due to the lack of sufficient knowledge, of how hard it is to understand the topics, unfamiliarity with the problem-solving process, and the negative notion of trigonometry. References: Amr, Kannan, "Learning through Games: Essential Features of an Educational Game" (2012). Instructional Design, Development and Evaluation - Dissertations.Paper 56. Boocock, Sarane S. and E. 0.Schild (eds. ). 1968. Simulation games in learning. Beverly Hills, Sage Publications. p. 279 46 Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.15, 2023 www.iiste.org Cavanagh, S. (2008). Playing games in classroom helping pupils grasp math.Education Week, 27(35),10.Retrieved from https://search.proquest.com/docview/202716701?accountid=141440 Chinazzi, M., Litvinova, M., Vespignani, A., Ajelli, M., Davis, J., Merler, S., et al. (2020). The effect of travel restrictions on the spread of the 2019 novel coronavirus (COVID-19) outbreak. Science , pp. 395-400. Cortez, C. P. (2020, May 17). Blended, Distance, Electronic and Virtual-Learning for the New Normal of Mathematics Education: A Senior High School Student’s Perception.Semantic Scholar .Dennis, J. M. & Stewart, J. (1979).Instructional Games and Computer using Teacher. 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Retrieved February 27, 2022, from http://conference.kuis.edu.my/icomm/5th/images/eproceeding2018/IC009.pdf Taspinar, B., Schmidt, W., &Schuhbauer, H. (2016).Gamification in Education: A Board Game Approach to Knowledge. International Conference on Knowledge Management. Vienna,Austria. Tomaso, P. (2014). A quantitative assessment of the effect of games on learning (Order No. 3628697).Available from ProQuest Central. (1560885980). Retrieved from https://search.proquest.com/docview/1560885980?accountid=141440 Treher, E. N. (2011). Learning with Board Games: Tools for Learning and Retention. Retrieved November 2018, from The Learning Key, Inc.: http://www.thelearningkey.com/pdf/Board_Games_TLKWhitePaper_May16_2011.pdf Tuna, A. (2013). A Conceptual Analysis of the Knowledge of |Prospective Mathematics Teaches about Degree and Radian. World J Educ. UNESCO. (2020). Education: From Disruption to Recovery UNESCO. 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Hindawi Publishing Corporation Journal of Diabetes Research Volume 2016, Article ID 1540267, 8 pages http://dx.doi.org/10.1155/2016/1540267 Review Article Physiological and Pharmacological Roles of FGF21 in Cardiovascular Diseases Peng Cheng,1,2,3 Fangfang Zhang,1,2,3 Lechu Yu,1 Xiufei Lin,1,2,3 Luqing He,1,2,3 Xiaokun Li,2,3 Xuemian Lu,1 Xiaoqing Yan,1,2,3 Yi Tan,1,2,4 and Chi Zhang1,2,3 1 The Chinese-American Research Institute for Diabetic Complications, Wenzhou Medical University, Wenzhou 325035, China Ruian Center of the Chinese-American Research Institute for Diabetic Complications, The Third Affiliated Hospital, Wenzhou Medical University, Wenzhou 325200, China 3 School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou 325035, China 4 Kosair Children Hospital Research Institute, Department of Pediatrics, University of Louisville School of Medicine, Louisville, KY 40202, USA 2 Correspondence should be addressed to Yi Tan; yi.tan@louisville.edu and Chi Zhang; zhangchi515@126.com Received 26 October 2015; Revised 26 February 2016; Accepted 18 April 2016 Academic Editor: Raffaella Mastrocola Copyright © 2016 Peng Cheng 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. Cardiovascular disease (CVD) is one of the most severe diseases in clinics. Fibroblast growth factor 21 (FGF21) is regarded as an important metabolic regulator playing a therapeutic role in diabetes and its complications. The heart is a key target as well as a source of FGF21 which is involved in heart development and also induces beneficial effects in CVDs. Our review is to clarify the roles of FGF21 in CVDs. Strong evidence showed that the development of CVDs including atherosclerosis, coronary heart disease, myocardial ischemia, cardiac hypertrophy, and diabetic cardiomyopathy is associated with serum FGF21 levels increase which was regarded as a compensatory response to induced cardiac protection. Furthermore, administration of FGF21 suppressed the above CVDs. Mechanistic studies revealed that FGF21 induced cardiac protection likely by preventing cardiac lipotoxicity and the associated oxidative stress, inflammation, and apoptosis. Normally, FGF21 induced therapeutic effects against CVDs via activation of the above kinases-mediated pathways by directly binding to the FGF receptors of the heart in the presence of 𝛽-klotho. However, recently, growing evidence showed that FGF21 induced beneficial effects on peripheral organs through an indirect way mediated by adiponectin. Therefore whether adiponectin is also involved in FGF21-induced cardiac protection still needs further investigation. 1. Introduction Cardiovascular diseases (CVDs) are the leading cause of death worldwide composed of heart and blood vessel diseases. In the recent years, the incidence of CVDs has been increasing at a sharp rate globally. According to the World Health Report 2010, CVDs contributed to 17.5 million deaths and these numbers are estimated to increase to 23.3 million by 2030 [1, 2]. Fibroblast growth factor (FGF) is a cytokine superfamily with pleiotropic biological functions including regulating cell growth, differentiation, development, and metabolism [3–7]. Human FGFs contain 22 members which can be divided into 7 subfamilies based on phylogeny and sequence [8–10]. Due to the lack of a heparin binding domain, FGF19 subfamily members (FGF19, FGF21, and FGF23) function in an endocrine manner rather than an autocrine manner as other subfamily members of FGFs [9]. Among them, FGF21 is a polypeptide with 209/210 (human/rodent) amino acid residues that is primarily produced and secreted by the liver, adipose tissue, and thymus [11]. FGF21 expression is mainly regulated by peroxisome proliferator-activated receptor 𝛼 (PPAR𝛼) in the liver [12] and PPAR𝛾 in adipocytes [13, 14]. FGF21 was firstly cloned in 2000 [11] and received global attention in recent years due to its outstanding ability on regulating carbohydrate and lipid metabolism including improving insulin sensitivity, lowering blood glucose, reducing hepatic/plasma triglycerides, inducing weight loss by increasing energy expenditure, and reducing fat mass [15–18]. Further studies indicated that FGF21 functions by binding 2 to (FGFR)1c and (FGFR)2c in the presence of coreceptor 𝛽-klotho and activation of downstream signaling pathway [19, 20]. Although FGF21 and other members of FGFs share the same FGF receptors, the coreceptors are different (𝛽klotho for FGF21 and heparin for others) which determined that they have different bioactivity due to activation of various pathways [21, 22]. Unlike traditional insulin therapy in clinics, FGF21 did not cause hypoglycemia [16]. The possible explanation is that FGF21 induces physiological role in healthy condition and pharmacological role under unhealthy condition [23, 24]. Additionally, FGF21 does not lead to carcinogenic event due to lack of mitogenic function which makes it possible to be administrated in vivo in clinics [16]. Therefore, FGF21 may hold promise as a clinically therapeutic option due to the abovementioned characters and advantages. In recent clinical and preclinical studies, CVDs have been closely associated with serum FGF21 which increased in the patients with atherosclerosis, coronary heart disease, myocardial ischemia, cardiac hypertrophy, and diabetic cardiomyopathy [25–27]. Therefore FGF21 has the potential to be considered as a biomarker for the above CVDs. Whether the increased serum FGF21 level is the basis for CVD pathogenesis or is induced to protect the heart form CVDs is still under discussion. However, growing evidence indicated that administration of exogenous FGF21 induces preventive effects on most of the above CVDs, suggesting that FGF21 not only is a simple marker of cardiovascular risk but also induces a protective effect on the cardiovascular system contributing to a reduction in risk (Table 1). In clinics, serum FGF21 levels were increased in patients with obesity or type 2 diabetes which was associated with high risk of CVDs. The paradoxical phenomenon was supposed to be explained by a compensatory response to induce cardiac protection or resistance to FGF21 which impaired its bioactivity [28, 29]. In animal study, we found that at the early-stage of diabetes serum FGF21 level of mice was sharply increased compared with nondiabetic mice (C57BL/6J), while it was dramatically decreased at the late-stage of diabetes which further confirmed that early-stage increase of serum FGF21 was a compensatory response and induced beneficial effect on the heart; late-stage decrease may be the cause of diabetes-induced cardiac damage [30], since the above CVDs are always attributed to lipid metabolic disorder. Mechanistic studies indicated that FGF21-induced cardiac protection in CVDs is possibly attributed to the suppression of lipotoxicity since the above CVDs are always the consequences of lipotoxicity. This review tries to illuminate the underlying relationship between FGF21 and CVDs and the possible mechanisms. 2. FGF21 and Atherosclerosis and Coronary Heart Disease Atherosclerosis is a chronic, inflammatory disorder characterized by the deposition of excess lipids in the arterial intima [31]. The accrued evidence indicated that lipidlowering therapy limits the progression of atherosclerosis and reduces CAD events [32]. Since FGF21 plays an important role in the regulation of lipid metabolism, the effect of FGF21 in atherosclerosis is of interest. Clinical studies showed that Journal of Diabetes Research increased circulating FGF21 levels were discovered in atherosclerotic patients or the individuals with high risk of developing atherosclerosis [33, 34]. Additionally, an in vivo study demonstrated that increased serum FGF21 was observed in aortas of apoE−/− mice (C57BL/6J background) [35]. Strong evidence identified that administration of exogenous FGF21 significantly improved lipid metabolic disorders and reduced atherosclerotic plaque areas in these animals [36]. Moreover, Lin et al. also reported that FGF21 deficiency enhanced atherosclerotic deterioration and mortality in apoE−/− mice (C57BL/6J background) [35], implying that increased serum FGF21 in patients with atherosclerosis described previously induces beneficial effect rather than the basic for atherosclerotic pathogenesis. Mechanistic study indicated that FGF21-induced prevention of atherosclerosis was associated with suppression of endoplasmic reticulum stress-mediated apoptosis in apoE−/− mice (C57BL/6J background) [37]. Further mechanistic studies revealed that prevention of atherosclerosis by FGF21 was attributed to the fine-tuning of multiorgan cross talk among the liver, adipose tissue, and blood vessels, characterized by suppression of hepatic sterol regulatory element-binding protein-2 and induction of adiponectin in mice with atherosclerosis [35]. Although FGF21 functions in an endocrine manner, whether FGF21 can also induce a direct protection to the blood vessels remains unclear. For decades, lowering levels of low-density lipoprotein (LDL) cholesterol and increasing level of high-density lipoprotein (HDL) have formed the cornerstone of management of patients with atherosclerotic cardiovascular disease. Strong evidence demonstrated that FGF-21 dramatically improved the condition of atherosclerosis in Wistar rats by decreasing serum LDL levels and increasing serum HDL levels. Moreover, FGF-21-induced antioxidative function is also involved in its therapeutic effect in atherosclerotic Wistar rat characterized by increased levels of superoxide dismutase, reduced glutathione, and reduced malondialdehyde [38]. Along with the development of atherosclerosis, the artery’s lining becomes hardened, stiffened, and swollen with all sorts of “gunge,” including fatty deposits and abnormal inflammatory cells, to form a plaque and then eventually deteriorate into coronary heart disease [39–41]. Strong evidence indicated that cardiac endothelial cell dysfunction may be an early initiating factor for atherosclerosis which facilitates the development of coronary heart disease [42]. Oxidized LDL (ox-LDL) is a proatherogenic lipoprotein that accumulates in the vascular wall and contributes to vascular dysfunction at the early-stage of atherosclerosis development [43–53]. Enhanced serum ox-LDL and antibodies against its epitopes are predictive for endothelial dysfunction and subsequent coronary heart disease [43]. Previous in vitro study indicated that both FGF21 mRNA and protein expressions were increased in response to ox-LDL treatment in cardiac endothelial cells and this was protective against apoptosis caused by ox-LDL [54]. Also, FGF21 has been reported to prevent high glucose induced cell damage and endothelial nitric oxide synthase dysfunction through an AMP-activated protein kinase- (AMPK-) dependent pathway in endothelial Apolipoprotein E(−/−) mice Atherosclerosis Coronary artery ligation (ischemia/reperfusion) Isoproterenol infusion-induced cardiac hypertrophy/LPS-induced cardiac hypertrophy Multiple low-dose STZ-induced type 1 diabetes Myocardial ischemia Cardiac hypertrophy Diabetic cardiomyopathy Coronary heart disease Model Heart disease Outcomes Atherosclerotic lesion area collagen composition ↓ Recombinant murine FGF21 was given daily Total cholesterol ↓ intraperitoneally for 16 weeks Hypertriglyceridemia ↓ Circulating adiponectin ↑ Cell apoptosis ↓ Mouse FGF21 full length protein was given for 24 or Oxidative stress ↓ 48 hours NO production ↑ eNOS phosphorylation ↑ Recombinant mouse FGF21 was administered Activity of caspase-3 ↓ intravenously immediately after myocardial injury Degree of myocardial infarction ↓ every 12 hrs for 3 days Left ventricular function ↑ Cardiomyocyte size ↓ FGF21 was injected intraperitoneally for 7 days or Δ heart weight/body weight ↓ given for 24 hours in neonatal cardiomyocytes Inflammation ↓ Cardiac oxidative stress ↓ Oxidative stress ↑ Knockout FGF21 in type 1 diabetic mouse model Lipid accumulation ↑ Cardiac dysfunction and remodeling ↑ Methods Table 1: Summary of major pharmacological studies of FGF21 in heart disease. [66] [64, 65] [59] [55] [63] Ref. Journal of Diabetes Research 3 4 cells [55]. Therefore the relationship between FGF21 and coronary heart disease is of interest. Shen et al. reported that serum FGF21 level was positively associated with coronary heart disease in clinics [56, 57]. Our previous work confirmed that serum levels of FGF-21 are increased in patients with coronary heart disease independently associated with adverse lipid profiles [33]. In contrast, another study indicated that serum FGF21 has been associated with hypertriglyceridemia, hyperinsulinemia, and pericardial fat accumulation but not associated with coronary heart disease [58]. This paradox may be explained by decreased body mass index of healthy controls compared to patients with coronary heart diseases. 3. FGF21 and Myocardial Ischemia Myocardial ischemia, a disorder causing cardiomyocytes injury and myocardial infarction and malfunction, activates adaptive responses enhancing myocardial tolerance to ischemia. Liu et al. indicated that, in response to myocardial ischemia in the C57BL/6J mouse, liver- and adipocytesderived FGF21 was upregulated and secreted into the circulation. After interacting with FGFR1 in cardiomyocytes in the presence of 𝛽-klotho, FGF21 activates its downstream kinases and proteins including phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB/AKT), and Bcl2 antagonist of cell death (BAD), thereby reducing myocardial ischemiainduced apoptosis characterized by reduction of caspase-3 activity [59]. However, the adaptive response was not found in FGF21-deficient mice. Reversely, myocardial ischemic size was significantly smaller in FGF21 transgenic mice than that in wild type mice [59], suggesting that upregulated endogenous FGF21 derived from the liver and adipose tissue in response to myocardial injury induced cardiac protection mediated by activation of FGFR1/𝛽-klotho-PI3K-Akt1-BAD signaling pathway. Although various growth factors and cytokines were upregulated during myocardial ischemia, the expression and secretion of cardiac FGF21 had no alteration, implying FGF21 induces cardiac protection against myocardial ischemia in an endocrine rather than an autocrine manner [59, 60]. To date, a question of whether administration of exogenous FGF21 can also induce cardiac protection during myocardial ischemia and if so whether the protection of exogenous FGF21 against myocardial ischemia can be direct to the heart or cardiomyocytes appears. This question was answered by Patel group [61]. They found that administration of exogenous FGF21 induced significant cardioprotection and restored cardiac function following global ischemia in Langendorff perfused rat hearts. Further study revealed that inhibition of AKT, extracellular signal-regulated kinase (ERK1/2), and AMPK impaired FGF21-induced antimyocardial ischemia effect in the hearts of obese Wistar rats, suggesting that the above kinases are involved in this cardioprotection of FGF21 [61]. Our previous in vitro study also confirmed that administration of exogenous FGF-21 attenuated ischemia-reperfusion induced damage in H9c2 cells characterized by inhibition of oxidative stress and apoptosis [62]. The mechanistic study revealed that FGF21-induced protection against ischemia-reperfusion injury in cardiac Journal of Diabetes Research cells mainly depended on the activation of Akt-GSK-3𝛽caspase-3 signaling pathway by preventing oxidative stress and recovery of the energy supply [62]. 4. FGF21 and Cardiac Hypertrophy Hypertrophic remodeling characterized by enlarged cardiomyocytes is an adaptive response of the heart to certain stresses. And it is also the leading cause of multiple cardiovascular problems including hypertension, myocardial ischemia, valvular disease, and cardiomyopathy [67–69]. Mature cardiomyocytes are considered to be terminally differentiated cells with no regenerative ability [70–72]. Under stresses, cardiac hypertrophy is characterized by cardiomyocytes enlargement, rather than cells division [73, 74], and this phenomenon is accompanied by the increase of extracellular matrix and fibroblasts inside the heart [75, 76]. Recently, cardiac hypertrophy was reported to induce FGF21 gene expression in the cardiomyocytes of mouse, and this was subjected to transcriptional regulation of the hepatic silent mating type information regulation 2 homolog 1/PPAR𝛼 pathway [64]. In turn, FGF21 knockout mice had greater heart weights and more severe cardiac dysfunction in response to isoproterenol infusion along with induction of hypertrophic inflammatory markers [64]. However, administration of recombinant FGF21 significantly prevented isoproterenol-induced cardiac hypertrophy damage in mice [64]. Mechanistic studies indicated that FGF21 prevented cardiac hypertrophy by activating mitogen-activated protein kinase (MAPK) signaling via activation of FGFR1c/𝛽-klotho [64, 77]. Additionally, FGF21 prevented cardiac hypertrophy by promoting multiple antioxidant genes expressions (e.g., uncoupling proteins 2 and 3, also superoxide dismutase-2) and inhibiting the formation of reactive oxygen species in an autocrine manner [65]. 5. FGF21 and Diabetic Cardiomyopathy Diabetic patients develop the diabetic cardiomyopathy independent of coronary artery disease and hypertension [78, 79]. Diabetic cardiomyopathy is attributed to multiple pathogenic factors, including hyperglycemia, hyperlipidemia, and inflammation [80–82]. Cardiomyopathy is a late consequence of diabetes-induced early cardiac responses especially the myocardial apoptosis [83, 84]. Thus, treatments to reduce cardiac apoptosis may help control diabetic cardiomyopathy. Recently, we reported that cardiac FGF21 mRNA expression was positively associated with the development of diabetes in the type 1 diabetic mice, suggesting that the increased cardiac FGF21 expression may be beneficial to the heart in this regard [30]. In the study we also observed cardiac apoptosis in early diabetic mice, which was remarkably prevented by administration of recombinant FGF21 [30]. Similar protection by FGF21 was observed in mice with cardiac lipotoxicity induced by fatty-acid [30]. Mechanistic studies indicated that FGF21-induced antiapoptotic effects in vitro and in vivo were mediated by ERK1/2-p38-MAPK-AMPK signaling pathway [30]. Thus, FGF21-induced cardioprotection in diabetic mice is mainly attributed to prevention of Journal of Diabetes Research 5 FGF21 FGFR1c Cell membrane 𝛽-klotho 𝛼 AR PP AMPK PI3K p85 Erk1/2 Sirt1 P38 MAPK Cell damage Endothelial dysfunction Atherosclerosis Akt BAD Inflammatory oxidative stress Cardiac hypertension Coronary heart disease AMPK Lipotoxicity Oxidative stress Apoptosis Myocardial ischemia Diabetic cardiomyopathy Figure 1: FGF21 induces preventive effect on CVDs through multiple signaling pathways. As a classical cytokine, FGF21 functions as a metabolic regulator by binding with its receptor FGFR1 or FGFR2 in the presence of 𝛽-klotho. Growing studies demonstrated that FGF21 also induced beneficial effects on CVDs probably due to inhibition of glucose or lipid metabolic disorders. For instance, FGF21 prevented atherosclerosis and the subsequent CHD by inhibition of lipogenesis which was also the possible mechanism of FGF21-induced preventive effect on CH. Additionally, FGF21 also prevented MI and DC by activation of Akt- and AMPK-mediated signaling pathway which were usually involved in maintaining glucose and lipid homeostasis. lipotoxicity by FGF21. Also, long-term treatment of FGF21 prevented diabetic-induced cardiac dysfunction and fibrosis mediated by the same signaling pathway as above [30]. Our work also revealed that FGF21 deletion-aggravated cardiac lipid accumulation is likely mediated by cardiac Nrf2-driven CD36 upregulation in type 1 diabetic mice, which contributes to increased cardiac oxidative stress and remodeling, and eventual development of diabetic cardiomyopathy [66]. cardiomyopathy via Akt- or AMPK-mediated signaling pathways which regulate lipid and glucose metabolisms (Figure 1). Since serum FGF21 increases in several kinds of CVDs, serum FGF21 levels might be regarded as a potential biomarker not only for diagnosis of metabolic disorders but also for diagnosis of CVD in clinics. And supplementation of exogenous FGF21 might also induce beneficial effect in patients with CVD based on the conclusion of preclinical studies. 6. Summary Disclosure CVD includes atherosclerosis, coronary heart disease, myocardial ischemia, cardiac hypertrophy, and diabetic cardiomyopathy which are all closely associated with severe lipid metabolic disorders [85–87]. FGF21, a metabolic regulator of carbohydrates and lipids, has been shown to improve insulin sensitivity and glucose uptake and suppress lipogenesis and lipid oxidation [15–18]. Clinical studies indicated that serum FGF21 changes were positively associated with the development of atherosclerosis, coronary heart disease, myocardial ischemia, cardiac hypertrophy, and diabetic cardiomyopathy, which implies that upregulated endogenous FGF21 may improve CVDs. Specifically, FGF21 prevented atherosclerosis and subsequent coronary heart disease was attributed to multiorgan cross talk among the liver, adipose tissue, and blood vessels and was characterized by suppression of lipid accumulation and increased lipid oxidation [63]. Similarly, FGF21 prevented stress-induced CH via enhancing lipid oxidation mediated by the ERK1/2-CREB-PGC-1𝛼 signaling pathway [64]. FGF21 also prevented myocardial ischemia and diabetic The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the paper. Competing Interests The authors declare that there are no competing interests regarding the publication of this paper. Authors’ Contributions Peng Cheng, Fangfang Zhang, and Lechu Yu are equal contributors to the review. Acknowledgments This study was supported in part by grants from the National Science Foundation of China (81370917, to Chi Zhang; 81471045, to Xuemian Lu; 81573435, 81273509, and 30971209 to Yi Tan), the Research Development Fund of Wenzhou 6 Medical University (QTJ13005, to Chi Zhang; QTJ13007 to Yi Tan), the Medical and Healthy Technological Grant of Zhejiang Province (201472233, to Chi Zhang), the Project of Public Welfare of Wenzhou (2014Y0416, to Chi Zhang), and the Project for Selected Overseas Chinese Supported by Zhejiang Technology Foundation (to Chi Zhang) and the Key New Drug Development Grant 2012ZX09103-301-016 (to Xiaokun Li and Yi Tan) and the grants from the American Diabetes Association and Juvenile Diabetes Research Foundation (1-13-JF-53 and 1-INO-2014-122-A-N to Yi Tan). References [1] Y. Yang, W. Duan, Y. Li et al., “Novel role of silent information regulator 1 in myocardial ischemia,” Circulation, vol. 128, no. 20, pp. 2232–2240, 2013. [2] C. D. Mathers and D. 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Lorier, “Glycaemic control, cardiovascular disease, and mortality in type 2 diabetes,” The Lancet, vol. 384, no. 9958, pp. 1906–1907, 2014. MEDIATORS of INFLAMMATION The Scientific World Journal Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Gastroenterology Research and Practice Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Hindawi Publishing Corporation http://www.hindawi.com Diabetes Research Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal of Journal of Endocrinology Immunology Research Hindawi Publishing Corporation http://www.hindawi.com Disease Markers Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Volume 2014 Submit your manuscripts at http://www.hindawi.com BioMed Research International PPAR Research Hindawi Publishing Corporation http://www.hindawi.com Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Volume 2014 Journal of Obesity Journal of Ophthalmology Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Evidence-Based Complementary and Alternative Medicine Stem Cells International Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Oncology Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Parkinson’s Disease Computational and Mathematical Methods in Medicine Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 AIDS Behavioural Neurology Hindawi Publishing Corporation http://www.hindawi.com Research and Treatment Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Oxidative Medicine and Cellular Longevity Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 

W2320895514.txt

Downloaded from http://trngl.lyellcollection.org/ at West Virginia University on July 11, 2015 MACNAIR—LIMESTONES IN THE GLASGOW DISTRICT. No. 37 V.—THE STRATIGRAPHY OF THE LIMESTONES LYING IMMEDIATELY ABOVE THE CALCIFEROUS LAVAS IN THE GLASGOW DISTRICT. By PETER MACNAIR, F.R.S.E., F.G.S., and HARRY R. J. CONACHER. [Read 13th March, 1913.] THE group of sediments under discussion is that commonly known as the Lower Limestones, with the addition of those which occupy the space between the base of the Hurlet Seam and the top of the lavas, that is, from the Hosie Limestones at the top to below the Hollybush. These limestones crop out practically all around the basin in which Glasgow lies, and are seen in burn sections, quarries, and railway cuttings along the base of the hills of Calciferous lava which form its rim. The strata below the Hurlet Seam are grouped by the officers of the Geological Survey with the Calciferous series, but as there is no essential break at this horizon in our district such as exists in the Lothians, we propose to deal with all these beds as one series. The district dealt with may be defined as including Hurlet, Howood, Paisley, Bridge of Weir, Duntocher, Bearsden, Campsie, Kilsyth. The literature of the subject is meagre. There are references in the Geological Survey's publications dealing with Sheet No. 31 of their 1-inch Geological Map. In the Transactions of our Society there is a paper by Mr. Hugh M'Phail dealing with the Barrhead-Paisley District; there is Dr. Young's paper of 1860 on Campsie and the Glasgow District; and there is the Memoir of the Geological Survey, published in October, 1911.4 1 2 3 1 Memoirs of t h e Geological Survey, Scotland. Explanation of Sheet 3 1 (1879). On t h e Carboniferous Sections of the Levern V a l l e y , Renfrewshire. Glas. Geol. Soc. Transactions, vol. iii., p. 254. On t h e Geology of the Campsie District. Trans. Geol. Soc. Glas. vol. i., p. 1, 1863. Memoirs of t h e Geological Survey of Scotland. T h e Geology of t h e -Glasgow District, 1911. 2 3 9 4 Downloaded from http://trngl.lyellcollection.org/ at West Virginia University on July 11, 2015 38 TRANSACTIONS GEOLOGICAL SOCIETY OP GLASGOW. Prior to the issue of the last-named publication, no definite co-relation of the beds under discussion had been published.. It was while going over the ground with that book in hand that we came on the points which we now bring before you. Hurlet District.—In the Memoir, the following horizons are recognised and described in the district, and may be examined at the localities named: — Hosie Limestone—River Levern at Barrhead. Blackhall Limestone—Jenny's Well, Paisley, and Levernside,. Crookston. Hurlet Limestone—Not now seen, except at Arkleston Cutting,. north-east of Paisley. Blackbyre Limestone—Railway Cutting at Barrhead. Hollybush Limestone—Hollybush Quarries, and Hawkhead Dam. With regard to the Hosie Limestone at Barrhead, we are unable to offer any opinion or criticism of this identification. The Blackhall Limestone, as seen at Jenny's Well (first described by Mr. John Smith in vol. xii. of our Transactions) shows definite and constant characters which enable it to be reoognised with certainty throughout the district. It is in several beds, the top one marine, the lower fresh water, and contains a parting of entomostracan oil shale. It can be seen to overlie a series of clay-band ironstones, which, again, are above the Hurlet Seam, as shown by old workings. The Blackbyre Limestone, as seen in the railway cutting at Blackbyre Farm, is also in several beds, the upper composed of small encrinite fragments, and the lower slabby and crowded with small producti and containing occasional plant remains. The Hollybush Seam is accessible with difficulty in the old quarries at Hollybush and below high-water level in the Hawkhead Dam. In both localities its most striking characteristic is the abundance of large producti (giganteus, latissimus, and semi-reticulatus), and branching and solitary corals. The stratigraphical relations of the Blackbyre and Hollybush Limestones cannot be certainly seen in the type district, but the positions assigned to them in the Memoir are confirmed by neighbouring sections, which are now described for the first time. r Downloaded from http://trngl.lyellcollection.org/ at West Virginia University on July 11, 2015 MACNAIR—LIMESTONES I N THE GLASGOW DISTRICT. 39" Nethercraigs.—At Nethercraigs, south of Paisley, there is a small syncline, truncated to the south by the fault which brings up the lavas of the Ferneze Hills, and in this basin— no more than half a mile across—we see the Hurlet Lime stone the Blackbyre, and the Hollybush. The Hurlet Limestone, which at Hurlet is stated to be no more than 4 to 5 feet thick, has here increased, by the addition of limey shales, to about 30 feet. This is a surprising phenomenon, but it is also seen to occur at Howood, in the G-ryfe, and at Campsie. The y Fig. 1.—Plan a n d Section across Limestones a t Nethercraigs, Paisley. (a) S t r a t a below Hollybush L i m e s t o n e ; (b) Hollybush L i m e s t o n e ; (c) Hollybush Sandstone ; (d) Blackbyre L i m e s t o n e ; (e) A s h y m u d s w i t h limey n o d u l e s ; ( / ) Hurlet Limestone. alum shale and coal, which usually underlie the encrinital Hurlet Limestone, are not seen here, but a very characteristic brecciated limestone bed with ashy mud, recognised by us in the same position at Howood, Bridge of Weir, and Campsie, is well displayed in old quarry section and in the neighbouring burn. Below it comes a thickness of sandstone whose lower boundary is a line of unconformity, which is also seen in the Campsie district. The Blackbyre Limestone comes in at this Downloaded from http://trngl.lyellcollection.org/ at West Virginia University on July 11, 2015 40 TRANSACTIONS GEOLOGICAL SOCIETT OP GLASGOW. point and is, as elsewhere, a typically marine deposit. The beds underlying it are seen in the burn section to consist mainly of sandstone with thin coal (the "Lady Anne"), and then comes the Hollybush Limestone. This seam is not well seen m section, but it contains the typical large producti and corals. At Nethercraigs the burn section goes only a short distance below the Hollybush, showing nothing but sandstone, but at Hollybush we get a glimpse in burn and railway sections of volcanic muds and oolitic beds which seem every­ where to occupy a position immediately above the lavas. There are also some ironstones containing plant remains. Howood.—In old quarries here, and in a burn section at Meikle Corseford, we find the Hurlet Limestone exhibiting the Nethercraigs characteristics and immediately underlain by a shale and coal. Some parts of the shale contain a marine fauna (producti, &c), while others are matted with a lamellibranch which appears to be Cypricardia (Sanguinolites) tricostata? The peculiar brecciated limestone is seen in the burn, and it here contains a few marine fossils, and is overlain by an ashy bed with nodules, like that of Nethercraigs. The equivalent of the Blackbyre Seam is also exposed, but differing somewhat in character from that of the type. Below it sandstone appears, overlying limey beds which are crowded witE giganteid producti and lithostrotion, which we regard as the equivalent of the Hollybush. Unfortunately the section gives no certain information beyond this point, and the boundary fault comes in. The modification of the characters of the Blackbyre Lime­ stone seen in this section is interesting as confirming the identification of the small, isolated exposure of limestone seen in the railway cutting at Auchinback, east of Barrhead, providing a link in the transition of this bed from the characters seen in the type section to those prevailng at Thorntonhall and in north Ayrshire. Bridge of Weir.—In the Gryfe, to the north-east of Bridge of Weir, there is an excellent section which, with but one important gap, is complete from the top of the lavas to the top Hosie Limestone. Beginning at Bridge of Weir and working down stream, there is an interesting section of muds and conglomerate of lava pebbles lying in hollows on the Downloaded from http://trngl.lyellcollection.org/ at West Virginia University on July 11, 2015 M A C N A I R — L I M E S T O N E S I N THE GLASGOW DISTRICT. 41 eroded l a v a surface. Above this comes ashy fireclay, with rootlets, irregular coal, and greenish sandstone. A horizon of fossiliferous nodules and shales then comes in, which is found t o develop into a fairly compact limestone in places, crowded with l a r g e producti and branching corals. I t can b e examined at intervals along the b a n k s of the Gryfe from the c o t t a g e on the west b a n k to the ford. A t the latter place the Fig. 2 — P l a n a n d Section along the Gryfe, from Bridge of Weir t o Crosslee. Lettering s a m e a s tables in t e x t (see page 42). nodular p h a s e is well seen. This limey horizon overlies a thin coal. A sill penetrates the s t r a t a , and the sequence is obscured b y small disturbances which run parallel to the line of section. F r o m the ford to the turn in the stream a t the old limestone workings, there are no exposures, but below water level a t the sharp bend, the nodular green limestone can be seen, contain­ i n g corals and encrinites. The coal and a l u m shale a r e not now exposed, b u t fragments can b e picked u p in the b i n g s . T h e evidence for identifying this seam as the Hurlet is fairly Downloaded from http://trngl.lyellcollection.org/ at West Virginia University on July 11, 2015 42 TRANSACTIONS—GEOLOGICAL SOCIETY OF GLASGOW. complete, and as already mentioned, this section also displays the augmentation of its thickness by the addition of limey shales, while the more typical encrinital part, 4 to 5 feet thick, has been worked out. The gap in this section occurs below the nodular green bed, just described, and we believe that at this point the Blackbyre Limestone exists, which opinion is confirmed by the evidence of the following bore put down at Goldilee and quoted in the Transactions of the High­ land and Agricultural Society : — BORE AT GOLDILEE, BRIDGE OF WEIR, RENFREWSHIRE. CORRELATED WITH THE HURLET SECTION. Feet. Inches. Clay, Flag, Limestone, Till, Coal, Fire-clay, Limestone, Fire-clay, Black Till, Coal, Grey Till, Greenstone, Grey Till, Freestone, Coal Till, Coal, Fire-clay, Whinstone, - - 12 20 7 1 2 5 4 2 19 13 5 5 5 1 5 - 0 0 0 6 ^ 10 } J g Hurlet Limestone. / A l u m Shale. e Hurlet Coal. d A s h y Muds w i t h Limey Nodules. c Blackbyre Limestone. b Equivalent of Hollybush Sandstone. Intrusive Sill. a Hollybush Limestone. 9 3 Lavas. The limey beds, containing large producti and corals, we correlate with the Hollybush Seam. Proceeding with the stream, a small fault is crossed, which brings down the top of the Hurlet, and then the shales and ironstones, with a thin coal, are met. These are overlain by a very interesting series of strata, of which we give a detailed section— 18" encrinital limestone. 14" strong shale. 12" shale or fireclay w i t h stigmaria and entomostracan shale. 12" entomostracan limestone. 12" shale. 4" coal. Shale w i t h ironstones (containing Lithostrotion in places) and coal. Downloaded from http://trngl.lyellcollection.org/ at West Virginia University on July 11, 2015 Trans. Geol. Soc. of Glasgow. Vol. XV., Plate V F i g 3. Plan and section across the South Hill of Campsie from Craigenglen to Baldow Glen, Lettering same as Table in text, see page 45. Downloaded from http://trngl.lyellcollection.org/ at West Virginia University on July 11, 2015 MACNAIR—LIMESTONES I N THE GLASGOW DISTRICT. 43 There can be no doubt that this represents the Blackhall Limestone with some interesting variations. Above these strata comes a fresh series of shales with iron­ stones, some of which contain entomostraca and almost develop into limestone, and then we reach the two encrinital bands of the Hosie, with shale containing Posidonomya corrugatcfi (marked i in section). We consider that the Nethercraigs, Howood, and Gryfe sections together afford important evidence bearing upon the co-relation of the Hurlet section with those in the north of Ayrshire. Thus it seems to us that the lower division of the main limestone, as seen at Broadstone, Beith, and elsewhere, with its underlying coal is the equivalent of the Hollybush seen in the three Renfrewshire localities. The fragmental deposits lying between the lower and upper divisions of the main limestone of Ayrshire, as seen at Broadstone, is the representative of the Hollybush Sandstone; while the upper division of the main limestone, as seen at Dockra, is the equivalent of the Blackbyre Limestone, the position of this limestone being fixed over a large part of the north of Ayrshire by the capping of ashy muds with limey nodules, which, we have shown, is so characteristic of this horizon in the Renfrew­ shire sections. The Hurlet in the north of Ayrshire has dwindled to less than its thickness in the type section, and the top of the Lower Limestone Series is marked by beds of limey shale with Posidonomya corrugata, which have a very striking resemblance to the shales and limestones of Calderwood Glen. We hope, however, to deal with this matter in more detail next session. Crossing now to the north side of the Clyde, we take the Campsie district first, because there we find the clearest sections. There are no sections so complete as that on the Gryfe, but, in the numerous small burns, exposures occur which can be pieced together. In the Memoir it is stated that the Hollybush Limestone is not seen north of the Clyde, but we 6 See Trans. Geol. Soc. Glas. vol. xiv., part iii., page 309, " On t h e Dis­ tribution of Posidonomya corrugata, Ether. J u n . , in the Carboniferous Limestone of t h e Glasgow District," by Peter Macnair, F . R . S . E . , F . G . S . , and Harry R. J. Conacher. f Downloaded from http://trngl.lyellcollection.org/ at West Virginia University on July 11, 2015 44 TRANSACTIONS—GEOLOGICAL SOCIETY OF GLASGOW. believe its exposures there are more plentiful than those of any other beds. South Brae of Campsie.—In Craigen Glen there is a section of a series of shales which are famous for the variety of their fossils. These beds are correlated by the Survey with the Blackbyre Seam (Productus Shales), but we are of opinion that they represent the Hollybush horizon. The section, beginning at the base, shows an oolite in fireclay-like beds overlain by sandstone containing a thin coal. The limey shales contain several bands of calcareous ironstone nodules, which were formerly mined; these nodules, as well as the shales, contain producti and corals which, in some cases, have been completely removed by percolating water, leaving very striking casts of the calcareous parts of the organism. The section passes up into sandstone and fireclay apparently underlying the Hurlet, whose position is traceable, but the section at this point is obscure. In Glen Wynd, immediately to the west, the Craigen Glen shales can be identified passing under sandstone which is overlain by a limestone, consisting of beds of small producti, with plant remains, and an encrinital upper part. These characters at once suggest the Blackbyre Limestone of Barrhead, and it also has an unconformity above it, as noted at Nethercraigs. Above this unconformity is a peculiar deposit, weathering with a characteristic red crust; if sampled near its base it is found to be a hard sandstone with rootlets and spirorbis, but the upper portions are distinctly limey, and in other sections it is found to be a calmy limestone with many entomostraca. It can be examined to advantage at the Linn of Baldernock, where thin shale bands subdivide it, as is also the case elsewhere. In Glen Wynd this limestone is not well seen, and the succeeding strata are also either worked out or hidden from sight, with the exception of a black shale, baked by a sill of white trap, whose identity is betrayed by its fauna as the alum shale of Lennoxtown district. North-west of Glen Wynd a small pine wood is fronted by extensive signs of old workings, and at one point, where a small burn falls into the old waste, a few feet of an encrinital limestone, with a few small producti, is seen overlain by black shales with small ironstones. These old workings, with occasional exposures of Downloaded from http://trngl.lyellcollection.org/ at West Virginia University on July 11, 2015 MACNAIR—LIMESTONES I N THE GLASGOW DISTRICT. 45 the limestone, can be traced practically right round the hill, and at Boyd's Burn and Culloch Slap work is still going on. For a short distance to the south of the wood just mentioned the tirring of old opencasts encumbers the ground, but above an old mine mouth in the former bed of the stream we see again the sandy base of the Baldernock Limestone, with its line of unconformity, and it is possible to recognise below it the same beds as seen in Glen Wynd. The structure in this glen (Borraston) is complicated by a number of small faults, which repeat part of the sequence. At Baldernock Linn, or higher in the burn which forms it, the following section is exposed: — Black shale with small ironstone nodules. Thin limestone, with small encrinites, &c, and pebbles of dolomite. (Shields bed). Black shales with small ironstone nodules. Encrinital t \ -i • » • here, i ~ - v bedj • m t EN. i Limestone. 1 Dolerite intrusion seen in Black Shale. V •> . *. ,of burn and in old quarry. Entomostracan Limestone. Sandy Limestone. Unconformity. Shales with plant remains and Lingula. Encrinital Limestone. c u A This section is much distorted by the dolerite intrusion near the linn, and a small dyke and sill of white trap also appear higher up. West of here, and near to the Auld Wives' Lifts, the sill is again seen, with fragments of limestone, &c, sufficient to identify it, and its outcrop across the slope to the south-east is marked by the depression left by old workings. No doubt this is the locality referred to in "The Geology of Campsie" where the author speaks of an encrinital limestone passing under the Craigmaddie Sandstones; in reality it is thrown against them by a fault. Passing now to the north face of the South Brae, we get several glimpses of our encrinital limestone—sometimes accom­ panied by the sill—in old workings. At Culloch Slap (above Lennoxtown), where work is still going on, we see that this limestone, 4 to 5 feet thick, overlies alum shale, coal, and Downloaded from http://trngl.lyellcollection.org/ at West Virginia University on July 11, 2015 46 TRANSACTIONS—GEOLOGICAL SOCIETY OP GLASGOW. entomostracan limestone, and in Baldow Glen we see the position of these strata in the sequence. Beginning at the road to Lennox Castle, we see, in ascending order, a Sandstones. b Fossiliferous limey shales, w i t h nodules and some more solid b e d s Productus giganteus (Craigen Glen beds). (Hollybush.) c Sandstones and fire-clays. d L i m e y shales crowded w i t h small Producti and t w o p o s t s of limestone composed of small encrinite fragments (Glen W y n d beds). (Blackbyre.) e Shale w i t h Lingula. f Unconformity. g Sandy Limestone. h Entomostracan Limestone (Baldernock). i Fireclay. j Gap. (Campsie Main Limestone worked out.) (Hurlet.) Jc Black shales w i t h small ironstone nodules. I Limestone band w i t h dolomitic pebbles, encrinites, &c. (Shields bed.) m Black shales w i t h large C.B. ironstones at top. n Limestone, in five beds, top one encrinital, others entomostracan and red crusted, partly oolitic, shale partings, one an oilshale (Baldow). (Blackhall.) o Shale w i t h nodules (Posidonomya becheri bed at base). The rest of this section shows only black shale and ironstone nodules. It is stated by the miners that in some parts of this district there is a 14-inch coal below the entomostracan limestone {Baldernock Limestone), but we have seen no trace of this. In the new Memoir, reference is made to a blue limestone cropping out in this glen, now no longer visible. This we suppose to be the limey shales, &c, which have yielded Productus giganteus. The exposure is very small and obscured by drift, but we identify it as the representative of the Craigen Glen shales, and we feel no doubt also that the productus shales with encrinital beds are the same as those seen in Glen Wynd. The upper part of the Baldernock Limestone here contains a nodular brecciated band with rootlets, whose appearance at once suggests that already described from Thorntonhall, Gryfe, and Nethercraigs. In Baldow, the Hurlet Coal and Limestone are not seen, and the alum shale is barely visible, but all can be examined in neighbouring workings. In €ulloch Slap and Boyd's Burn the alum shale contains numerous Downloaded from http://trngl.lyellcollection.org/ at West Virginia University on July 11, 2015 MACNAIR—LIMESTONES I N THE GLASGOW DISTRICT. 47 limestone nodules which closely resemble, when seen lying broken and weathered, the Baldernock Limestone, but they contain no entomostraca, but frequently a marine fauna, and are intricately brecciated like septarian nodules, which indeed they are. The alum shale contains a varied fauna; at Culloch Slap, Boyd's Burn, and Glen Wynd beds occur which are crowded with Leppodomus costellatus, while others bear only Lingula, small Producti, or Bellerophcm, and an occasional plant print (Sph&nopteris?), and in parts fish remains and ento­ mostraca are abundant. At Boyd's Burn and Glen Wynd, and in old bings east of Craigen Glen, we have found an occasional Posidonomya corrugata (one specimen from each locality). In Baldow Glen, at the sharp bend above the Hurlet outcrop, there is well exposed the striking bed which we refer to as the " Shields Bed." This bed is seen with approximately the same features in the burn above Baldernock Linn, in diminished size in the burn at Flutcher, in the wood between Langshot and Newlands (in small burn at east side of wood), and in Glen Whapple (north-west of Upper Carlston). In the last-named locality it has become partly oolitic and sandy enough to contain annelid burrows. In the type locality (Shields Burn, Milton of Campsie), it is a 3- to 4-feet bed of sandy encrinital limestone; while it is constant in some form throughout this district, the distance above the Hurlet Limestone varies. The section of the Baldow Limestone in Baldow Glen is the best in the district, but it is also seen in Glen Whapple, where it has been wrought, and its position is marked by old workings and numerous fine fragments at the mouth of Craigen Glen, to which position it is brought by a fault. On the South Hill, no higher strata than the Hosie Sandstone •occur, and this is to be seen in one or two small, old quarries south of Clochcore and at Newlands. 7 7 I n the alum shale which comes between the Cobbinshaw limestone and coal at Cobbinshaw and a t W e s t Mains there occurs an exactly similar fauna t o t h a t j u s t described, and i t is certainly very striking t h a t in t w o places so widely separated there should be found such a close resemblance i n t h e strata and their contents ; in both districts the descending sequence i s encrinital limestone, alum shale, pyritous coal, fire-clay, and entomostracan limestone. Downloaded from http://trngl.lyellcollection.org/ at West Virginia University on July 11, 2015 48 TRANSACTIONS GEOLOGICAL SOCIETY OF GLASGOW. North Brae of Campsie, &c.—Leaving the South Hill and crossing to Balgrochan, north of Lennoxtown, we find 4 to 5 feet of encrinital limestone capped by many feet of flaggy limestone, with pecten, productus, &c, and limey shales, a state of affairs similar to that at Nethercraigs, Gryfe, and Howood. Below this seam occurs the alum shale and coal, then the Baldernock Limestone, and a long section of the Glen Wynd Limestone is found in burns at Balgrochan and east of Bencloich Mains. In the burn at the latter place the Shields bed can be seen in its more typical form, and higher up the Baldow Limestone comes in. Above it at this point the shales contain an interesting fauna, not yet fully examined, including Posidonomya becheri (first noticed in this position by Mr. Reoch at Corrie Burn), orthoceras, and nautilus. This fauna we have also found in the same position in Baldow Glen and at Shields Cottage, but where the lime­ stone crops in Glen Whapple the shales are too much crushed by an adjacent fault to yield fossils. The Baldow Limestone is also exposed in the Bught Glen at Maiden Castle, where it is curious to note that the burn has sunk its ravine many feet right through that structure. In the burn east of Shields Farm we get our first sight of the Hosie Limestone, where it is of a cement-like nature in two bands about 8 feet apart, accompanied by black shales and one or two ironstone and kingle ribs. A few feet above the upper limestone the Posidonomya corrugafebeds occur in good form. The group is not again seen .early until we reach the Corrie district, but there the burn west of Burnhead gives an excellent section. Above the Hosie Limestone along the south face of the Campsie Hills between Corrie and Spouthead there occurs a. mass of sandstone alternating with shales and fakes, the alter*nation being repeated several times with remarkable regularity. Near the top of the series there occurs a sandstone with annelid burrows and Spirophytan caudagalli and small encrinities, and the latter bed is also to be seen near Burnhead. At Corrieburn there is a very interesting section, which passes from the zone of the volcanic detritus, with rough conglomerates and fine muds to the strata above the Upper Downloaded from http://trngl.lyellcollection.org/ at West Virginia University on July 11, 2015 Vol. XV., Plate VI. Trans. Geol. Soc. of Glasgow. Fig. 4. Plan and section across Limestones at Corrieburn, K i l s y t h . Lettering same as Table in t e x t , see page 49. Downloaded from http://trngl.lyellcollection.org/ at West Virginia University on July 11, 2015 MACNAIR—LIMESTONES I N THE GLASGOW DISTRICT. 49 Hosie Limestone. The details of the section as observed by us differ from those given in the Survey Memoir, and we set them out side by side below— Survey Memoir. Black oily shale w i t h Lingula. Limestone w i t h Bellerophon (Hosie Limestone). Strata w i t h thick yellow sandstone (Hosie Sandstone). Black shale w i t h septarian ironstone nodules. Marine fossils near base. Limestone, partly marine w i t h encrinites, partly dolomitic and oolitic w i t h entomostraca (Black­ hall). Strata—black shales and sandstones w i t h I . S . band. Hurlet j Upper hard band, -r« , i Calmy band. Limestone. | / L o w r h a r d b a n d Large growth forms of brachiopods and mollusca. Strata w i t h thin coal. Coral limestone. Black shales, sandstone bands, iron­ stones and calcareous n o d u l e s ; w i t h alternations of purplish volcanic detritus and rolled fragments. Lingula in shales and volcanic muds. Thick volcanic muds and con­ glomerates resting on lavas (pp. 15 and 24 of Memoir). Present Authors. z Posidonomya corrugata beds and Lingula shale. y Black limestone w i t h Bellerophon. x Black shales w i t h Leptodomus costellatus, Pinna, &c. w Limey shale. v Black limestone w i t h Bellerophon and Orthoceras. u Shale. t Sandstone. s Shale with nodules ( P . becheri bed at base). r Encrinital limestone w i t h small solitary corals. q Brown - crusted entomostracan limestone. p Sandstone, flaggy and ripplemarked, limey and oolitic in parts. 0 Limey shales w i t h large encrinites and Polyzoa. n Encrinital limestone (Hurlet). m Black shale w i t h Leptodomus costellatuSy and small ironstone nodules. 1 Coal. k Fire-clay. j Nodular green limestone w i t h corals. i Coral limestone, eroded in places (Hollybush). h Limey shales w i t h Rhynconella and nodules containing corals and brachiopods. g Shale with ironstone. /Naiadites crassa bed. e Sphenopteris (?) bed. d Beds containing young of Naia­ dites crassa. c Shales grading down into volcanic mud w i t h oolitic ironstone nodules. b Sandstone. a Muds and conglomerate. The Hurlet Limestone appears to be correctly identified here, and the overlying beds at once betray their identity, but below the coal there is difficulty. We are inclined to think that here the line of erosion, elsewhere noted above the Glen VOL. XV., PT. I. Downloaded from http://trngl.lyellcollection.org/ at West Virginia University on July 11, 2015 50 TRANSACTIONS—GEOLOGICAL SOCIETY OF GLASGOW. Wynd Limestone, has cut out that seam, and that the coral limestone and the accompanying very fossiliferous beds are equivalent to the Craigen Glen shales. East of Corrie there are only one or two unimportant, isolated exposures of the Baldow Limestone at Berryhill, hints of it at Linns, while the Hosie is seen at Drumnessie. At North Bardowie, the Hurlet group can be traced by old workings, at Flutcher the Shields bed is seen, and at Dowans there is an excellent section of the Craigen Glen beds involved in a dolerite intrusion, the intervening strata being hidden. West of Milngavie, the Hurlet group is not seen, but old workings mark its outcrop. At Hardgate Quarry, in the burn at Duntocher Mill and at Auchentoshan and on and near the golf course at Lawmuir, are exposures of what is evidently the same calcareous series, containing corals and large producti. The character of these beds, their relation to the Hurlet outcrop, and the nature of the adjacent strata (sandstone with thin coal above and below), cause us to regard them as the equivalent of the Craigen Glen beds. From the foregoing, it will be seen that throughout the Glasgow district the limestones are remarkably constant in occurrence, and that those modifications which take place can be traced in their development from one locality to another, showing that the Blackhall, Hurlet, Blackbyre, and Hollybush Limestones of the type locality have their counterparts north of the Clyde in the district stretching from Kilsyth to Dun­ tocher in the Baldow, Campsie Main, Glen Wynd, and Craigen Glen beds respectively. 

W4294190135.txt

Journal of Educational and Psychological Sciences ‫مجلة العلوم‬ ‫التربوية والنفسية‬ Volume (6), Issue (36): 30 Aug 2022 P: 57 - 73 ‫م‬2022 ‫ أغسطس‬30 :)36( ‫ العدد‬،)6( ‫املجلد‬ 73 - 57 :‫ص‬ ISSN: 2522- 3399 The obstacles which are facing mathematics teachers in activating professional learning communities from their point of view Fahad Ibrahim Mokli Ahmed Abdullah Zaylaee Sabya Education Department || Ministry of Education || KSA Abstract: The study aimed to identify the obstacles facing mathematics teachers in activating professional learning communities in Sabya's Educational directorate. The questionnaire was used in the study. which was consisting of three dimensions. (administrative obstacles, physical obstacles, and personal obstacles), the number of study sample members reached (716) teachers. the study found a set of obstacles, most notably: high number of classes given to the teacher, lack of training programs on professional learning communities, and teachers' low conviction in the process of change and development in their performance. The study also concluded that there were no statistically significant differences at the level (0.05) between the opinions of the sample members about the three dimensions of the study due to years of experience. The study recommended a number of recommendations. Keywords: Obstacles, professional learning communities, mathematics teachers. ً ‫املعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات التعلم املهنية وفقا آلرائهم‬ ‫فهد بن إبراهيم موكلي‬ ‫أحمد بن عبد هللا زيلعي‬ ‫إدارة التعليم بمحافظة صبيا || وزارة التعليم || اململكة العربية السعودية‬ ‫ هدفت الدراسة إلى التعرف على املعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات التعلم املهنية بإدارة تعليم‬:‫املستخلص‬ ‫ املعيقات الشخصية) وبلغت عينة‬،‫ املعيقات املادية‬،‫ وقد تم استخدام استبانة مكونه من ثالثة محاور وهي (املعيقات اإلدارية‬،‫صبيا‬ ً )716( ‫الدراسة‬ ‫ قلة البرامج التدريبية حول‬،‫ ارتفاع أنصبة املعلمين‬:‫ وقد توصلت الدراسة إلى مجموعة من املعوقات من أبرزها‬،‫معلما‬ ‫ كما توصلت الدراسة إلى أنه ال توجد فروق ذات‬.‫ ضعف قناعة املعلمين بعملية التغيير والتطوير في أدائهم‬،‫مجتمعات التعلم املهنية‬ ‫ وقد أوصت الدراسة‬،‫) بين آراء أفراد العينة حول محاور الدراسة الثالثة تعزى إلى سنوات الخبرة‬0.05( ‫داللة إحصائية عند مستوى‬ .‫بمجموعة من التوصيات‬ ‫ معلمي الرياضيات‬-‫ مجتمعات التعلم املهنية‬-‫ معيقات‬:‫الكلمات املفتاحية‬ .‫ املقدمة‬-1 ‫ من‬،‫فرضت التغيرات املتسارعة في شتى مناحي الحياة إحداث مدخالت تطويرية حديثة في العملية التعليمية‬ ً ً ‫عامال‬ ‫ واالهتمام بتحقيق الجودة في أداء‬،‫مؤثرا في تحسين مخرجات التعليم‬ ‫شأنها تطوير األداء املدرس ي الذي يعد‬ .‫ وبما يمكن من مواكبة التغيرات‬،‫املعلمين‬ DOI: https://doi.org/10.26389/AJSRP.H030122 )57( Available at: https://www.ajsrp.com ‫المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس ‪2022‬م‬ ‫ويستلزم تطوير التعليم االهتمام بالتنمية املهنية املستدامة للمعلمين وتحسين ثقافتهم املهنية واألكاديمية‬ ‫من خالل تدريبيهم على سد الفجوات بين واقع ممارساتهم التعليمية وبين متطلبات النظم الحديثة لإلصالح املدرس ي‪،‬‬ ‫والتجديدات التربوية (السليمي والزلفي‪.)2019 ،‬‬ ‫وتعد مجتمعات التعلم املهنية إحدى املدخالت التطويرية الحديثة في التعليم واإلصالح املدرس ي التي تتبنى‬ ‫التنمية املهنية املستدامة للمعلمين من خالل تزويدهم باملعارف واملهارات‪ ،‬وتحسين ممارساتهم التدريسية‪ ،‬وزيادة‬ ‫ً‬ ‫إيجابيا من خالل استخدام عدة استراتيجيات فعالة‪ ،‬كما تحد من العمل الفردي بين‬ ‫فاعليتهم‪ ،‬والتأثير عليهم‬ ‫املعلمين‪ ،‬وتتغلب على ثقافة االنعزال‪ ،‬األمر الذي يؤدي إلى تطوير األداء املدرس ي (حسن بت‪2016 ،‬؛ إبراهيم‬ ‫وحسان‪2018 ،‬؛ البرعمي وعبد الرشيد‪2020 ،‬؛ ‪.)Herrington, 2011‬‬ ‫وتقوم فكرة مجتمعات التعلم املهنية على تشكيل مجموعات تخصصية تجتمع باستمرار لتبادل األفكار‪،‬‬ ‫وتحسين التدريس (الزايد وعمر‪ .)2016 ،‬كما جاءت متمحورة حول زيادة املعرفة املهنية للمعلمين من خالل‬ ‫اندماجهم وتفاعلهم في هذه املجتمعات وتأملهم للخبرات واملمارسات اليومية التي يتشاركونها فيما بينهم (شركة تطوير‬ ‫للخدمات التعليمية‪.)2014 ،‬‬ ‫وتدعم مجتمعات التعلم املهنية املناخ املدرس ي والثقافة التنظيمية‪ ،‬باإلضافة إلى أنها تدمج املعلمين في‬ ‫املشروعات واألنشطة التعاونية‪ ،‬وتزيد من قدراتهم وإمكاناتهم على العمل‪ ،‬مما يسهم في وجود بيئة مدرسية منتجة‪،‬‬ ‫وتحسين عملية التعليم والتعلم (‪.)Kalkan, 2016‬‬ ‫وتهدف مجتمعات التعلم املهنية إلى مساعدة املعلمين ليكونوا أكثر فاعلية في عملهم مع الطالب‪ ،‬وبالتالي‬ ‫التركيز على التعلم والنتائج‪ ،‬وكذلك دعم زمالئهم واالهتمام باملمارسات التدريسية وتأملها ونقدها وذلك من خالل بناء‬ ‫ثقافة تعزيز التعاون بين العاملين في املدرسة (‪.)Battersby & Verdi, 2015‬‬ ‫ً‬ ‫وتعرف مجتمعات التعلم املهنية على أنها مجاال لتطوير األداء املدرس ي‪ ،‬يركز على تمكين املدرسة من تحديد‬ ‫املشكالت والتحديات التي تقابلها والقدرة على مواجهتها بفاعلية‪ ،‬وذلك من خالل تحويل املدرسة إلى مجتمعات تعلم‬ ‫مهنية لها أنشطة تستهدف تنمية قدرات املعلمين وتحسين مستويات التعلم لدى طالبهم (إبراهيم واملرزوقي‪.)2018 ،‬‬ ‫إن االفتراض الذي تقوم عليه مجتمعات التعلم املهنية هو ارتباط تحسين تعلم الطالب بالتنمية املهنية‬ ‫املستدامة ملعلميهم)‪ . (Dufour et. al, 2016‬وقد أكدت دراسة الصالحية والهاشم (‪ )2018‬على دور مجتمعات التعلم‬ ‫املهنية في رفع املستوى التحصيلي للطالب‪.‬‬ ‫وتناولت األدبيات مجتمعات التعلم املهنية‪ ،‬وأكدت على االهتمام بها‪ ،‬والتركيز عليها‪ ،‬وأشارت إلى أهميتها‪،‬‬ ‫ً‬ ‫اتجاها ع ً‬ ‫امليا إلصالح التعليم في كثير من الدول‪ :‬كأمريكا‪ ،‬وبريطانيا‪ ،‬وفرنسا‪،‬‬ ‫حيث أصبحت مجتمعات التعلم املهنية‬ ‫وأملانيا‪ ،‬والصين (ناصف‪.)2012 ،‬‬ ‫كما تؤكد دراسة دوقان (‪ )Dogan et al, 2017‬أن مجتمعات التعلم املهنية من الركائز الرئيسية في‬ ‫مشروعات التطوير املنهي للمعلمين في كثير من دول العالم حيث تؤثر بصورة إيجابية في تنمية قدرات املعلمين‬ ‫ومهاراتهم وتركز على جهود التعاون والرؤية املشتركة بينهم‪.‬‬ ‫وقد وقفت اململكة العربية السعودية على عدد من التجارب الناجحة‪ ،‬واالستعانة بالعديد من الدراسات‬ ‫العاملية التي تناولت األنظمة التربوية والتعليمية (املطيري‪ .)2018 ،‬واحتلت مجتمعات التعلم املهنية ً‬ ‫حيزا ً‬ ‫كبيرا ضمن‬ ‫ً‬ ‫ً‬ ‫رئيسيا في نموذج تطوير‬ ‫ومنطلقا‬ ‫تطوير التعليم في اململكة العربية من خالل مشروع امللك عبد هللا لتطوير التعليم‪،‬‬ ‫املدارس‪ ،‬وداعمة لعمليتي التعليم والتعلم (أنموذج تطوير املدارس‪.)1436 ،‬‬ ‫موكلي‪ ،‬زيلعي‬ ‫(‪)58‬‬ ‫المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات‬ ‫التعلم المهنية وفقًا آلرائهم‬ ‫المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس ‪2022‬م‬ ‫وقد أجريت العديد من الدراسات العربية واألجنبية حول مجتمعات التعلم املهنية‪ ،‬ومنها دراسة سونق‬ ‫(‪ )Song,2012‬التي توصلت إلى أن تطبيق مجتمعات التعلم املهنية يساعد املدارس على تحسين التعلم‪ ،‬وعلى جعل‬ ‫املعلمين أكثر تقبال لإلصالحات التربوية‪ ،‬كما توصلت دراسة فولر (‪ )Fuller, 2014‬إلى األثر اإليجابي ملجتمعات التعلم‬ ‫املهنية على ممارسات املعلمين‪ ،‬وكذلك على التحصيل الدراس ي للطالب‪ ،‬وتوصلت دراسة األخناوي (‪ )2016‬إلى ضعف‬ ‫تطبيق مجتمعات التعلم املهنية‪ ،‬وأن هناك معوقات تحول دون تفعيلها‪ ،‬كما توصلت دراسة محمد (‪ )2019‬إلى دور‬ ‫مجتمعات التعلم املهنية في تحسين األداء األكاديمي في مدارس التعليم العام‪ ،‬وقد أوصت هذه الدراسات بضرورة‬ ‫تفعيل مجتمعات التعلم املهنية‪ ،‬وتذليل الصعوبات التي تعيق تفعيلها‪.‬‬ ‫وقد اقتصر الباحثان في هذه الدراسة على معلمي الرياضيات؛ كون الرياضيات مجال تخصص الباحثين‪.‬‬ ‫ويمكن تحقيق مجتمعات التعلم املهنية ملعلمي الرياضيات من خالل تشكيل مجتمع التعلم املنهي ملعلمي‬ ‫الرياضيات‪ ،‬وتحديد املعلم املشرف‪ ،‬وإعطاء الوقت الجتماع املعلمين فيما بينهم‪ ،‬وبناء الخطة التطويرية لتنمية‬ ‫معرفتهم بمحتوى الرياضيات وطرق تدريسه‪ ،‬حيث إن إتاحة الفرصة لتطوير معرفة معلمي الرياضيات وممارساتهم‬ ‫التدريسية وفق احتياجاتهم‪ ،‬ومعالجة القصور لديهم يؤثر في تعلم الطالب‪ ،‬وبالتالي في تحصيلهم (موكلي‪.)2019 ،‬‬ ‫قصورا واضحاً‬ ‫ً‬ ‫وبالرغم من أهمية مجتمعات التعلم املهنية في تحسين تعليم وتعلم الرياضيات إال أن هناك‬ ‫في تفعيلها من قبل معلمي الرياضيات في الوقت الذي يؤكد املشروع الوطني لتطوير التعليم في اململكة العربية‬ ‫السعودية على ضرورة تطبيق مجتمعات التعلم املهنية لنجاح عملية تطوير األداء املدرس ي‪ ،‬وتحسين نتائج الطالب‪،‬‬ ‫لذلك كان من األهمية بمكان القيام بدراسة للتعرف على املعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات‬ ‫التعلم املهنية‪.‬‬ ‫مشكلة الدراسة‪:‬‬ ‫تسهم مجتمعات التعلم املهنية في تطوير كفايات املعلمين ورفع أدائهم التدريس ي وتحسين عمليات التعليم‬ ‫والتعلم‪ ،‬كما تعد ر ً‬ ‫افدا للمدرسة في التنبؤ بمشكالتها املستقبلية‪ ،‬وقدرتها على التحسين والتطوير‪.‬‬ ‫وقد أكدت نتائج دراسة (هلل‪ ،)2013 ،‬ودراسة بيرتش )‪ (Bertsch,2013‬أن مجتمعات التعلم املهنية تسهم‬ ‫في التقليل من عزلة املعلم‪ ،‬وتزيد من التزامه بمهامه وتحقيق أهداف املدرسة‪ .‬كما أكدت دراسة (الزايد‪ )2018 ،‬على‬ ‫ً‬ ‫إيجابيا باملمارسات التأملية في مجتمعات التعلم‬ ‫أن الجانب املعرفي‪ ،‬واملمارسات الصفية‪ ،‬واملعتقدات التربوية تأثرت‬ ‫املهنية‪ .‬وتؤكد دراسة محروس (‪ )2015‬أن مجتمعات التعلم املهنية استراتيجية فعالة تغيير وتطوير املدارس‪ ،‬ومدخل‬ ‫قوي لإلصالح املدرس ي املنشود‪.‬‬ ‫وقد أكدت دراسة كل من‪ :‬الحازمي (‪ ،)2016‬وناصف (‪ ،)2012‬على أهمية تفعيل مجتمعات التعلم املهنية‬ ‫داخل املدرسة‪ ،‬وأوصت دراسة (العجالن‪ )2016 ،‬بتعميم تجربة مجتمعات التعلم املهنية على جميع املدارس باململكة‬ ‫العربية السعودية‪ ،‬وأوصت دراسة (الزايدي‪ )2013 ،‬بنشر ثقافة تحول املدارس إلى مجتمعات تعلم مهنية من خالل‬ ‫تصميم برامج تدريبية لجميع منسوبي املدارس في السعودية‪ .‬وتؤكد دراسة توفيق (‪ )2017‬على أهمية تفعيل‬ ‫مجتمعات التعلم املهنية لتحقيق اإلصالح والتطوير التعليمي‪.‬‬ ‫وبالرغم من أهمية مجتمعات التعلم املهنية ودورها في عمليتي التعليم والتعلم إال أن الباحثين من خالل‬ ‫عملهما مشرفين تربويين في وحدة تطوير املدارس‪ ،‬ومتابعتهما لتطبيق مجتمعات التعلم املهنية الحظا أن هناك ً‬ ‫ضعفا‬ ‫في تفعيل مجتمعات التعلم املهنية لدى املعلمين‪ ،‬وكذلك ما توصلت له الدراسات السابقة من وجود ضعف في‬ ‫موكلي‪ ،‬زيلعي‬ ‫(‪)59‬‬ ‫المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات‬ ‫التعلم المهنية وفقًا آلرائهم‬ ‫المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس ‪2022‬م‬ ‫تفعيل مجتمعات التعلم املهنية‪ ،‬ووجود معيقات تحول دون تفعيلها‪ ،‬وبذلك يمكن تحديد مشكلة الدراسة في محاولة‬ ‫التعرف على املعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات التعلم املهنية ً‬ ‫وفقا آلرائهم‪.‬‬ ‫أسئلة الدراسة‪:‬‬ ‫بناء على ما سبق؛ تتحدد مشكلة هذه الدراسة في السؤال الرئيس اآلتي‪:‬‬ ‫ً‬ ‫ما املعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات التعلم املهنية وفقا آلراء املعلمين؟‬ ‫ويتفرع منه األسئلة التالية‪:‬‬ ‫ً‬ ‫‪ -1‬ما املعيقات اإلدارية التي تحد معلمي الرياضيات من تفعيل مجتمعات التعلم املهنية وفقا آلرائهم؟‬ ‫‪ -2‬ما املعيقات املادية التي تحد معلمي الرياضيات من تفعيل مجتمعات التعلم املهنية ً‬ ‫وفقا آلرائهم؟‬ ‫‪ -3‬ما املعيقات الشخصية التي تحد معلمي الرياضيات من تفعيل مجتمعات التعلم املهنية ً‬ ‫وفقا آلرائهم؟‬ ‫‪ -4‬هل توجد فروق ذات داللة احصائية عند مستوى (‪ )α≤0.05‬بين آراء معلمي الرياضيات حول املعيقات التي‬ ‫تواجه معلمي الرياضيات في تفعيل مجتمعات التعلم املهنية تعزى للخبرة التدريسية؟‬ ‫أهداف الدراسة‬ ‫سعت الدراسة لتحقيق األهداف التالية‪:‬‬ ‫ً‬ ‫‪ .1‬التعرف على املعيقات اإلدارية التي تحد معلمي الرياضيات من تفعيل مجتمعات التعلم املهنية وفقا آلراء‬ ‫املعلمين‪.‬‬ ‫‪ .2‬التعرف على املعيقات املادية التي تحد معلمي الرياضيات من تفعيل مجتمعات التعلم املهنية ً‬ ‫وفقا آلراء‬ ‫املعلمين‪.‬‬ ‫ً‬ ‫‪ .3‬التعرف على املعيقات الشخصية التي تحد معلمي الرياضيات من تفعيل مجتمعات التعلم املهنية وفقا آلراء‬ ‫املعلمين‪.‬‬ ‫‪ .4‬الكشف عن وجود فروق ذات داللة احصائية عند مستوى (‪ (α≤0.05‬بين آراء معلمي الرياضيات حول‬ ‫املعيقات التي تواجههم في تفعيل مجتمعات التعلم املهنية تعزى للخبرة التدريسية‪.‬‬ ‫أهمية الدراسة‬ ‫تكمن أهمية الدراسة في أنها قد تفيد في‪:‬‬ ‫ تقديم تغذية راجعة للمسئولين في وزارة التعليم باململكة العربية السعودية للتعرف على املعيقات التي تواجه‬‫معلمي الرياضيات في تفعيل مجتمعات التعلم املهنية‪.‬‬ ‫ االسهام في تقديم الحلول لتالفي املعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات التعلم املهنية‪.‬‬‫حدود الدراسة‪.‬‬ ‫اقتصرت الدراسة على الحدود اآلتية‪:‬‬ ‫ً‬ ‫‪ ‬الحدود املوضوعية‪ :‬املعيقات التي تحول دون تفعيل معلمي الرياضيات ملجتمعات التعلم املهنية وفقا آلرائهم‪.‬‬ ‫‪ ‬الحدود املكانية‪ :‬املدارس الحكومية للبنين في إدارة تعليم صبيا‪.‬‬ ‫‪ ‬الحدود الزمانية‪ :‬طبقت الدراسة في الفصل الدراس ي الثاني من العام الدراس ي ‪ 1442 -1441‬هـ‬ ‫موكلي‪ ،‬زيلعي‬ ‫(‪)60‬‬ ‫المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات‬ ‫التعلم المهنية وفقًا آلرائهم‬ ‫المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس ‪2022‬م‬ ‫مصطلحات الدراسة‬ ‫ مجتمعات التعلم املهنية‪ :‬هي مجموعات تخصصية يشكلها مدير املدرسة ويقوم بدعمها‪ ،‬ويتم فيها بناء الخطة‬‫التطويرية للمجموعة‪ ،‬وتبادل التجارب بين أفرادها‪ ،‬وتشارك الخبرات املهنية‪ ،‬وتأمل املمارسات التدريسية‬ ‫ونقدها‪ ،‬ومناقشة املحتوى الرياض ي‪ ،‬وتقويم الطالب‪.‬‬ ‫‪ -2‬منهجية الدراسة وإجراءاتها‪.‬‬ ‫منهج الدراسة‪:‬‬ ‫ً‬ ‫نظرا لهدف الدراسة وهو التعرف على املعيقات التي تحول دون تفعيل معلمي الرياضيات ملجتمعات التعلم‬ ‫املهنية‪ .‬فقد تم استخدام املنهج الوصفي املسحي منهجا للدراسة ملالءمته لهدفها‪.‬‬ ‫مجتمع الدراسة‪:‬‬ ‫ً‬ ‫تألف مجتمع الدراسة من جميع معلمي الرياضيات في مدارس إدارة تعليم صبيا والبالغ عددهم (‪ )716‬معلما‬ ‫للعام الدراس ي ‪ 1440 -1439‬هـ‪.‬‬ ‫عينة الدراسة‪:‬‬ ‫ً‬ ‫شملت عينة الدراسة (‪ )279‬معلما‪ ،‬أي ما يمثل (‪ )%39‬من مجتمع الدراسة‪ ،‬حيث تم االختيار بالطريقة‬ ‫العشوائية‪.‬‬ ‫توزيع أفراد العينة‪:‬‬ ‫أ‪ -‬توزيع أفراد العينة ملتغير سنوات الخبرة التدريسية‬ ‫جدول (‪ )1‬عدد أفراد العينة وفقا ملتغير سنوات الخبرة التدريسية‬ ‫النسبة املئوية‬ ‫التكرار‬ ‫سنوات الخبرة التدريسية‬ ‫‪%7.53‬‬ ‫‪21‬‬ ‫أقل ‪ 5‬سنوات‬ ‫‪%8.60‬‬ ‫‪24‬‬ ‫من ‪ 10 -6‬سنوات‬ ‫‪%83.87‬‬ ‫‪234‬‬ ‫أكثر من ‪ 10‬سنوات‬ ‫‪%100‬‬ ‫‪279‬‬ ‫املجموع‬ ‫ً‬ ‫يتضح من الجدول (‪ )1‬توزيع أفراد العينة وفقا ملتغير عدد سنوات الخبرة التدريسية‪ ،‬حيث يظهر أن عدد‬ ‫أفراد العينة التي تتراوح خبرتهم أقل من ‪ 5‬سنوات (‪ )21‬ويمثلون نسبة (‪ )%7.53‬من إجمالي أفراد العينة‪ ،‬ويبلغ عدد‬ ‫أفراد العينة الذين تتراوح خبرتهم من ‪ 10 -6‬سنوات (‪ )24‬ويمثلون نسبة (‪ )%8.60‬من إجمالي أفراد العينة‪ ،‬بينما يبلغ‬ ‫عدد أفراد العينة التي تزيد خبرتهم التدريسية عن ‪10‬سنوات (‪ )234‬ويمثلون نسبة (‪ )%83.87‬من إجمالي أفراد‬ ‫العينة‪.‬‬ ‫ب‪ -‬توزيع أفراد العينة وفقا ملتغير املؤهل‬ ‫جدول (‪ )2‬عدد أفراد العينة وفقا ملتغير املؤهل‬ ‫النسبة املئوية‬ ‫التكرار‬ ‫املؤهل‬ ‫‪%96.77‬‬ ‫‪270‬‬ ‫بكالوريوس‬ ‫موكلي‪ ،‬زيلعي‬ ‫(‪)61‬‬ ‫المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات‬ ‫التعلم المهنية وفقًا آلرائهم‬ ‫المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس ‪2022‬م‬ ‫النسبة املئوية‬ ‫التكرار‬ ‫املؤهل‬ ‫‪%3.23‬‬ ‫‪9‬‬ ‫الشهادات العليا‬ ‫‪%100‬‬ ‫‪279‬‬ ‫املجموع‬ ‫يتضح من جدول (‪ )2‬توزيع أفراد عينة الدراسة وفقا ملتغير عدد املؤهل العلمي‪ ،‬حيث يتبين أن (‪ )270‬من‬ ‫العينة الذين يمثلون ما نسبته (‪ )96.77‬من إجمالي عينة الدراسة كان مؤهلهم بكالوريوس‪ ،‬في حين أن (‪ )9‬من العينة‬ ‫الذين يمثلون ما نسبته (‪ )3.23‬من إجمالي العينة كان مؤهلهم دراسات عليا‪.‬‬ ‫أداة الدراسة‪:‬‬ ‫‪ ‬استبانة‬ ‫الهدف‪ :‬التعرف على املعيقات التي تحول دون تفعيل معلمي الرياضيات ملجتمعات التعلم املهنية‪.‬‬ ‫إجراءات بناء االستبانة‪:‬‬ ‫‪ -1‬قراءة الدراسات السابقة التي تناولت مجتمعات التعلم املهنية‪.‬‬ ‫‪ -2‬تحديد محاور االستبانة والتي تمثل معوقات مجتمعات التعلم املهنية‪ .‬وهي (إدارية‪ ،‬مادية‪ ،‬شخصية)‪.‬‬ ‫‪ -3‬كتابة مجموعة من العبارات املناسبة لكل محور‪.‬‬ ‫‪ -4‬عرض االستبانة على مجموعة من الخبراء في مجال تعليم الرياضيات‪.‬‬ ‫‪ -5‬كتابة األداة بالصورة النهائية‪ .‬حيث جاءت بالشكل التالي‪:‬‬ ‫ املحور األول‪ :‬املعوقات اإلدارية وتضمن (‪ )10‬عبارة‪.‬‬‫ املحور الثاني‪ :‬املعوقات املادية وتضمن (‪ )11‬عبارات‪.‬‬‫ املحور الثالث‪ :‬املعوقات الشخصية وتضمن (‪ )9‬عبارة‪.‬‬‫صدق االستبانة وثباتها‬ ‫ً‬ ‫أوال‪ :‬صدق االستبانة‬ ‫للتعرف على صدق أداة الدراسة‪ ،‬فقد تم عرضها على مجموعة من أعضاء هيئة التدريس املتخصصين في‬ ‫املناهج وطرق تدريس الرياضيات ومجموعة من املشرفين التربويين ومجموعة من الباحثين في موضوع مجتمعات‬ ‫ً‬ ‫التعلم‪ ،‬وفي ضوء آراء املحكمين أعيدت صياغة بعض الفقرات لغويا‪ ،‬وعدلت بعض الفقرات‪ ،‬وتم اعتماد الفقرات‬ ‫التي كان معدل اتفاق املحكمين حولها (‪ )%90‬فما فوق‪.‬‬ ‫ً‬ ‫ثانيا‪ -‬ثبات االستبانة‬ ‫للتأكد من ثبات االستبانة‪ ،‬قام الباحثان بحساب معامالت ارتباط درجة كل عبارة باملحور الذي تنتمي له‪،‬‬ ‫وذلك باستخدام معامل ارتباط (بيرسون) كما يوضحه الجدول (‪:)3‬‬ ‫جدول (‪ :)3‬درجة ارتباط كل عبارة باملحور الذي تنتمي له‬ ‫معامل االرتباط‬ ‫معامل االرتباط‬ ‫معامل االرتباط باملحور‬ ‫املفردة‬ ‫املفردة‬ ‫املفردة‬ ‫باملحور‬ ‫باملحور‬ ‫‪0.580‬‬ ‫‪22‬‬ ‫‪0.705‬‬ ‫‪11‬‬ ‫‪0.561‬‬ ‫‪1‬‬ ‫‪0.803‬‬ ‫‪23‬‬ ‫‪0.795‬‬ ‫‪12‬‬ ‫‪0.792‬‬ ‫‪2‬‬ ‫موكلي‪ ،‬زيلعي‬ ‫(‪)62‬‬ ‫المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات‬ ‫التعلم المهنية وفقًا آلرائهم‬ ‫المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس ‪2022‬م‬ ‫معامل االرتباط‬ ‫معامل االرتباط‬ ‫املفردة‬ ‫املفردة‬ ‫باملحور‬ ‫باملحور‬ ‫‪0.796‬‬ ‫‪24‬‬ ‫‪0.822‬‬ ‫‪13‬‬ ‫‪0.826‬‬ ‫‪3‬‬ ‫‪0.679‬‬ ‫‪25‬‬ ‫‪0.858‬‬ ‫‪14‬‬ ‫‪0.810‬‬ ‫‪4‬‬ ‫‪0.814‬‬ ‫‪26‬‬ ‫‪0.804‬‬ ‫‪15‬‬ ‫‪0.811‬‬ ‫‪5‬‬ ‫‪0.848‬‬ ‫‪27‬‬ ‫‪0.711‬‬ ‫‪16‬‬ ‫‪0.859‬‬ ‫‪6‬‬ ‫‪0.795‬‬ ‫‪28‬‬ ‫‪0.897‬‬ ‫‪17‬‬ ‫‪0.866‬‬ ‫‪7‬‬ ‫‪0.563‬‬ ‫‪29‬‬ ‫‪0.852‬‬ ‫‪18‬‬ ‫‪0.832‬‬ ‫‪8‬‬ ‫‪0.718‬‬ ‫‪30‬‬ ‫‪0.781‬‬ ‫‪19‬‬ ‫‪0.715‬‬ ‫‪9‬‬ ‫‪0.719‬‬ ‫‪20‬‬ ‫‪0.493‬‬ ‫‪10‬‬ ‫‪0.766‬‬ ‫‪21‬‬ ‫يتضح من الجدول (‪ )3‬أن جميع معامالت ارتباط العبارات مع محاورها دالة إحصائيا عند مستوى داللة‬ ‫(‪ ،)0.01‬حيث تراوحت ملحور املعوقات اإلدارية بين (‪ ،)0.866 -0.493‬بينما تراوحت للمعوقات املادية بين (‪-0.705‬‬ ‫‪ ،)0.897‬وتراوحت للمعوقات الشخصية بين (‪ ،)0.848 -0.563‬مما يدل على ارتباط كل عبارة من عبارات االستبانة‬ ‫باملحور الذي تنتمي له‪ ،‬مما يعني توافر درجة عالية من ثبات عبارات لالستبانة‪.‬‬ ‫كما يوضح جدول (‪ )4‬حساب معامل ألفا كرونباخ للثبات‪:‬‬ ‫جدول (‪ :)4‬قيم ألفا كرونباخ لثبات محاور االستبانة‬ ‫قيمة ثبات املحاور‬ ‫عدد العبارات‬ ‫محاور االستبانة‬ ‫املحور األول‬ ‫‪0.94‬‬ ‫‪10‬‬ ‫‪0.95‬‬ ‫‪11‬‬ ‫املحور الثاني‬ ‫‪0.92‬‬ ‫‪9‬‬ ‫املحور الثالث‬ ‫‪0.96‬‬ ‫‪30‬‬ ‫الثبات العام ملحاور الدراسة‬ ‫يتضح من جدول (‪ )4‬أن معامل الثبات ملحاور األداة (االستبانة) مرتفع‪ ،‬حيث يتراوح بين (‪ )0.95~0.92‬تم‬ ‫حساب معامل ألفا كرونباخ‪ ،‬حيث بلغت نسبة ثبات األداة (‪ .)0.96‬وهذا يدل على أن األداة تتمتع بدرجة عالية من‬ ‫الثبات‪ ،‬ويمكن االعتماد عليها والوثوق بنتائج الدراسة‪.‬‬ ‫املفردة‬ ‫معامل االرتباط باملحور‬ ‫‪ -3‬عرض النتائج ومناقشتها‪.‬‬ ‫‪ ‬النتائج املتعلقة بالسؤال الفرعي األول‪" :‬ما املعيقات اإلدارية التي تحد معلمي الرياضيات من تفعيل مجتمعات‬ ‫التعلم املهنية ً‬ ‫وفقا آلرائهم؟‬ ‫ولإلجابة تم حساب التكرارات والنسب املئوية واملتوسطات الحسابية واالنحرافات املعيارية لكل مفردة من‬ ‫مفردات األداة والجدول (‪ )5‬التالي يوضح تلك النتائج‪.‬‬ ‫موكلي‪ ،‬زيلعي‬ ‫(‪)63‬‬ ‫المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات‬ ‫التعلم المهنية وفقًا آلرائهم‬ ‫المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس ‪2022‬م‬ ‫جدول (‪ :)5‬املعيقات اإلدارية التي تحد معلمي الرياضيات من تفعيل مجتمعات التعلم املهنية‬ ‫أوافق بشدة‬ ‫‪%‬‬ ‫ك‬ ‫م‬ ‫العبارة‬ ‫‪1‬‬ ‫يبني املدير خطة‬ ‫املدرسة دون إشراك‬ ‫املعلمين‬ ‫‪2‬‬ ‫‪3‬‬ ‫‪4‬‬ ‫‪5‬‬ ‫‪6‬‬ ‫‪7‬‬ ‫‪8‬‬ ‫‪9‬‬ ‫‪1‬‬ ‫‪0‬‬ ‫غياب دعم مدير‬ ‫املدرسة إلتاحة الوقت‬ ‫املناسب الجتماع‬ ‫املعلمين‬ ‫غياب دعم مدير‬ ‫املدرسة إلتاحة املكان‬ ‫املناسب الجتماع‬ ‫املعلمين‬ ‫غياب العمل من قبل‬ ‫مدير املدرسة على‬ ‫تشكيل فرق مجتمعات‬ ‫التعلم املهنية‬ ‫ضعف تهيئة مدير‬ ‫املدرسة للمناخ املالئم‬ ‫لعقد الحوارات‬ ‫واملناقشات الهادفة‬ ‫ضعف متابعة مدير‬ ‫املدرسة لعمل فرق‬ ‫مجتمعات التعلم املهنية‬ ‫ضعف قناعة مدير‬ ‫املدرسة بأهمية‬ ‫مجتمعات التعلم املهنية‬ ‫ودورها في تحسين‬ ‫عملية التعلم‬ ‫ضعف العالقة بين‬ ‫مدير املدرسة واملعلمين‬ ‫في املدرسة‬ ‫ضعف العالقة بين‬ ‫املعلمين داخل املدرسة‬ ‫قلة البرامج التدريبية‬ ‫حول مجتمعات التعلم‬ ‫املهنية‬ ‫‪72‬‬ ‫أوافق‬ ‫‪%‬‬ ‫ك‬ ‫‪54‬‬ ‫‪25.81‬‬ ‫‪19.35‬‬ ‫إلى حد ما‬ ‫‪%‬‬ ‫ك‬ ‫‪60‬‬ ‫‪21.51‬‬ ‫ال أوافق‬ ‫‪%‬‬ ‫ك‬ ‫‪66‬‬ ‫‪23.66‬‬ ‫ال أوافق أبدا‬ ‫‪%‬‬ ‫ك‬ ‫‪27‬‬ ‫‪9.68‬‬ ‫املتوسط‬ ‫الحسابي‬ ‫‪2.72‬‬ ‫االنحراف‬ ‫املعياري‬ ‫‪0.791‬‬ ‫الترتيب‬ ‫‪10‬‬ ‫‪45‬‬ ‫‪16.13‬‬ ‫‪39‬‬ ‫‪13.98‬‬ ‫‪54‬‬ ‫‪19.35‬‬ ‫‪87‬‬ ‫‪31.18‬‬ ‫‪54‬‬ ‫‪19.35‬‬ ‫‪3.24‬‬ ‫‪0.835‬‬ ‫‪6‬‬ ‫‪30‬‬ ‫‪10.75‬‬ ‫‪54‬‬ ‫‪19.35‬‬ ‫‪69‬‬ ‫‪24.73‬‬ ‫‪75‬‬ ‫‪26.88‬‬ ‫‪51‬‬ ‫‪18.28‬‬ ‫‪3.23‬‬ ‫‪0.590‬‬ ‫‪7‬‬ ‫‪42‬‬ ‫‪15.05‬‬ ‫‪45‬‬ ‫‪16.13‬‬ ‫‪39‬‬ ‫‪13.98‬‬ ‫‪102‬‬ ‫‪36.56‬‬ ‫‪51‬‬ ‫‪18.28‬‬ ‫‪3.27‬‬ ‫‪0.807‬‬ ‫‪5‬‬ ‫‪45‬‬ ‫‪16.13‬‬ ‫‪36‬‬ ‫‪12.90‬‬ ‫‪54‬‬ ‫‪19.35‬‬ ‫‪84‬‬ ‫‪30.11‬‬ ‫‪60‬‬ ‫‪21.51‬‬ ‫‪3.28‬‬ ‫‪0.818‬‬ ‫‪4‬‬ ‫‪48‬‬ ‫‪17.20‬‬ ‫‪33‬‬ ‫‪11.83‬‬ ‫‪66‬‬ ‫‪23.66‬‬ ‫‪81‬‬ ‫‪29.03‬‬ ‫‪51‬‬ ‫‪18.28‬‬ ‫‪3.19‬‬ ‫‪0.810‬‬ ‫‪8‬‬ ‫‪30‬‬ ‫‪10.75‬‬ ‫‪57‬‬ ‫‪20.43‬‬ ‫‪48‬‬ ‫‪17.20‬‬ ‫‪81‬‬ ‫‪29.03‬‬ ‫‪63‬‬ ‫‪22.58‬‬ ‫‪3.32‬‬ ‫‪0.743‬‬ ‫‪3‬‬ ‫‪39‬‬ ‫‪13.98‬‬ ‫‪36‬‬ ‫‪12.90‬‬ ‫‪48‬‬ ‫‪17.20‬‬ ‫‪81‬‬ ‫‪29.03‬‬ ‫‪75‬‬ ‫‪26.88‬‬ ‫‪3.42‬‬ ‫‪0.838‬‬ ‫‪2‬‬ ‫‪33‬‬ ‫‪11.83‬‬ ‫‪57‬‬ ‫‪20.43‬‬ ‫‪78‬‬ ‫‪27.96‬‬ ‫‪66‬‬ ‫‪23.66‬‬ ‫‪45‬‬ ‫‪16.13‬‬ ‫‪3.12‬‬ ‫‪0.562‬‬ ‫‪9‬‬ ‫‪66‬‬ ‫‪23.66‬‬ ‫‪81‬‬ ‫‪29.03‬‬ ‫‪72‬‬ ‫‪25.81‬‬ ‫‪39‬‬ ‫‪13.98‬‬ ‫‪21‬‬ ‫‪7.53‬‬ ‫‪3.53‬‬ ‫‪0.769‬‬ ‫‪1‬‬ ‫‪3.23‬‬ ‫‪0.48‬‬ ‫املتوسط الحسابي العام‬ ‫يتضح من جدول (‪ )5‬ما يلي‪:‬‬ ‫موكلي‪ ،‬زيلعي‬ ‫(‪)64‬‬ ‫المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات‬ ‫التعلم المهنية وفقًا آلرائهم‬ ‫المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس ‪2022‬م‬ ‫‪-1‬‬ ‫‪-2‬‬ ‫‪-‬‬ ‫‪-‬‬ ‫‪-3‬‬ ‫‬‫‬‫‬‫‬‫‬‫‬‫‬‫‬‫‬‫‬‫‪-4‬‬ ‫هناك تقارب في استجابات عينة الدراسة ملحور املعيقات اإلدارية التي تحد معلمي الرياضيات من تفعيل‬ ‫مجتمعات التعليم املهنية‪ ،‬حيث تراوحت متوسطات استجاباتهم على املحور األول ما بين (‪2.72‬إلى ‪ )3.53‬وهي‬ ‫متوسطات تقع في الفئتين‪ :‬الثالثة (متوسط) والرابعة (مرتفع) من درجات املقياس املتدرج الخماس ي املشار‬ ‫ً‬ ‫إليه سابقا (في أداة الدراسة)‪ ،‬ويالحظ أن متوسط املوافقة العام على فقرات املحور بلغ (‪ 3.23‬درجة من ‪)5‬‬ ‫مما يعني أن هناك (موافقة متوسطة) من أفراد العينة على عبارات هذا املحور‪.‬‬ ‫ً‬ ‫ً‬ ‫ترتيب عبارات هذا املحور حسب درجة املوافقة عليها ترتيبا تنازليا كما يلي‪:‬‬ ‫كانت استجابات أفراد العينة على الفقرة رقم (‪ )10‬وهي‪( :‬قلة البرامج التدريبية حول مجتمعات التعلم‬ ‫املهنية) باملرتبة األولى بين الفقرات بمتوسط الحسابي (‪ ،)3.53‬وهذا يعني أن هناك موافقة بين أفراد العينة‬ ‫أن من أهم املعيقات اإلدارية هي‪( :‬قلة البرامج التدريبية حول مجتمعات التعلم املهنية)‪.‬‬ ‫جاءت استجابات أفراد العينة على الفقرات املتبقية‪ )8( :‬و (‪ )7‬و (‪ )5‬و (‪ )4‬و (‪ )2‬و (‪ )3‬و (‪ )6‬و (‪ )9‬و (‪)1‬‬ ‫بمتوسطات حسابية على التوالي‪،)3.12( ،)3.19( ،)3.23( ،)3.24( ،)3.27( ،)3.28( ،)3.32( ،)3.42( :‬‬ ‫(‪)2.72‬؛ وهذا يعني أن هناك موافقة بدرجة (مرتفعة) للفقرة (‪ )8‬وموافقة متوسطة للفقرات (‪،2 ،4 ،5 ،7‬‬ ‫‪ )1 ،9 ،6 ،3‬بين أفراد العينة على تلك املعيقات؛ ومن ثم تعد تلك الفقرات معيقات بدرجة متوسطة‪.‬‬ ‫ً‬ ‫ترتيب املعيقات بحسب درجة تحققها وفقا لآلتي‪:‬‬ ‫قلة البرامج التدريبية حول مجتمعات التعلم املهنية‪.‬‬ ‫ضعف العالقة بين مدير املدرسة واملعلمين في املدرسة‪.‬‬ ‫ضعف قناعة مدير املدرسة بأهمية مجتمعات التعلم املهنية ودورها في تحسين عملية التعلم‪.‬‬ ‫ضعف تهيئة مدير املدرسة للمناخ املالئم لعقد الحوارات واملناقشات الهادفة‪.‬‬ ‫غياب العمل من قبل مدير املدرسة على تشكيل فرق مجتمعات التعلم املهنية‪.‬‬ ‫غياب دعم مدير املدرسة إلتاحة الوقت املناسب الجتماع املعلمين‪.‬‬ ‫غياب دعم مدير املدرسة إلتاحة املكان املناسب الجتماع املعلمين‪.‬‬ ‫ضعف متابعة مدير املدرسة لعمل فرق مجتمعات التعلم املهنية‪.‬‬ ‫ضعف العالقة بين املعلمين داخل املدرسة‪.‬‬ ‫يبني املدير خطة املدرسة دون إشراك املعلمين‪.‬‬ ‫يالحظ أن هناك اتفاقا بين أفراد العينة في أن قلة البرامج التدريبية حول مجتمعات التعلم املهنية للمعلمين‬ ‫يعد معيقا بدرجة مرتفعة؛ وهذا معيق كبير وال شك‪ ،‬فقلة البرامج التدريبية حول مجتمعات التعلم املهنية ال‬ ‫يتيح تفعيل مجتمعات التعلم املهنية بالصورة املطلوبة‪.‬‬ ‫‪ ‬النتائج املتعلقة بالسؤال الفرعي الثاني‪" :‬ما املعيقات املادية التي تحد معلمي الرياضيات من تفعيل مجتمعات‬ ‫التعلم املهنية ً‬ ‫وفقا آلرائهم؟‬ ‫ولإلجابة تم حساب التكرارات والنسب املئوية واملتوسطات الحسابية واالنحرافات املعيارية لكل مفردة من‬ ‫مفردات األداة والجدول (‪ )6‬التالي يوضح تلك النتائج‪.‬‬ ‫موكلي‪ ،‬زيلعي‬ ‫(‪)65‬‬ ‫المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات‬ ‫التعلم المهنية وفقًا آلرائهم‬ ‫المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس ‪2022‬م‬ ‫جدول (‪ :)6‬املعيقات املادية التي تحد معلمي الرياضيات من تفعيل مجتمعات التعلم املهنية‬ ‫م‬ ‫‪1‬‬ ‫‪2‬‬ ‫‪3‬‬ ‫‪4‬‬ ‫العبارة‬ ‫أوافق بشدة‬ ‫ك‬ ‫نقص الكوادر املؤهلة‬ ‫علميا ومهنيا لقيادة‬ ‫‪84‬‬ ‫أنشطة مجتمعات التعلم‬ ‫املهنية داخل املدرسة‬ ‫ارتفاع أنصبة املعلمين‬ ‫‪183‬‬ ‫التدريسية‬ ‫زيادة األعباء غير‬ ‫التدريسية على املعلم‬ ‫‪159‬‬ ‫غياب التجهيزات واملرافق‬ ‫‪141‬‬ ‫في املدرسة لتفعيل‬ ‫مجتمعات التعلم املهنية‬ ‫ضعف الحوافز املادية‬ ‫‪168‬‬ ‫واملعنوية لتفعيل‬ ‫مجتمعات التعلم املهنية‬ ‫ضعف الدعم املالي‬ ‫املخصص ملجتمعات‬ ‫‪156‬‬ ‫التعلم املهنية في امليزانية‬ ‫التشغيلية‬ ‫أوافق‬ ‫ك‬ ‫‪%‬‬ ‫‪%‬‬ ‫إلى حد ما‬ ‫‪%‬‬ ‫ك‬ ‫ال أوافق‬ ‫ك‬ ‫‪%‬‬ ‫ال أوافق أبدا‬ ‫ك‬ ‫‪%‬‬ ‫املتوسط االنحراف‬ ‫الحسابي املعياري‬ ‫الترتيب‬ ‫‪30.11‬‬ ‫‪81‬‬ ‫‪29.03‬‬ ‫‪66‬‬ ‫‪23.66‬‬ ‫‪30‬‬ ‫‪10.75‬‬ ‫‪18‬‬ ‫‪6.45‬‬ ‫‪2.83‬‬ ‫‪0.646‬‬ ‫‪9‬‬ ‫‪65.59‬‬ ‫‪51‬‬ ‫‪18.28‬‬ ‫‪27‬‬ ‫‪9.68‬‬ ‫‪9‬‬ ‫‪3.23‬‬ ‫‪9‬‬ ‫‪3.23‬‬ ‫‪4.11‬‬ ‫‪0.525‬‬ ‫‪1‬‬ ‫‪56.99‬‬ ‫‪69‬‬ ‫‪24.73‬‬ ‫‪36‬‬ ‫‪12.90‬‬ ‫‪15‬‬ ‫‪5.38‬‬ ‫‪0‬‬ ‫‪0.00‬‬ ‫‪4.04‬‬ ‫‪0.812‬‬ ‫‪4‬‬ ‫‪50.54‬‬ ‫‪75‬‬ ‫‪26.88‬‬ ‫‪42‬‬ ‫‪15.05‬‬ ‫‪9‬‬ ‫‪3.23‬‬ ‫‪12‬‬ ‫‪4.30‬‬ ‫‪3.91‬‬ ‫‪0.68‬‬ ‫‪7‬‬ ‫‪60.22‬‬ ‫‪63‬‬ ‫‪22.58‬‬ ‫‪24‬‬ ‫‪8.60‬‬ ‫‪18‬‬ ‫‪6.45‬‬ ‫‪6‬‬ ‫‪2.15‬‬ ‫‪4.05‬‬ ‫‪0.57‬‬ ‫‪3‬‬ ‫‪55.91‬‬ ‫‪75‬‬ ‫‪26.88‬‬ ‫‪30‬‬ ‫‪10.75‬‬ ‫‪12‬‬ ‫‪4.30‬‬ ‫‪6‬‬ ‫‪2.15‬‬ ‫‪3.98‬‬ ‫‪0.842‬‬ ‫‪6‬‬ ‫‪7‬‬ ‫كثافة مقررات‬ ‫الرياضيات ومتطلباتها‬ ‫‪162‬‬ ‫‪58.06‬‬ ‫‪66‬‬ ‫‪23.66‬‬ ‫‪33‬‬ ‫‪11.83‬‬ ‫‪6‬‬ ‫‪2.15‬‬ ‫‪12‬‬ ‫‪4.30‬‬ ‫‪3.99‬‬ ‫‪0.670‬‬ ‫‪5‬‬ ‫‪8‬‬ ‫كثافة أعداد الطالب في‬ ‫الفصول‬ ‫‪135‬‬ ‫‪48.39‬‬ ‫‪78‬‬ ‫‪27.96‬‬ ‫‪33‬‬ ‫‪11.83‬‬ ‫‪24‬‬ ‫‪8.60‬‬ ‫‪9‬‬ ‫‪3.23‬‬ ‫‪3.84‬‬ ‫‪0.641‬‬ ‫‪8‬‬ ‫‪9‬‬ ‫قلة أعداد معلمي‬ ‫الرياضيات في املرحلة‬ ‫الدراسية‬ ‫‪168‬‬ ‫‪5‬‬ ‫‪6‬‬ ‫‪66‬‬ ‫‪60.22‬‬ ‫‪23.66‬‬ ‫‪30‬‬ ‫‪10.75‬‬ ‫املتوسط الحسابي العام‬ ‫‪9‬‬ ‫‪3.23‬‬ ‫‪6‬‬ ‫‪2.15‬‬ ‫‪4.09‬‬ ‫‪0.808‬‬ ‫‪3.87‬‬ ‫‪0.39‬‬ ‫يتضح من جدول (‪ )6‬ما يلي‪:‬‬ ‫‪ -1‬هناك تقارب في استجابات عينة الدراسة ملحور املعيقات املادية التي تحد معلمي الرياضيات من تفعيل‬ ‫مجتمعات التعليم املهنية وفقا آلراء املعلمين‪ ،‬حيث تراوحت متوسطات استجاباتهم على املحور األول ما بين‬ ‫(‪ 2.83‬إلى ‪ )4.11‬وهي متوسطات يقع في الفئتين الثالثة (متوسط) والرابعة (مرتفع) من درجات املقياس‪،‬‬ ‫ويالحظ أن متوسط املوافقة العام على فقرات املحور بلغ (‪ )3,87‬مما يعني أن هناك (مرتفعة) من أفراد العينة‬ ‫على عبارات هذا املحور‪.‬‬ ‫ً‬ ‫ً‬ ‫‪ -2‬ترتيب عبارات هذا املحور حسب درجة املوافقة عليها ترتيبا تنازليا كما يلي‪:‬‬ ‫موكلي‪ ،‬زيلعي‬ ‫(‪)66‬‬ ‫المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات‬ ‫التعلم المهنية وفقًا آلرائهم‬ ‫‪2‬‬ ‫المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس ‪2022‬م‬ ‫‪-‬‬ ‫‪-‬‬ ‫‪-3‬‬ ‫‬‫‬‫‬‫‬‫‬‫‬‫‬‫‬‫‬‫‪-4‬‬ ‫كانت استجابات أفراد العينة على الفقرة رقم (‪ )2‬وهي‪( :‬ارتفاع أنصبة املعلمين التدريسية) باملرتبة األولى بين‬ ‫الفقرات بمتوسط الحسابي (‪ ،)4.11‬وهذا يعني أن هناك موافقة بين أفراد العينة أن من أهم املعيقات‬ ‫املادية هي‪( :‬ارتفاع أنصبة املعلمين التدريسية)‪.‬‬ ‫جاءت استجابات أفراد العينة على الفقرات املتبقية‪ )9( :‬و (‪ )5‬و (‪ )3‬و (‪ )7‬و (‪ )6‬و (‪ )4‬و (‪ )8‬و (‪)1‬‬ ‫بمتوسطات حسابية على التوالي‪)2.83( ،)3.84( ،)3.91( ،)3.98( ،)3.99( ،)4.04( ،)4.05( ،)4.09( :‬؛ وهذا‬ ‫يعني أن هناك موافقة بدرجة (مرتفعة) بين أفراد العينة على تلك املعيقات؛ ومن ثم تعد تلك الفقرات‬ ‫معيقات بدرجة مرتفعة‪.‬‬ ‫ً‬ ‫ترتيب املعيقات بحسب درجة تحققها وفقا لآلتي‪:‬‬ ‫ارتفاع أنصبة املعلمين التدريسية‪.‬‬ ‫قلة أعداد معلمي الرياضيات في املرحلة الدراسية باملدرسة‪.‬‬ ‫ضعف الحوافز املادية واملعنوية لتفعيل مجتمعات التعلم املهنية‪.‬‬ ‫زيادة األعباء غير التدريسية على املعلم‪.‬‬ ‫كثافة مقررات الرياضيات ومتطلباتها‪.‬‬ ‫ضعف الدعم املالي املخصص ملجتمعات التعلم املهنية في امليزانية التشغيلية‪.‬‬ ‫غياب التجهيزات واملرافق في املدرسة لتفعيل مجتمعات التعلم املهنية‪.‬‬ ‫كثافة أعداد الطالب في الفصول‪.‬‬ ‫نقص الكوادر املؤهلة علميا ومهنيا لقيادة أنشطة مجتمعات التعلم املهنية داخل املدرسة‪.‬‬ ‫يالحظ أن هناك اتفاقا بين أفراد العينة في أن ارتفاع أنصبة املعلمين التدريسية يعد معيقا بدرجة مرتفعة؛‬ ‫وهذا معيق كبير وال شك‪ ،‬فارتفاع أنصبة املعلمين التدريسية ال يتيح تفعيل مجتمعات التعلم املهنية بالصورة‬ ‫املطلوبة‪.‬‬ ‫‪ ‬النتائج املتعلقة بالسؤال الفرعي الثالث‪" :‬ما املعيقات الشخصية التي تحد معلمي الرياضيات من تفعيل‬ ‫مجتمعات التعلم املهنية ً‬ ‫وفقا آلرائهم؟‬ ‫ولإلجابة تم حساب التكرارات والنسب املئوية واملتوسطات الحسابية واالنحرافات املعيارية لكل مفردة من‬ ‫مفردات األداة والجدول التالي يوضح تلك النتائج‪.‬‬ ‫جدول (‪ :)7‬املعيقات الشخصية التي تحد معلمي الرياضيات من تفعيل مجتمعات التعلم املهنية‬ ‫أوافق بشدة‬ ‫م‬ ‫العبارة‬ ‫‪1‬‬ ‫ضعف الثقة املتبادلة‬ ‫بين أعضاء مجتمع‬ ‫التعلم املنهي‬ ‫‪48‬‬ ‫‪2‬‬ ‫غياب وعي املعلمين‬ ‫بمفهوم مجتمعات‬ ‫التعلم املهنية‬ ‫‪60‬‬ ‫ك‬ ‫‪%‬‬ ‫‪17.20‬‬ ‫‪21.51‬‬ ‫موكلي‪ ،‬زيلعي‬ ‫أوافق‬ ‫ك‬ ‫‪66‬‬ ‫‪93‬‬ ‫‪%‬‬ ‫‪23.66‬‬ ‫‪33.33‬‬ ‫أوافق إلى حد‬ ‫ما‬ ‫‪%‬‬ ‫ك‬ ‫‪78‬‬ ‫‪84‬‬ ‫‪27.96‬‬ ‫‪30.11‬‬ ‫(‪)67‬‬ ‫ال أوافق‬ ‫ك‬ ‫‪69‬‬ ‫‪30‬‬ ‫‪%‬‬ ‫‪24.73‬‬ ‫‪10.75‬‬ ‫ال أوافق أبدا املتوسط االنحراف‬ ‫الحسابي املعياري‬ ‫‪%‬‬ ‫ك‬ ‫‪18‬‬ ‫‪12‬‬ ‫‪6.45‬‬ ‫‪4.30‬‬ ‫‪2.45‬‬ ‫‪2.43‬‬ ‫‪0.603‬‬ ‫‪0.761‬‬ ‫المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات‬ ‫التعلم المهنية وفقًا آلرائهم‬ ‫الترتيب‬ ‫‪6‬‬ ‫‪7‬‬ ‫المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس ‪2022‬م‬ ‫م‬ ‫العبارة‬ ‫أوافق بشدة‬ ‫ك‬ ‫‪3‬‬ ‫‪4‬‬ ‫‪5‬‬ ‫‪6‬‬ ‫‪7‬‬ ‫‪8‬‬ ‫‪9‬‬ ‫‪10‬‬ ‫‪11‬‬ ‫ضعف قناعة املعلمين‬ ‫بأهمية مجتمعات التعلم‬ ‫املهنية في تحسين‬ ‫العملية التعليمية‬ ‫ضعف قناعة املعلمين‬ ‫بعملية التغيير والتطوير‬ ‫في أدائهم‬ ‫قلة رغبة املعلمين في‬ ‫ممارسة العمل الجماعي‬ ‫غياب تقبل املعلمين‬ ‫للمقترحات والنقد بينهم‬ ‫بروح ايجابية‬ ‫ضعف رغبة املعلمين في‬ ‫تفعيل مجتمعات التعلم‬ ‫املهنية‬ ‫ضعف معرفة املعلمين‬ ‫بأدوارهم في مجتمعات‬ ‫التعلم املهنية‬ ‫نقص الوعي بمتطلبات‬ ‫تطبيق مجتمعات التعلم‬ ‫املهنية‬ ‫غياب التعاون بين‬ ‫معلمي املدرسة لتفعيل‬ ‫مجتمعات التعلم املهنية‬ ‫ضعف القدرة على‬ ‫تخطيط مجتمعات‬ ‫التعلم املهنية‬ ‫أوافق إلى حد‬ ‫ما‬ ‫‪%‬‬ ‫ك‬ ‫أوافق‬ ‫ك‬ ‫‪%‬‬ ‫‪%‬‬ ‫ال أوافق‬ ‫ك‬ ‫‪%‬‬ ‫ال أوافق أبدا املتوسط االنحراف‬ ‫الحسابي املعياري‬ ‫‪%‬‬ ‫ك‬ ‫الترتيب‬ ‫‪57‬‬ ‫‪20.43‬‬ ‫‪87‬‬ ‫‪31.18‬‬ ‫‪78‬‬ ‫‪27.96‬‬ ‫‪45‬‬ ‫‪16.13‬‬ ‫‪12‬‬ ‫‪4.30‬‬ ‫‪2.53‬‬ ‫‪0.828‬‬ ‫‪3‬‬ ‫‪54‬‬ ‫‪19.35‬‬ ‫‪78‬‬ ‫‪27.96‬‬ ‫‪78‬‬ ‫‪27.96‬‬ ‫‪51‬‬ ‫‪18.28‬‬ ‫‪18‬‬ ‫‪6.45‬‬ ‫‪2.57‬‬ ‫‪0.684‬‬ ‫‪1‬‬ ‫‪78‬‬ ‫‪27.96‬‬ ‫‪78‬‬ ‫‪27.96‬‬ ‫‪81‬‬ ‫‪29.03‬‬ ‫‪24‬‬ ‫‪8.60‬‬ ‫‪18‬‬ ‫‪6.45‬‬ ‫‪2.38‬‬ ‫‪0.768‬‬ ‫‪11‬‬ ‫‪63‬‬ ‫‪22.58‬‬ ‫‪72‬‬ ‫‪25.81‬‬ ‫‪87‬‬ ‫‪31.18‬‬ ‫‪39‬‬ ‫‪13.98‬‬ ‫‪18‬‬ ‫‪6.45‬‬ ‫‪2.56‬‬ ‫‪0.580‬‬ ‫‪2‬‬ ‫‪60‬‬ ‫‪21.51‬‬ ‫‪84‬‬ ‫‪30.11‬‬ ‫‪75‬‬ ‫‪26.88‬‬ ‫‪48‬‬ ‫‪17.20‬‬ ‫‪12‬‬ ‫‪4.30‬‬ ‫‪2.41‬‬ ‫‪0.695‬‬ ‫‪9‬‬ ‫‪63‬‬ ‫‪22.58‬‬ ‫‪87‬‬ ‫‪31.18‬‬ ‫‪87‬‬ ‫‪31.18‬‬ ‫‪33‬‬ ‫‪11.83‬‬ ‫‪9‬‬ ‫‪3.23‬‬ ‫‪2.42‬‬ ‫‪0.737‬‬ ‫‪8‬‬ ‫‪66‬‬ ‫‪23.66‬‬ ‫‪75‬‬ ‫‪26.88‬‬ ‫‪108‬‬ ‫‪38.71‬‬ ‫‪15‬‬ ‫‪5.38‬‬ ‫‪15‬‬ ‫‪5.38‬‬ ‫‪2.40‬‬ ‫‪0.759‬‬ ‫‪10‬‬ ‫‪66‬‬ ‫‪23.66‬‬ ‫‪69‬‬ ‫‪24.73‬‬ ‫‪96‬‬ ‫‪34.41‬‬ ‫‪39‬‬ ‫‪13.98‬‬ ‫‪9‬‬ ‫‪3.23‬‬ ‫‪2.48‬‬ ‫‪0.809‬‬ ‫‪5‬‬ ‫‪57‬‬ ‫‪20.43‬‬ ‫‪75‬‬ ‫‪26.88‬‬ ‫‪102‬‬ ‫‪36.56‬‬ ‫‪36‬‬ ‫‪12.90‬‬ ‫‪9‬‬ ‫‪3.23‬‬ ‫‪2.52‬‬ ‫‪0.722‬‬ ‫‪4‬‬ ‫‪2.47‬‬ ‫‪0.48‬‬ ‫املتوسط الحسابي العام‬ ‫يتضح من جدول (‪ )7‬ما يلي‪:‬‬ ‫‪ -1‬هناك تقارب في استجابات عينة الدراسة ملحور املعيقات الشخصية التي تحد معلمي الرياضيات من تفعيل‬ ‫مجتمعات التعليم املهنية وفقا آلراء املعلمين‪ ،‬حيث تراوحت متوسطات استجاباتهم على املحور األول ما بين‬ ‫(‪ 2.38‬إلى ‪ )2.57‬وهي متوسطات يقع في الفئتين الثانية (متدنية) من درجات املقياس‪ ،‬ويالحظ أن متوسط‬ ‫املوافقة العام على فقرات املحور بلغ (‪ )2.48‬مما يعني أن هناك (متدنية) من أفراد العينة على عبارات هذا‬ ‫املحور‪.‬‬ ‫ً‬ ‫ً‬ ‫‪ -2‬ترتيب عبارات هذا املحور حسب درجة املوافقة عليها ترتيبا تنازليا كما يلي‪:‬‬ ‫موكلي‪ ،‬زيلعي‬ ‫(‪)68‬‬ ‫المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات‬ ‫التعلم المهنية وفقًا آلرائهم‬ ‫المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس ‪2022‬م‬ ‫‪-‬‬ ‫‪-‬‬ ‫‪-3‬‬ ‫‬‫‬‫‬‫‬‫‬‫‬‫‬‫‬‫‬‫‬‫‬‫‪-4‬‬ ‫كانت استجابات أفراد العينة على الفقرة رقم (‪ )4‬وهي‪( :‬ضعف قناعة املعلمين بعملية التغيير والتطوير في‬ ‫أدائهم) باملرتبة األولى بين الفقرات بمتوسط الحسابي (‪ ،)2.57‬وهذا يعني أن هناك موافقة بين أفراد العينة‬ ‫أن من أهم املعيقات الشخصية هي‪( :‬ضعف قناعة املعلمين بعملية التغيير والتطوير في أدائهم)‪.‬‬ ‫جاءت استجابات أفراد العينة على الفقرات املتبقية‪ )6( :‬و (‪ )3‬و (‪ )11‬و (‪ )10‬و (‪ )1‬و (‪ )2‬و (‪ )8‬و (‪ )7‬و‬ ‫(‪ )9‬و (‪ )5‬بمتوسطات حسابية على التوالي‪،)2.42( ،)2.43( ،)2.45( ،)2.48( ،)2.52( ،)2.53( ،)2.56( :‬‬ ‫(‪)2.38( ،)2.40( ،)2.41‬؛ وهذا يعني أن هناك موافقة بدرجة (متدنية) بين أفراد العينة على تلك املعيقات؛‬ ‫ومن ثم تعد تلك الفقرات معيقات بدرجة متدنية‪.‬‬ ‫ً‬ ‫ترتيب املعيقات بحسب درجة تحققها وفقا لآلتي‪:‬‬ ‫ضعف قناعة املعلمين بعملية التغيير والتطوير في أدائهم‪.‬‬ ‫غياب تقبل املعلمين املقترحات والنقد بينهم بروح ايجابية‪.‬‬ ‫ضعف قناعة املعلمين بأهمية مجتمعات التعلم املهنية في تحسين العملية التعليمية‪.‬‬ ‫ضعف القدرة على تخطيط مجتمعات التعلم املهنية‪.‬‬ ‫غياب التعاون بين معلمي املدرسة لتفعيل مجتمعات التعلم املهنية‪.‬‬ ‫ضعف الثقة املتبادلة بين أعضاء املجتمع التعلم املنهي‪.‬‬ ‫غياب وعي املعلمين بمفهوم مجتمعات التعلم املهنية‪.‬‬ ‫ضعف معرفة املعلمين بأدوارهم في مجتمعات التعلم املهنية‪.‬‬ ‫ضعف رغبة املعلمين في تفعيل مجتمعات التعلم املهنية‪.‬‬ ‫نقص الوعي بمتطلبات تطبيق مجتمعات التعلم املهنية‪.‬‬ ‫قلة رغبة املعلمين في ممارسة العمل الجماعي‪.‬‬ ‫يالحظ أن هناك اتفاقا بين أفراد العينة في أن ضعف قناعة املعلمين بعملية التغيير والتطوير في أدائهم يعد‬ ‫املعيق األعلى من بين املعيقات الشخصية؛ وهذا معيق وال شك‪ ،‬فضعف قناعة املعلمين بعملية التغيير‬ ‫والتطوير في أدائهم ال يتيح تفعيل مجتمعات التعلم املهنية بالصورة املطلوبة‪.‬‬ ‫‪ ‬النتائج املتعلقة بالسؤال الفرعي الرابع‪" :‬هل توجد فروق ذات داللة احصائية عند مستوى (‪ )α≤0.05‬بين آراء‬ ‫معلمي الرياضيات حول املعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات التعلم املهنية تعزى للخبرة‬ ‫التدريسية"؟‬ ‫جدول (‪ )8‬حساب تحليل التباين األحادي لداللة الفروق في متوسطات إجابات العينة على املحاور الثالثة تعزى‬ ‫لسنوات الخبرة‬ ‫املحور‬ ‫األول‬ ‫الثاني‬ ‫مصدر التباين‬ ‫بين املجموعات‬ ‫داخل املجموعات‬ ‫املجموع‬ ‫بين املجموعات‬ ‫داخل املجموعات‬ ‫املجموع‬ ‫موكلي‪ ،‬زيلعي‬ ‫درجات‬ ‫الحرية‬ ‫‪2‬‬ ‫‪276‬‬ ‫‪278‬‬ ‫‪2‬‬ ‫‪276‬‬ ‫‪278‬‬ ‫مجموع‬ ‫املربعات‬ ‫‪57.227‬‬ ‫‪9322.516‬‬ ‫‪9379.743‬‬ ‫‪61.543‬‬ ‫‪9754.783‬‬ ‫‪9816.326‬‬ ‫(‪)69‬‬ ‫قيمة ف‬ ‫(‪)F‬‬ ‫متوسط‬ ‫املربعات‬ ‫‪28.614‬‬ ‫‪33.777‬‬ ‫‪0.847‬‬ ‫‪30.772‬‬ ‫‪35.343‬‬ ‫‪0.871‬‬ ‫الداللة‬ ‫اإلحصائية‬ ‫‪0.624‬‬ ‫‪0.661‬‬ ‫المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات‬ ‫التعلم المهنية وفقًا آلرائهم‬ ‫المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس ‪2022‬م‬ ‫املحور‬ ‫مصدر التباين‬ ‫الثالث‬ ‫بين املجموعات‬ ‫داخل املجموعات‬ ‫املجموع‬ ‫درجات‬ ‫الحرية‬ ‫‪2‬‬ ‫‪276‬‬ ‫‪278‬‬ ‫مجموع‬ ‫املربعات‬ ‫‪49.768‬‬ ‫‪7592.743‬‬ ‫‪7642.511‬‬ ‫متوسط‬ ‫املربعات‬ ‫‪24.884‬‬ ‫‪27.507‬‬ ‫قيمة ف‬ ‫(‪)F‬‬ ‫‪0.905‬‬ ‫الداللة‬ ‫اإلحصائية‬ ‫‪0.543‬‬ ‫يتضح من جدول (‪ )8‬عدم وجود فروق ذات داللة إحصائية عند مستوى (‪ )0.05‬فأقل في آراء أفراد العينة‬ ‫حول جميع محاور الدراسة الثالثة ترجع إلى سنوات الخبرة؛ وهذا يعني أن جميع أفراد العينة بمختلف خبراتهم‬ ‫التدريسية يرون الرأي نفسه في املعيقات وال فرق بينهم في ذلك‪ ،‬وهذا يؤكد وجود املعيقات التي اتضحت أثناء‬ ‫مناقشة كل محور على حدة‪ ،‬وأن وجودها بالدرجة نفسها‪.‬‬ ‫مناقشة النتائج‪:‬‬ ‫بناء على ما تقدم يمكن الخلوص إلى النتائج اآلتية‪:‬‬ ‫‪ -1‬املعيقات وفقا آلراء املعلمين عالية الدرجة وعددها (‪ )10‬من (‪ )30‬معيقا أي ما نسبته (‪ )%33.33‬من املعيقات‬ ‫التي تحد من تفعيل مجتمعات التعلم املهنية في تعلم وتعليم الرياضيات هي‪:‬‬ ‫ ارتفاع أنصبة املعلمين التدريسية‪.‬‬‫ قلة أعداد معلمي الرياضيات في املرحلة الدراسية باملدرسة‪.‬‬‫ ضعف الحوافز املادية واملعنوية لتفعيل مجتمعات التعلم املهنية‪.‬‬‫ زيادة األعباء غير التدريسية على املعلم‪.‬‬‫ كثافة مقررات الرياضيات ومتطلباتها‪.‬‬‫ ضعف دعم مالي مخصص ملجتمعات التعلم املهنية في امليزانية التشغيلية‪.‬‬‫ غياب التجهيزات واملرافق في املدرسة لتفعيل مجتمعات التعلم املهنية‪.‬‬‫ كثافة أعداد الطالب في الفصول‪.‬‬‫ قلة البرامج التدريبية حول مجتمعات التعلم املهنية‪.‬‬‫ ضعف العالقة بين مدير املدرسة واملعلمين في املدرسة‪.‬‬‫‪ -2‬بقية املعيقات وفقا آلراء املعلمين متوسطة الدرجة وعددها (‪ )9‬من (‪ )30‬معيقا أي ما نسبته (‪ )%30‬من‬ ‫املعيقات‪ ،‬ومتدنية الدرجة وعددها (‪ )11‬من (‪ )30‬معيقا أي ما نسبته (‪ )%36.67‬من املعيقات‪.‬‬ ‫التوصيات واملقترحات‪.‬‬ ‫‪-1‬‬ ‫‪-2‬‬ ‫‪-3‬‬ ‫‪-4‬‬ ‫‪-5‬‬ ‫‪-6‬‬ ‫‪-7‬‬ ‫بالنظر إلى نتائج الدراسة يوص ي الباحثان ويقترحان بما يلي‪:‬‬ ‫ضرورة تدريب وتأهيل املعلمين لتطبيق مجتمعات التعلم املهنية‬ ‫تخفيض أنصبة املعلمين بما يتناسب مع تفعيل مجتمعات التعلم املهنية بالصورة املطلوبة‪.‬‬ ‫تحفيز املعلمين معنويا وماديا على تفعيل مجتمعات التعلم املهنية‬ ‫تخفيف األعباء غير التدريسية على املعلمين من أجل تفعيل مجتمعات التعلم املهنية‬ ‫تأمين تجهيزات ومرافق املدرسة لتطبيق مجتمعات التعلم املهنية‬ ‫تعاون مدير املدرسة ومعلميها وتقوية العالقة بينهم لتفعيل مجتمعات التعلم املهنية‬ ‫كما يقترح الباحثان إجراء الدراسات اآلتية‪:‬‬ ‫موكلي‪ ،‬زيلعي‬ ‫(‪)70‬‬ ‫المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات‬ ‫التعلم المهنية وفقًا آلرائهم‬ ‫المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس ‪2022‬م‬ ‫‪ .1‬دراسة معيقات تفعيل مجتمعات التعلم املهنية في التخصصات األخرى‪.‬‬ ‫‪ .2‬دراسة أثر مجتمعات التعلم املهنية على تحسن نتائج الطالب في مادة الرياضيات‪.‬‬ ‫‪ .3‬دراسة أثر مجتمعات التعلم املهنية على تحسن أداء املعلمين تدريسيا‪.‬‬ ‫قائمة املراجع‪.‬‬ ‫ً‬ ‫أوال‪ -‬املراجع بالعربية‪:‬‬ ‫ إبراهيم‪ ،‬حسام الدين؛ املرزوقي‪ ،‬أحمد سعيد (‪ .)2018‬املشكالت التي تواجه مجتمعات التعلم املهنية في املدارس‬‫في سلطنة عمان‪ .‬مجلة الفنون واألدب وعلوم اإلنسانيات واالجتماع‪ ،‬ع (‪.337 -306 ،)29‬‬ ‫ إبراهيم‪ ،‬محمد عبد الناصر؛ حسان‪ ،‬حسن محمد (‪ .)2018‬مجتمعات التعلم املهنية مدخل ملواجهة بعض‬‫مشكالت مدارس التعليم الثانوي الصناعي في مصر‪ .‬دراسة ميدانية‪ ،‬رسالة ماجستير غير منشورة‪ ،‬كلية التربية‪،‬‬ ‫جامعة املنصورة‪ ،‬مصر‪.‬‬ ‫ األخناوي‪ ،‬محمد السيد (‪ )2016‬متطلبات تجويد األداء املدرس ي بالتعليم الثانوي الصناعي في مصر في ضوء‬‫مدخل مجتمعات التعلم املهنية‪ .‬مجلة كلية التربية‪.162 -82 ،)64( 4 ،‬‬ ‫ البرعمي‪ ،‬يوسف أحمد؛ عبد الرشيد‪ ،‬ناصر سيد (‪ .)2020‬تصور مقترح لتفعيل اإلشراف التربوي املعتمد على‬‫مجتمعات التعلم املهنية‪ .‬مجلة البحوث التربوية والنفسية‪.247 -212 ،)65( 17 ،‬‬ ‫ البرنامج الوطني لتطوير املدارس باململكة العربية السعودية (‪ .)2016‬مجتمعات التعلم املهنية‪ .‬الرياض‪ ،‬مشروع‬‫امللك عبد هللا بن عبد العزيز لتطوير التعليم العام‪.‬‬ ‫ توفيق‪ ،‬فيفي أحمد (‪ .)2017‬سيناريو مستقبلي لتفعيل مجتمعات التعلم بمدراس التعليم العام بمحافظة‬‫سوهاج‪ .‬املجلة التربوية‪ ،‬كلية التربية بسوهاج‪.260 -113 ،)47( 47 ،‬‬ ‫ الحازمي‪ ،‬زكي رزيق (‪ .)2016‬املجتمعات املهنية للمعلمين "دراسة تطبيقية"‪ .‬بحث مقدم للمؤتمر الخامس إلعداد‬‫املعلم (إعداد وتدريب املعلم في ضوء مطالب التنمية ومستجدات العصر)‪ 4 -2 ،‬فبراير‪ ،‬كلية التربية جامعة أم‬ ‫القرى بمكة املكرمة‪.‬‬ ‫ حسن بت‪ ،‬حسين بن قاسم (‪ .)2016‬الكفايات املهنية املنشودة لدى القيادات املدرسية لتطبيق معايير‬‫مجتمعات التعلم بمؤسسات التعليم العام السعودية‪ :‬نموذج مقترح‪ ،‬الثقافة والتنمية‪ -‬مصر‪-1 ،)103( 16 ،‬‬ ‫‪.16‬‬ ‫ الزايد‪ ،‬زينب بنت عبد هللا (‪ .)2018‬تأثير املمارسة التأملية في مجتمعات التعلم املهنية في تعلم معلمات املرحلة‬‫الثانوية‪ .‬مجلة رسالة التربية وعلم النفس‪ ،‬جامعة امللك سعود‪.79 -55 ،)62( ،‬‬ ‫ الزائد‪ ،‬زينب عبد هللا؛ عمر‪ ،‬سوزان حسين (‪ .)2016‬تأثير مجتمعات التعلم املهنية على االنترنت في فهم معلمات‬‫العلوم لطبيعة العلم ومواسا تدريسها‪ .‬املجلة األردنية في العلوم التربوية ‪.362 -349 ،)3( 12‬‬ ‫ الزايدي‪ ،‬أحمد محمد (‪ .)2013‬متطلبات التنمية املهنية في مجتمعات التعلم مدخل إلصالح املدارس الثانوية‬‫نموذج مقترح‪ .‬مجلة كلية التربية بجامعة األزهر‪ ،‬مصر‪.406 -333 ،)152( 3 ،‬‬ ‫ السليمي‪ ،‬خالد بن سعد؛ الزلفي‪ ،‬وافي بن صالح (‪ .)2019‬التنمية املهنية املستدامة للمعلمين أثناء الخدمة‪.‬‬‫املؤتمر االول للجمعية السعودية للمعلم (جسم)‪ ،‬في الفترة بين ‪ 5 -4‬ديسمبر ‪ 2019‬جامعة امللك خالد‪ ،‬أبها‪،‬‬ ‫‪.446 -436‬‬ ‫موكلي‪ ،‬زيلعي‬ ‫(‪)71‬‬ ‫المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات‬ ‫التعلم المهنية وفقًا آلرائهم‬ ‫المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس ‪2022‬م‬ ‫‪-‬‬ ‫‪-‬‬ ‫‬‫‪-‬‬ ‫‬‫‬‫‬‫‬‫‪-‬‬ ‫الصالحية‪ ،‬فاطمة محمد؛ الهاشم‪ ،‬نور حياتي (‪ .)2018‬تطبيق مجتمعات التعلم املهنية ودورها في رفع مستوى‬ ‫التحصيل الدراس ي للطالب‪ .‬مجلة دراسات عربية في التربية وعلم النفس‪ ،‬رابطة التربويين العرب‪-447 ،)97( ،‬‬ ‫‪.472‬‬ ‫العجالن‪ ،‬فتحية محمد (‪ .)2016‬أثر برنامج املجتمعات التعلمية املهنية ‪ PLC‬في االرتقاء باملستويات التحصيلية‬ ‫لطالبات الصف الخامس االبتدائي في مادة الرياضيات في التعليم العام بمنطقه مكة املكرمة‪ .‬بحث مقدم‬ ‫للمؤتمر الخامس إلعداد املعلم‪ ،‬إعداد املعلم في ضوء مطالب التنمية ومستجدات العصر‪ ،‬كليه التربية‪ ،‬جامعة‬ ‫أم القرى‪.‬‬ ‫محروس‪ ،‬محمد األصمعي (‪ .)2015‬املتطلبات املهنية املأمولة لإلصالح املدرس ي املنشود‪ .‬املجلة التربوية‪ ،‬كلية‬ ‫التربية بسوهاج‪.588 -567 ،)40( 40 ،‬‬ ‫محمد‪ ،‬ماهر أحمد (‪ .)2019‬بناء مجتمعات التعلم املهنية كمدخل لتجويد األداء األكاديمي في مدارس التعليم‬ ‫العام بالسعودية‪ .‬مجلة الجامعة اإلسالمية للدراسات التربوية والنفسية‪ ،‬الجامعة اإلسالمية بغزة‪-62 ،)6( 27 ،‬‬ ‫‪.92‬‬ ‫مشروع امللك عبد هللا بن عبد العزيز لتطوير التعليم العام (‪ .)2013‬أنموذج تطوير املدارس‪ .‬مكتبة امللك فهد‬ ‫الوطنية‪ ،‬الرياض‪.‬‬ ‫املطيري‪ ،‬هيا عمر (‪ .)2018‬واقع مجتمعات التعلم املهنية ملعلمات العلوم ومتطلبات تطبيقها في املرحلة الثانوية‬ ‫بمنطقة القصيم‪ .‬رسالة ماجستير غير منشورة‪ ،‬كلية التربية‪ ،‬جامعة القصيم‪ ،‬السعودية‪.‬‬ ‫موكلي‪ ،‬فهد بن إبراهيم (‪ .)2019‬العالقة بين معرفة املعلم الرياضية وممارساته التدريسية‪ .‬ملتقى الرياضيات‬ ‫الثاني بإدارة تعليم صبيا "التحول في تعليم الرياضيات‪ .‬طرق وأساليب"‪1440/6/29 ،‬هـ‪.‬‬ ‫ناصف‪ ،‬محمد أحمد (‪ .)2012‬مجتمعات التعلم كمدخل إلصالح مدارس التعليم العام في مصر‪ :‬دراسة تحليلية‪،‬‬ ‫مجلة كلية التربية‪ ،‬جامعة طنطا‪ ،‬مصر‪ ،‬ع (‪.358 -269 ،)48‬‬ ‫هلل‪ ،‬شعبان أحمد (‪ .)2013‬مجتمعات التعلم بمدارس التعليم العام في مصر‪ .‬دراسة تحليلية‪ ،‬رسالة دكتوراه‬ ‫غير منشورة‪ ،‬كلية التربية‪ ،‬جامعة دمنهور‪ ،‬مصر‪.‬‬ ‫ً‬ ‫ثانيا‪ -‬املراجع باإلنجليزية‪:‬‬ ‫‪- Battersby, Sharyn L. & Verdi, Brian (2015) The Culture of Professional Learning Communities and‬‬ ‫‪Connections to Improve Teacher Efficacy and Support Student Learning, Arts Education Policy Review,‬‬ ‫‪116, 22- 29.‬‬ ‫‪- Bertsch, T. (2013). Teacher Concerns and Perceptions About the Implementation of Professional‬‬ ‫‪Learning Communities at The High- School Level, Un Published Ph. D. Dissertation, North Dakota‬‬ ‫‪State University., USA.‬‬ ‫‪- Dogan, S., Tatık, R. Ş., & Yurtseven, N. (2017). Professional learning communities assessment:‬‬ ‫‪Adaptation, internal validity, and multidimensional model testing in Turkish context. Educational‬‬ ‫‪Sciences: Theory & Practice, 17, 1203–1229.‬‬ ‫موكلي‪ ،‬زيلعي‬ ‫(‪)72‬‬ ‫المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات‬ ‫التعلم المهنية وفقًا آلرائهم‬ ‫م‬2022 ‫المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس‬ - DuFour, Richard ; DuFour, Rebecca; Eaker, Robert & Many, Thomas. (2016) Learning by Doing: A Handbook for professional Learning Communities at Work (3rd ed). Bloomington: Solution Three Press. - Fuller, S. (2014). The Impact of Professional Learning Communities on Teachers and Students in an Elementary School, Doctoral Thesis in Education, Taxas A&M University- United States. - Herrington, R. (2011). Professional learning communities: Perceptions of teacher quality and sustainability. Doctoral Dissertation, Walden University. - Kalkan, Fatma. (2016). The Relationship between professional learning community, bureaucratic structure and organisational trust in primary education schools. Educational Sciences: Theory & Practice, 16, 1619–1637. - Song, H. (2012). The Role of Teachers Professional Learning Communities in the Context of Curriculum Reform in High- School, Chinese Education & Society, 45 (4), 81- 95. ‫المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات‬ ‫التعلم المهنية وفقًا آلرائهم‬ )73( ‫ زيلعي‬،‫موكلي‬ 

W3166822786.txt

ADMJ (Ahmad Dahlan Medical Journal) VOL 2, No. 1, 10-18 http:// http://journal2.uad.ac.id/index.php/admj Albumin Description of Continuous Ambulatory Peritoneal Dialysis Patients in Sardjito Hospital Yogyakarta Barkah Purwanto1*, Dewi Yuniasih2, Metalia Puspitasari3 * Email (Corresponding Author): barkahdj@gmail.com of Internal Medicine, Ahmad Dahlan University 2Department of Pharmacology, Ahmad Dahlan University 3Department of Internal Medicine, Universitas Gadjah Mada 1Department ARTICLE INFO Article history Received 5 May 2021 Revised 17 May 2021 Accepted 29 May 2021 Keywords Albumin profile, quality of life, CAPD, Cross sectional ABSTRACT Albumin profile is an important sign of nutritional status as a qualityof life parameter, whereas hypoalbuminemia has been shown to be associated with a high mortality rate for patients with End Stage Renal Disease (ESRD) undergoing dialysis including CAPD. The study was conducted to determine serum albumin levels in patients with ESRD undergoing CAPD therapy. The study was conducted in a crosssectional manner on ESRD patients who were still actively undergoing CAPD in the outpatient Installation of Dr. Sardjito Hospital Yogyakarta until November 2018. Subjects were examined for serum albumin levels. There were 57 research subjects with the category of albumin levels <3.5 g / dL as many as 19 (33.3%), between 3.5-4.0 g / dL as many as 27 (47.3%) and albumin levels> 4.0 g / dL of 11 (19.3%). There were no significant differences in all age groups, BMI and duration of CAPD. Albumin profile can be an independent parameter of quality of life and even a predictor of mortality in CAPD patients because it does not depend on age group, BMI and length of CAPD. This is an open access article under the CC–BY-SA license. Pendahuluan Penyakit Ginjal Kronik (PGK) terjadi akibat penurunan fungsi ginjal progresif sehingga ginjal tidak dapat mengendalikan hemostasis dalam tubuh (El Nahas and Bello, 2005; Polonsky and Bakris, 2019). Peningkatan prevalensi PGK menjadi kekhawatiran global dengan berbasis data semakin meningkatnya jumlah pasien End Stage Renal Disease (ESRD) atau stadium Gagal Ginjal Terminal (GGT) yang ditandai dengan peningkatan jumlah pasien yang melakukan transplantasi ginjal (Mencarelli, Busutti and Montini, 2015; Anand et al., 2017; Dhondup et al., 2018; Muhammed, Kibria and Crispen, 2020; Pu et al., 2020). Dengan rata-rata peningkatan jumlah global pasien GGT hingga dua kali lipatnya maka fenomena GGT akan menjadi beban sistem layanan kesehatan di semua negara dunia (Mencarelli, Busutti and Montini, 2015; Polonsky and Bakris, 2019). Kebijakan yang lebih baik pada tatalaksana terapi pasien GGT dengan segala komplikasinya menjadi pertimbangan yang urgen bagi tiap negara agar menurunkan beban layanan kesehatan. 10 ADMJ Vol. 2. No 1, May 2021 p. 10-18 Modalitas terapi pengganti ginjal (TPG) pada pasien GGT yang terbanyak adalah hemodialisis (HD) selanjutnya peritoneal dialisis (PD) dan transplantasi ginjal. Di Indonesia, pasien PD umumnya dengan continuous ambulatory peritoneal dialysis (CAPD) atau Dialisis Peritoneal Mandiri Berkesinambungan (DPMB). Pasien GGT yang menjalani CAPD mempunyai kebebasan yang lebih baik, rutinitas hidup tanpa mesin, perasaan lebih nyaman dan biaya yang relatif lebih murah dibanding melalui modalitas hemodialisis (Widyatmoko, A., 2009). Modalitas CAPD dinilai semakin baik dan pengalaman secara kolektif semakin banyak, tetapi angka mortalitas pasien GGT yang menjalani dialisis di Indonesia tetap tinggi (Bandiiara et al., 2012). Berbagai penelitian mengaitkan tingginya prevalensi mortalitas pasien dialisis ini akibat kondisi malnutrisi pasien GGT yang menjalani terapi dialisis (Tortorelli, Fanelli and Luciani, 2005; Al-othman et al., 2016; Naini et al., 2016). Penentuan tingkat malnutrisi pasien secara biokimia tetap menjadi tantangan dunia medis karena penentuan indikator malnutrisi sulit ditentukan. Salah satu indikator malnutrisi pasien GGT yang menjalani TPG adalah dengan penentuan kadar albumin serum (Gama-axelsson et al., 2012; Tortorici et al., 2020). Kajian tentang pentingnya profil albumin serum perlu diperhatikan karena penurunan kadar albumin serum dari standar adalah satu tanda progres inflamasi (Sridhar and Josyula, 2013). Konsekuensinya, rendahnya serum albumin berefek negatif pada pasien hingga potensi terjadinya mortalitas (Amaral et al., 2015; Sun et al., 2020; Tortorici et al., 2020). Gambaran tingkat keparahan (severity level) dari pasien dibandingkan dengan nilai normal albumin serum standar dengan kriteria sangat parah (Q1) < 3,5 g/ dl, parah (Q2) 3,5 g/dL – 4,0 g/dL dan normal (Q3) > 4,0 g /dL. (Amaral et al., 2015). Profil albumin serum pasien CAPD diperlukan sebagai informasi awal agar terapi terhadap pasien terkontrol secara efektif dan lebih menyeluruh. Profil albumin serum juga diperlukan sebelum pasien menjalani TPG untuk mencegah efek negatif setelah pasien menjalani CAPD dengan cara meningkatkan nutrisi pasien untuk mendapatkan manfaat optimal yang diharapkan (Szeto Cheuk et al., 2000; Levin and Kotanko, 2006). Berdasar latar belakang tersebut, peneliti bermaksud melakukan penelitian untuk mengetahui kadar albumin serum sebagai parameter status nutrisi dan kualitas hidup pada pasien GGT yang menjalani terapi CAPD. Metode Penelitian Penelitian dilakukan di Instalasi Rawat Jalan CAPD Unit Penyakit Dalam RSUP Dr Sardjito Yogyakarta pada bulan Oktober 2018 sampai dengan bulan November 2018. Populasi target adalah pasien GGT dengan CAPD sebagai terapi pengganti ginjal. Populasi terjangkau adalah pasien GGT dengan CAPD yang menjalani pemeriksaan rutin selama periode penelitian dan bersedia mengikuti Albumin Description of Continuous Ambulatory Peritoneal Dialysis Patients in Sardjito Hospital Yogyakarta (Barkah Purwanto, et.al.) 11 ADMJ Vol 2. No 1, May 2021 p. 10-18 penelitian ini dengan menandatangani informed consent. Sampel penelitian adalah pasien GGT dengan CAPD yang memenuhi kriteria inklusi dan ekslusi. Kriteria inklusi adalah pasien GGT yang menjalani CAPD, berusia antara 18 sampai 65 tahun, kontrol rawat jalan di RSUP Dr Sardjito, menjalani pemeriksaan kadar albumin, kurun waktu dari pemasangan tenckhof lebih dari 1 bulan, dan bersedia mengikuti penelitian dengan menandatangani informed consent. Kriteria eksklusi meliputi pasien yang sedang mengalami sepsis, peritonitis, infeksi, overload cairan dan hasil pemeriksaan tidak lengkap. Gambaran tingkat keparahan (severity level) kadar albumin berdasar kategori sangat parah (Q1) < 3,5 g/ dl, parah (Q2) antara 3,5 – 4,0 g/dL dan normal (Q3) > 4,0 g /dL. Hasil Penelitian Selama periode penelitian didapatkan 82 pasien CAPD yang kontrol rutin. Sebanyak 25 subyek tidak diikutkan dalam penelitian terdiri dari 11 subyek usia kurang dari 18 tahun, 3 subyek mengalami overload cairan, 3 subyek tidak bersedia ikut penelitian, 5 subyek tidak dapat dihubungi dan 3 subyek hasil pemeriksaan tidak lengkap. Tabel 1. Karakteristik data dasar 57 subyek CAPD Variabel Jenis kelamin Laki-laki Perempuan Usia (tahun) Diabetes Melitus Ya tidak Hipertensi Ya Tidak Lama CAPD BMI (kg/m2) Under weight Normal Overweight Obese Kadar Albumin < 3,5 3,5 – 4,0 > 4,0 n (%) 35 (61,4) 22 (38,6) Rerata ±SB 37,84±16,16 4 (7,0) 53 (93,0) 14 (24,6) 43 (75,4) 40.44 + 28.63 22.19 + 3.33 11 (19,3) 35 (61,4) 9 (15,8) 2 (3,51) 19 (33,3) 27 (47,3) 11 (19,3) Analisis dilakukan pada 57 subyek, didapatkan kadar albumin 3,62±0,56 < 3,5 sebanyak 19 (33,3%), antara 3,5-4,0 sebanyak 27 (47,3%) dan kadar albumin > 4,0 sebanyak 11 (19,3%) (Gambar 2). Subyek terdiri dari 35 (61,4 %) laki-laki dan 22 (38,6 %) perempuan. Rerata usia 37,84±16,16 tahun dengan rerata lama CAPD 40.44 ± 28.63 bulan. Subyek dengan diabetes melitus sebanyak 7% dan hipertensi 24,6%. Rerata BMI 22,19±3,33 dengan BMI normal sebanyak 61,4%, 12 E-ISSN 2774-3837 ADMJ Vol. 2. No 1, May 2021 p. 10-18 underweight 19,3%, overweight 15,8% dan obese 3,5%. (Tabel 1). Gambar 1. Gambaran tingkat keparahan pasien CAPD berdasarkan kadar albumin Sementara itu, gambaran tingkat keparahan pasien CAPD berdasarkan kadar albumin (gambar 1), didapatkan hasil 47,3% berada di level Q2 atau parah, diikuti 33,3% berada pada tingkatan sangat parah (Q1) dan terakhir 19,3% pasien CAPD pada level normal. Tabel 2. Perbedaan variabel berdasarkan kadar Albumin serum Kadar Albumin Serum Karakteristik Usia BMI Lama CAPD Total (n=57) Q1 (< 3.5) N = 19 rerata + SD 37.84 + 16.16 37.58 + 15.353 22.19 + 3.33 40.44 + 28.63 21.52 + 2.87 45.11 + 36.52 Q2 (3.5 - 4,0) N = 27 rerata + SD 37.41 + 16.768 22.32 + 3.19 38.30 + 26.42 Q3 (>4.0) N = 11 rerata + SD 39.36 + 17.449 23.03 + 4.37 37.64 + 17.64 P value 0.943 0.476 0.691 Pada table 2, tampak secara statistik tidak ada perbedaan yang bermakna antara rata-rata usia, BMI dan lama CAPD terhadap albumin (p > 0.05). Pembahasan Profil Albumin Pasien CAPD Jumlah pasien GGT yang menjalani terapi CAPD di RSUP Dr. Sardjito lebih banyak berjenis kelamin laki-laki. Selain itu, pasien pada kategori Q1 dan Q2 dengan kadar serum albumin di bawah standar juga didominasi pasien laki-laki, sementara pada kategori Q3 dengan kadar albumin normal jumlahnya berimbang. Fenomena pasien CAPD dengan gender laki-laki lebih banyak dari pada perempuan umumnya karena pola hidup lelaki yang lebih tinggi faktor risikonya, misalkan pekerja keras, perokok dan faktor lingkungan lainnya (Wu, Wu and Tarng, 2014; Napierala et al., 2019; Muhammed, Kibria and Crispen, 2020). Albumin Description of Continuous Ambulatory Peritoneal Dialysis Patients in Sardjito Hospital Yogyakarta (Barkah Purwanto, et.al.) 13 ADMJ Vol 2. No 1, May 2021 p. 10-18 Profil Albumin sebagai penanda status nutrisi pasien CAPD Profil albumin relevan sebagai penanda penting tingkat nutrisi pasien dialisis, termasuk CAPD (Joseph et al., 1996; Kim et al., 1999; Swartzendruber, 2020). Selain itu, profil kadar albumin pada pasien gagal ginjal kronik sangat penting diketahui, terutama pasien yang menderita kekurangan albumin atau hipoalbuminemia karena komplikasi penyakit dan terapi yang dijalani. Pada kajian ini, pasien lebih banyak pada kondisi hipoalbuminemia sebanyak 80,7 %. Fenomena ini merupakan prevalensi komplikasi umum yang ditemui pada penyakit ginjal kronik (Sridhar and Josyula, 2013). Karenanya, tingkat relevansi mendapatkan profil serum albumin pada pasien CAPD juga sangat penting karena akan berpengaruh juga terhadap optimalisasi adekuasi CAPD (Joseph et al., 1996; Cueto-manzano et al., 1997; Yang et al., 1997; Kim et al., 1999; Szeto Cheuk et al., 2000; Sezer, Tutal and Haberal, 2005). Risiko malnutrisi pasien CAPD di RSUP Dr Sardjito Secara umum pasien CAPD berpotensi mengalami risiko malnutrisi. Penyebab malnutrisi pada pasien dialisis antara lain karena minimnya asupan energi akibat anoreksia, kehilangan nutrien selama terapi dialisis, pembatasan makanan/diet sebelum dialisis, peningkatan katabolisme protein dan asidosis (Tortorelli, Fanelli and Luciani, 2005; Al-othman et al., 2016; Naini et al., 2016). Berdasarkan profil serum albumin pada penelitian ini, kekurangan serum albumin pada pasien CAPD RSUP Sardjito dapat berdampak progres negatif pasien yang sedang melakukan terapi gagal ginjal tersebut. Selain adanya konsekuensi malnutrisi, memungkinkan juga terjadinya inflamasi progresif (Gelder et al., 2018). Albumin menentukan tekanan osmotik plasma darah, karenanya penurunan albumin dalam peredaran darah akan menggeser cairan dalam ruang intravaskuler (Sridhar and Josyula, 2013). Pada penelitian ini diperoleh gambaran bahwa hanya 19,3% pasien CAPD memiliki kadar albumin serum normal (>4 g/ dL), sementara sebanyak 80,7% pasien lainnya memiliki kadar albumin kurang dari kondisi normal. Dari pasien yang mengalami hipoalbuminemia, terindikasi 33,3 % pasien pada kriteria Q1 (sangat parah) dan 47,3 % pada level Q2 (parah). Bila memperhatikan kajian retrospektif dari Sun et al, 2020, profil albumin serum pasien CAPD di RSUP Dr. Sardjito ini perlu mendapatkan perhatian serius karena pada pasien yang memiliki rentang kadar albumin serum tingkat Q1 dan Q2 memiliki faktor risiko all-cause mortality (ACM) relatif tinggi, masing-masing dengan probabilitas 28% pada tingkat Q1, 20% pada tingkat Q2 dan 8% pada level Q3. Pasien GGT dengan terapi CAPD pada mayoritas penelitian mengalami hipoalbuminemia yang berpotensi memperburuk kelangsungan hidupnya pasca terapi (Cueto-manzano et al., 1997; Sezer, Tutal and Haberal, 2005; Al-othman et al., 2016) seperti pada pasien dengan hemodialisis, 14 E-ISSN 2774-3837 ADMJ Vol. 2. No 1, May 2021 p. 10-18 kadar albumin kurang dari 3,5 gram/dl termasuk faktor risiko utama mortalitas (Pifer, Cullough, Port and Dadvid A G Goodkin, 2002; Pifer, Cullough, Port and David A G Goodkin, 2002; Sridhar and Josyula, 2013; Gelder et al., 2018). Kajian-kajian lain juga menekankan perhatian pada terapi pasien gagal ginjal kronik yang mengalami penurunan kadar serum albumin. Penelitian-penelitian tersebut memaparkan relevansi rendahnya serum albumin dengan peningkatan prevalensi mortalitas (Ustace et al., 2004b; Levin and Kotanko, 2006; Herselman et al., 2010; Sridhar and Josyula, 2013; Amaral et al., 2015; Gelder et al., 2018). Penurunan albumin pada pasien GGT yang menerima terapi CAPD di RSUP Sardjito tersebut harus mendapatkan terapi peningkatan albumin (Of et al., 2004; Gelder et al., 2018). Di masa yang akan datang, perlu pengkajian tatalaksana yang lebih optimal terkait terapi albumin bagi pasien CAPD. Pada penelitian ini didapatkan bahwa kadar albumin serum dapat menjadi prediktor independen terhadap kualitas hidup bahkan mortalitas karena tidak didapatkan perbedaan bermakna pada kelompok usia, Body-Mass-Index (BMI) dan lama CAPD. Selanjutnya, tantangan penelitian berikutnya di bidang ini adalah untuk menggali teori lebih detail tentang mekanisme terjadinya penurunan level albumin pasca CAPD. Kehilangan albumin pada pasien CAPD sudah dikaji sebelumnya, yang menguatkan fakta bahwa pasien CAPD rentan malnutrisi karena potensi kehilangan albumin selama terapi (Iktzler et al., 1994; Ustace et al., 2004a; Herselman et al., 2010; Gelder et al., 2018; Tanaka et al., 2020). Perlu dilakukan penelitian lebih detail tentang mekanisme dan tahapan terjadinya kehilangan albumin pada pasien CAPD selama mengikuti terapi sehingga potensi hipoalbuminemia dikurangi dan menghasilkan penanganan pasien lebih baik selama terapi (Cueto-manzano et al., 1997; Sezer, Tutal and Haberal, 2005; Guest, 2013; Sridhar and Josyula, 2013). Kesimpulan Penelitian telah mengkaji profil albumin pada responden pasien CAPD di RSUP Sardjito. Albumin merupakan indikator dari kondisi nutrisi pasien. Hasil penelitian menunjukkan bahwa sebanyak 80,7% pasien CAPD yang ditangani di RSUP Dr. Sardjito memiliki kadar albumin lebih rendah dari standar normal. Secara gender, pasien laki-laki lebih banyak dari pasien wanita. Kadar albumin rendah /hipoalbuminemia seperti pada responden penelitian ini adalah indikasi dari malnutrisi. Dari hasil kajian penelitian sebelumnya menguatkan korelasi positif hipoalbuminemia dengan peningkatan prevalence mortalitas pasien GGT. Karenanya, terapi peningkatan kadar albumin pasien CAPD RSUP Dr. Sardjito yang mengalami hipoalbuminemia perlu dilakukan. Fokus penelitian ini hanya pada pemaparan profil albumi pasien CAPD, karenanya perlu dilakukan kajian berikutnya untuk memberikan kontribusi lebih banyak lagi pada kajian klinis dan akademis di Albumin Description of Continuous Ambulatory Peritoneal Dialysis Patients in Sardjito Hospital Yogyakarta (Barkah Purwanto, et.al.) 15 ADMJ Vol 2. No 1, May 2021 p. 10-18 bidang ini. Beberapa topik penelitian berikutnya yang menjadi perhatian pada kajian ini diuraikan diakhir pembahasan dengan besar harapan menjadi kontribusi untuk penelitian-penelitian berikutnya di bidang ini yang lebih detail sehingga membantu terapi pasien CAPD yang lebih optimal. Terimakasih Penulis mengucapkan banyak terimakasih kepada RS Sarjito Yogyakarta dan seluruh pasien yang telah berkontribusi dalam penelitian ini. Daftar Pustaka Al-othman, A. M. et al. 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Ramadani L., Rashiti N., Shkodra M., Heta G. Influence of some anthropometric and motor parameters on triple jump. Journal of Education, Health and Sport. 2021;11(12):38-46. eISSN 2391-8306. DOI http://dx.doi.org/10.12775/JEHS.2021.11.12.003 https://apcz.umk.pl/JEHS/article/view/JEHS.2021.11.12.003 https://zenodo.org/record/5759663 The journal has had 5 points in Ministry of Science and Higher Education parametric evaluation. § 8. 2) and § 12. 1. 2) 22.02.2019. © The Authors 2021; This article is published with open access at Licensee Open Journal Systems of Nicolaus Copernicus University in Torun, Poland Open Access. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author (s) and source are credited. This is an open access article licensed under the terms of the Creative Commons Attribution Non commercial license Share alike. (http://creativecommons.org/licenses/by-nc-sa/4.0/) which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited. The authors declare that there is no conflict of interests regarding the publication of this paper. Received: 15.11.2021. Revised: 25.11.2021. Accepted: 05.12.2021. Influence of some anthropometric and motor parameters on triple jump Ramadani L.1, Rashiti N.2, Shkodra M.2, Heta G.3 University of Prishtina, Faculty of Physical Education and Sport, Prishtina, Kosova Abstract Athletics disciplines are characterized by motor movements that can be successfully applied during the educational process or through other forms of exercise, which significantly affect the development of general psychophysical abilities of individuals. Each of the disciplines of athletics acts on the development of individual abilities, but also on the general psychophysical status of man and for this reason athletics is called the "queen of sports". Athletic activities have a characteristic of participant inclusion. The values of the paper will be based on the purpose of anthropometric, basic motor and specific motor characteristics of students aged 21 years ± 6 months. A total of 70 male students, 7 anthropometric variables, 7 motor variables and 1 specific motor variable that were tested during the period of May, of the academic year, 2020/2021 were included in the research. In the basic parameters students, based on the results achieved in this anthropometric variable, are presented as a moderately homogeneous group. Although it is seen that the acquired flexibility and convexity (Skewness and Kurtosis) have a pronounced asymmetry, in the vast majority of anthropometric variables. Although it is seen that the acquired flexibility and convexity (Skewness and Kurtosis) have a pronounced asymmetry, in the vast majority of motor and criterion variables. Thus the statistically significant coefficients of the variables with the highest degree of statistical inference (p <0.01) are denoted by two asterisks. With easier statistical conclusion criterion (p <0.05) correlation coefficients. All variables have statistically significant correlation to all anthropometric, basic and criterion motor variables. Keywords: anthropometric variables, motor, specific, student, regression. INTRODUCTION Athletic jumps are exercises, characterized by an extended phase without support, running over obstacles as a result of momentum and energetic beating on the ground with one foot. [1] These types of exercises are associated with great neuromuscular strain, within a short time [2] As such they develop with priority explosive force and speed as well as sensations of orientation in space 38 and time. Long and triple jumps are natural forms of movement, in general, and athletics in particular. These types constitute one of the basic tools of all-round training: physical, functional and psychological. This is the main reason why they are constantly appreciated, especially in the context of peak exercise for sporting achievement. [3] The data show that the triple jump is the discipline of the old Olympic Games and the first result found is 16 m, but this result is assumed to be the sum of the results of three separate jumps, to jump in a row. The triple jump is part of the group of monostructural movements and is quite complex. Momentum, as the first phase, is cyclic motion, while the other phases belong to acyclic motions. [1,2,4] During the jump, large loads (300-500 kg) are exerted on the moving system of the jumpers, so the three-step jump is approached after the students have reached a proper quantum of basic motor properties, such as speed, strength, mobility and proper condition. Speed and strength mainly dominate in this discipline. The sporting result depends on the size of the initial flight speed, the flight angle as well as the height of the overall center of gravity. that the jumper makes in space on every flight. [5,6] High ump is a complex movement. Its structure consists of: momentum as cyclic motion, thrust, flight and fall belong to the acyclic group of motions. High jump belongs to the monostructural group of movements where the determining element is strength and speed. [7,9,10] The complexity of this discipline increases with the increase of the sports result which is characterized by the high degree of connectivity of these elements as well as by the high degree of harmony and coordination of movements in space. THE PURPOSE OF THE STUDY The problem which is addressed in this paper is as important as it is current, because all students have different characteristics, so even in the sport of athletics there is an increasing need for objectification of indicators of the impact of physical exercise and especially the various jumps, can safely and efficiently diagnose, program, directly manage and control transformational processes in the subject of athletics and sports in general at Faculty of Physical Education and Sports. The main purpose of this research is to determine the impact of some anthropometric, motor and criterion variables in the triple jump to the students of the Faculty of Physical Education and Sports at the University of Prishtina "Hasan Prishtina" - Prishtina, male students in the first year of study of the academic year 2020/2021, aged 19 years ± 6 months. It also aims to verify the anthropometric, motor and motor specific characteristics of the Faculty of Physical Education and Sports students. The basic criteria for participation in the research was: a) that during the year they were always present in athletics classes, b) they were present in the teaching process, c) they were not sick during the test. VARIABLES SAMPLE 1. Anthropometric variables The following anthropometric parameters have been applied to identify anthropometric variables: Body height (BH), Arm length (AL), Leg length (LL), Body weight (BW), Subcutaneous abdominal fat tissue (SAFT), adipose tissue of the arm (ATA), adipose tissue of the thigh (ATTH). 39 2. Specific-motor variables Standing long jump with two legs (SLJTL), Standing high jump (SHJ), Standing long jump with the left foot (SLJLF), Standing long jump with the right foot. (SLJRF), Hand tapping (HT), Foot tapping (FT), Flexibility - body bending forward (BBF) and running 30 meters high start (RHS 30 m.). 3. Specific motor variables Triple jump with momentum METHODS FOR PROCESSING THE RESULTS Taking into account all these facts, for the purposes of this research, procedures have been chosen that are considered to correspond to the nature of the problems being researched. For each variable, the following values will be calculated: 1. Central basic and Distribution Parameters: Arithmetic Mean (AM), Standard Deviation (SD), Minimum Value (Min), Maximum Value (Max). 2. The distribution curve is tested by the coefficient of asymmetry (Skewness), and the degree of curvature of the vertex of the distributed results curve (distribution height) through the flat coefficient (Kurtosis). 3. Interconnections reports between variables in the manifest space, as well as correlations between the systems of variables. 4. To determine the relationship between predictor variables (anthropometric and motor characteristics) and criterion variables (triple jump with momentum), regression analysis was applied in the manifest space. DESCRIPTIVE ANALYSIS OF ANTHROPOMETRIC AND MOTOR SPECIFIC VARIABLES In Table 1 descriptive analysis of anthropometric and motor specific variables is presented to the 19-year-old students of the Faculty of Physical Education and Sports. The sample included a group of 60 male students, where the arithmetic mean values, minimum value, maximum value, standard deviation, distribution or asymmetry parameters (Skewness - tilt, asymmetry) and the degree of extension of the curvature peak of the distribution of results (Kurtosis - convexity). 40 Table 1. Basic statistical parameters of anthropometric and motor specific variables in students of the Faculty of Physical Education and Sports. The arithmetic mean of the anthropometric variable, body height (BH), (Table 1 and Histogram 1) is (178.67 cm). The minimum result (165.50 cm) and the maximum result (197.60 cm) of the variable, body height shows that there is a significant difference between students in this anthropometric variable, and a slight tilt of the variables has the lowest results, because in the asymmetry test dominates the results in between, the asymmetry test is mesocortical (mesokurtical). Students based on the results achieved in this anthropometric variable are presented as a moderately homogeneous group with a larger number of values towards the lower ones. Although there is a marked difference between the minimum and maximum result, it is seen that the acquired flexibility and convexity (Skewness and Kurtosis) have pronounced asymmetry. All variables presented in table 1 are homogeneous; no variable presented in this table is heterogeneous. INTERCORRELATION MATRIX BETWEEN BASIC AND SPECIFIC ANTHROPOMETRIC AND MOTOR VARIABLES Intercorrelation coefficients of basic and specific anthropometric and motor variables in students of year (I) of the Faculty of Physical Education and Sports Table 2 presents the intercorrelation matrix of anthropometric and motor-specific variables in Faculty of Physical Education and Sports students. Due to the better reflection of the table and to more clearly observe the intercorrelation coefficients, the significant coefficients of the different connection levels are identified with the asterisk. Thus the statistically significant coefficients of the variables with the highest statistical conclusion (p <0.01) are marked with two asterisks. With the easiest statistical conclusion criterion (p <0.05) the correlation coefficients are marked with 41 an asterisk. The interpretation of simple linear correlation coefficients as it is known in most cases depends on the number of tested, respectively more precise on the degree of freedom. The sample of 70 students tested, the degree of freedom number is 48. With the inspection of the table of limited values, with Pearson coefficients it is noticed that the value of the limit for 48 degrees of freedom (df (N-2) is r = 0.250 with the easiest criterion (p = 0.05) respectively r = 0.325 with stricter statistical inference criteria (p = 0.01) (Bala, 1990). Table 2. Intercorrelation coefficients of basic and specific anthropometric and motor variables in students of year (I) of the Faculty of Physical Education and Sports By inspecting the intercorrelation coefficients of anthropometric variables in students we notice that all anthropometric variables among themselves have a high correlation and that that coefficient or all correlation coefficients are statistically significant at the level of p <0.001. Especially the correlation coefficients that belong to the longitudinal dimension (arm lengths) and those that belong to the dimension of body mass and body volume are higher. r = 0.989; p <0.01. leg length. r = 0.758; p <0.01., body lengths r = 0.501; p <0.01, arm adipose tissue r = 0.481; p <0.01, adipose tissue of the thigh r = 0.440; p <0.01 etc. By inspecting the intercorrelation coefficients of the basic motor variables in basketball players (table 3) are grouped among themselves. In the first group we have the correlation coefficients belonging to the explosive forces of the lower extremities, standing long jump and standing high jump r = 0.278; p <0.05. In the second group we have the correlation coefficients belonging to the running speed 30 meters r = 0.780; p <0.01. triple jump r = 0.345; p <0.01, etc. 42 Table 3. Regression analysis - correlation and influence of anthropometric and motor variables as independent (predictor) variables on the dependent (criterion) variable - triple jump with momentum (TJM). Model R 1 .607a Model Regression 1 Model Summary R Square Adjusted R Std. Error of the Square Estimate .369 .193 .71194 ANOVAa Sum of Squares df Mean Square 15.983 15 1.066 Residual 27.370 54 Total 43.354 69 F 2.102 Sig. .024b .507 a. Dependent Variable: K3HAP b. Predictors: (Constant), MV30M, AGJKEM, MPPARA, IDHKRA, MKMAJ, MTKEM, APTRU, MTDOR, AIDHKOF, KLVEN, AIDHBA, MKGJVE, AGJKRA, MKDJA, ALATR Coefficientsa Model 1 Standardized Coefficients Beta Unstandardized Coefficients B Std. Error (Constant) 15.386 7.572 ALATR AGJKRA AGJKEM APTRU AIDHBA IDHKRA AIDHKOF MKGJVE KLVEN MKMAJ MKDJA MTDOR MTKEM MPPARA MV30M .099 -.137 -.056 .001 -.109 .127 .025 -.785 -.063 .027 .107 .015 .012 -.010 -1.196 .117 .747 .244 -.487 .031 -.341 .021 .011 .056 -.351 .063 .298 .032 .117 .749 -.194 .032 -.347 .057 .091 .048 .487 .031 .065 .035 .045 .017 -.074 1.273 -.180 a. Dependent Variable: K3HAP t Sig. 2.032 .047 .847 -.563 -1.839 .057 -1.948 2.027 .779 -1.047 -1.965 .482 2.221 .486 .337 -.600 -.939 .401 .576 .071 .955 .057 .048 .439 .300 .055 .632 .031 .629 .737 .551 .352 The regression analysis in students shows that the value of the correlation between the group of independent predictor variables (basic anthropometric and motor variables with the criterion dependent variable - Triple jump with momentum (TJM) has been verified. Connectivity of the whole system of independent predictOR variables (basic anthropometric and motor variables) - Triple jump with momentum (TJM). Connectivity of the whole system of independent predictor variables (basic anthropometric and motor variables): Body height (BH), Arm length (AL), Leg length (LL), Body weight (BW), Abdominal subcutaneous adipose tissue (ASAT), Adipose tissue of the arm (ATA), Adipose Tissue of the Thigh (ATT), Standing Long 43 Jump (SLJ), Standing High Jump (SHJ), Standing Long Jump with the Left Foot (SLJLF), Standing Long Jump with the Right Foot (SLJRF), Hand Tapping (HT) , Foot Tapping (FT), Forward Body Bend Flexibility (FBBF) with the criterion variable - Triple Jump with Momentum (TJM), these have been proven with multiple correlations. The multiple correlation coefficient has the value R = 0.607, which explains the common variability between the system of predictor variables and the criterion variable about 40% (R Square = 0.369). The distribution (F) is obtained as the quotient distribution of the two variances, and in these cases it is always necessary to assign the two degrees of freedom. The first degree of freedom is equal to the number of predictor variables (df = n) respectively (df = 15), while the second is performed so that the number of subjects (70) is reduced by the number of predictive variables minus 1 (df = N - n - 1) respectively (df = 70 - 15– 1). We can emphasize that the test (F-test) is always more valuable if the multiple correlation is greater, in the concrete case of this paper is significant (Sig = 0.024), because the value of the F-test is 3.926. In this research, after obtaining a statistically significant multiple correlation (R = 0.642), it is necessary to look for the coefficient in the column (Beta) as well as the value of (t-test), which show the impact of each predictor variable ( independent) in the dependent or criterion variable (Table 3). CONCLUSION Athletic jumps are exercises, characterized by an extended phase without support, as a result of momentum and energetic beating on the ground with one foot. These types of exercises are associated with great neuromuscular strain, within a short time. As such they develop with priority explosive force and speed as well as sensations of orientation in space and time. In the formation of anthropological features of a population, the genetic factor has a primary influence, but the influence of external factors, thus ecological factors should not be left aside. This paper aimed to ascertain the impact of anthropometric, motor - specific parameters on Faculty of Physical Education and Sports students aged 19 years. A total of 70 students were included in this research. The measurements were executed during the month of May in the physical education hall of the Faculty of Physical Education and Sports. Based on the scope and problem of the research and in accordance with the purpose and statistical methods for processing the results, four hypotheses are presented. A total of 15 motor variables and 1 specific motor variable were applied. All these applied variables have been subjected to basic statistical processing, on the basis of which it was proved that all these variables met the application conditions and have had satisfactory metric characteristics. Descriptive analysis were applied in order to determine the arithmetic mean, standard deviation and to determine the asymmetry and curve. In order to determine the factors in psychomotor variables regression analysis has been applied. Regression analysis shows that student results are presented as homogeneous results. The intercorrelation coefficients of the basic motor and specific motor parameters of some variables, the obtained values have a significant statistical correlation, which has been proven based on the research so far according to many authors. The arguments obtained show that the most realistic picture regarding the selection of students for the sport of athletics based on psychomotor skills are processed through regression analysis. 44 The application of scientific research methods, in a good way creates the information base in order to valorize the values and program contents during the holding of classes in athletics and other sports as well as training processes. The results of the research for pedagogues and trainers will present important information on the impact of anthropometric and basic motor characteristics on the performance of specific motor tasks in the sport of athletics in order to dose the volume and intensity of loads during regular hours of training processes, special hours and various recreational activities. Based on what was said above, it can be concluded that the results and conclusions of the research will be in function of the identification, monitoring, programming and control of students of this age group who regularly attend the subject of athletics in the Faculty of Physical Education and Sports. The whole research is presented in tabular form of results. I think that this paper will be a small window in this field of science and sports in the subject of athletics. Based on what was said above, it can be concluded that the results and conclusions of the research will be in function of the identification, monitoring, programming and control of students of this age group who regularly attend the subject of athletics at the Faculty of Physical Education and Sports. REFERENCES [1] Rashiti N., Meta A ., “Studime Sportive” , “Funkcioni i aparatit vestibular në veprimtarit sportive”, Vol. 5, No. 2. Pp. 7, 2002 Tiranë. [2] Rashiti N., Elezi A., Maliqi A., “Националната спортна академия “, “Impact of some motor abilities on the manifestation of endurance with highschool students at the age 16 years”, “В. Левски” broji 1 -10, 2010, Sofia [3] Rashiti N., Nika F., Sylejmani B., “MONTENEGRIN SPORT ACADEMY” “Prediktivna vrjednost baterije motoricnih testova na tercanje kratki i srednjih staze” Vol. 7, No. 3, pp.15,2011, Montenegro. [4] Rashiti V., Rashiti N,. “Journal of Sport Sciences “, “Atletska Ajrobia” , Zbornik Naucnih i Strucnih Radova” Tuzla, ISSN 1840-4790,Vol.5, pp.2-7, 2013 (10), 1019-1026 [4] Ramadani L., Heta G., Bekolli L., Rashiti N., Millaku A., “Journal of Education, Health and Sport”, “The impact of short run distances to the final results of the decathlon at the 2019 Athletic World Championships”, 2019;9(12):101-109. e ISSN 2391-8306. 2020 [5] Rashiti V., Ramadani L., Rashiti N., “St. Kliment Ohridski University”, “The pole of the students educational background at elementary schools and their tesults”, Faculty of Education – Bitola, Vol. 9, No. 1, God. 2020/2 [6] G Heta., L Bekolli., L Ramadani., Q Ramabaja., N Rashiti.,” Difference between midfield and forward football players in active and passive Body Mass Index as well as in the aerobic capacity” Open Access ISSN 2391-8306 formerly ISSN: 1429-9623 / 2300- 665X, 2020 [7] Rashiti N., Myrtaj N., Xhemajli N., “НАУЧНИ ТРУДОВЕ НА РУСЕНСКИЯ УНИВЕРСИТЕТ”, Canon anthropometric relationship between the characteristics and specific motor abilities in football club in Gjilan, 2/ 2011 том 50, серия 8.2 [8] Rhea M. R., “Determining the magnitude of treatment effects in strength training research through the use of the effect size,” Journal of Strength and Conditioning Research, vol. 18, no. 4, pp. 918-920, 2004. DOI: 10.1519/14403.1 [9] Rashiti N., Ajvazi V., Shkodra M., “Characteristicas anthopomertic and motor test for short running 100 meters (famales) ISSN 1311-3321, RUSE, Bugaria 45 [10] Rashiti N., Nika F., Bekolli L., Heta G., “Jurnal of physical activity and sports” “Difference Betwen finalists in antopometric parameters and 100 meters running of top athlete” vol. 2, no. 3, pp. 1–8, 2017. bot.2017.172689 [11] Spurrs, R.W., Murphy, A.J., & Watsford, M.L. (2003). The effect of plyometric training on distance running performance. Eur J Appl Physiol, 89(1), pp.1-7 [12] Kaynak K., Eryılmaz S. K., Aydoğan S., Mihailov D., “The effects of 20-m repeated sprint training on aerobic capacity in college volleyball players,” Biomedical Human Kinetics, vol. 9, no. 1, pp. 43–50, 2017. DOI: 10.1515/bhk-2017-0007 [13] Kotzamanidis C., “The effect of sprint training on running performance and vertical jumping in prea-dolescent boys,” Journal of Human Movement Studies, vol. 44, no. 3, pp. 225240, 2003. [14] Mero A., Komi P. V., Gregor R. J., “Biomechanics of sprint running. A review,” Sports Medicine, vol. 13, no. 6, pp. 376–392, 1992. DOI: 10.2165/00007256-199213060-00002 [14] Wellenkotter J, Kernozek TW, Meardon SA, Suchomel TJ. The Effects of Running Cadence Manipulation on Plantar Loading in Healthy Runners. International Journal of Sports Medicine. 2014;35(9):1–6. [15] Messier SP, Messier SP, Mihalko SL, Ip E, DeVita P, Cannon DW, et al. A 2-Year Prospective Cohort Study of Overuse Running Injuries: The Runners and Injury Longitudinal Study (TRAILS). The American Journal of Sports Medicine. 2018;46(9):2211–2221. [16] Kessler DF. Running FASTER: Changing Running Technique to Reduce Stress Injuries. International Journal of Athletic Therapy and Training. 2020;25:49–53. [16] Mokha M, Gatens D. Hip and Pelvis Biomechanics during Running as Risk Factors for Injury in Collegiate Runners: A Prospective Study. Journal of Exercise and Nutrition. 2018;1(4):1–6. Correspondence to: Prof.Dr. Rashiti Naser, Ph.D. University Prishtina Faculty of Physical Education and Sport Kosova Phone number: +383 49 666 619 E-mail: naser.rashiti@uni-pr.edu 46 

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BMC Cancer Xu et al. BMC Cancer (2023) 23:636 https://doi.org/10.1186/s12885-023-11135-3 Open Access R E T R AC T I O N N OT E Retraction Note: A synthetic dsRNA, as a TLR3 pathwaysynergist, combined with sorafenib suppresses HCC in vitro and in vivo Yu-Yin Xu1,2†, Li Chen1*†, Gui-Lan Wang1, Jia-Ming Zhou1, Yi-Xin Zhang3, Yin-Ze Wei3, Yuan-Yuan Zhu4 and Jing Qin1 Retraction Note: BMC Cancer 13, 527 (2013) https://doi.org/10.1186/1471-2407-13-527 The Editor has retracted this article because it appears that some of the text and figures have been previously published by some of the same authors. Specifically: •  there is substantial text overlap with results section in [1] •  Figure 2B appears to present the same data as Fig. 1C in [1] •  It appears the same FACS plots have been presented in Fig. 3B (for srfn, poly (I:C) and control) as in Fig. 4A in [1] •  Figure 3C (sifn and Poly(I:C) ) and 3D appear to show the same images as those published previously in [1] in Fig. 3A Author Yu-Yin Xu does not agree to this retraction. None of the other authors have responded to any correspondence from the editor or publisher about this retraction References 1. Chen, L., Xu, Y., Zhou, J., Wu, Y., E, Q., Zhu, Y. “TLR3 dsRNA agonist inhibits growth and invasion of HepG2.2.15 HCC cells”. Oncology Reports 28.1 (2012): 200–206. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. † Equal contributors. The online version of the original article can be found at https://doi. org/10.1186/1471-2407-13-527 *Correspondence: Li Chen bl1@ntu.edu.cn 1 Department of Pathological Anatomy, Nantong University, Nantong, China 2 Department of Nephrology, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China 3 Department of Pathology, Affiliated Tumor Hospital, Nantong University, Nantong, China 4 Biomics (Nantong) Co., Ltd, Nantong, China © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://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. 

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Scientific and practical journal EJCRP&P № 1(2) 2023 5 Психолого-педагогические исследования МНРТИ 15.01.79; 14.35.01 УДК 159.99; 37.082 https://doi.org/10.46914/2959-3999-2023-1-1-59-65 Г.О. Абдуллаева,*¹ к.пед.н., ассоц. профессор. *e-mail: g.abdullayeva@turan-edu.kz ORCID ID: 0000-0002-8359-3117 И. Сулейменов,¹ магистрант. e-mail: 22232256@turan-edu.kz ORCID ID: 0009-0006-8299-4070 1 Университет «Туран», г. Алматы, Казахстан Карьерные ориентации студентов: подходы к исследованию Аннотация В статье раскрывается актуальность и существующие подходы к изучению карьерных ориентаций студентов, определены перспективы и стратегия дальнейшего исследования. Отражены методологические основания исследования, среди которых философские положения о диалектическом методе познания, сущности деятельности, участие в которой преобразует личность; теории системно-комплексного, личностно-деятельностного подхода; идеи формирования личности, становление профессионала; связи теории с практикой. Представлен анализ подходов к пониманию карьеры и карьерных ориентаций в психологии, а также показана авторская позиция, которая выражается в том, что карьерные ориентации включены в систему профессиональной направленности личности и закладывают основу построения карьеры. Ключевые слова: карьера, карьерные ориентации, профессиональное самоопределение, профессиональная направленность, студенты вуза. Введение Постоянно усиливающийся темп жизни, инновации и преобразования в социально-экономической сфере, бурное развитие информационных технологий, ускоряющиеся процессы глобализации и интернационализации, неспокойная геополитическая обстановка в мире оказывают серьезное влияние на жизнь молодежи и заставляют ее адекватно реагировать на проявляющиеся вызовы. Прежде всего это отражается на профессиональном самоопределении и построении карьеры молодых людей. Сегодня, чтобы быть конкурентоспособным на рынке труда, важны не только hard skills, но особую значимость приобретают soft skills. Благодаря сформированности так называемых «мягких навыков» современные специалисты могут успешно решать сквозные профессиональные задачи в русле непосредственной профессиональной деятельности: эффективно коммуницировать с разными людьми, гибко реагировать на изменения в деятельности, уметь работать в ситуации многозадачности, выстраивать профессиональную деятельность в ситуациях неопределенности и т.п. Все это способствует успешному построению карьеры современного специа­ листа. Чем раньше человек начнет осознанно задумываться о выборе профессионального и карьерного пути, тем большей ответственностью и качеством будет отличаться его учебно-профессиональная деятельность, например, в ходе обучения в вузе. 59 Scientific and practical journal EJCRP&P № 1(2) 2023 В период обучения по выбранному профессиональному направлению студент пытается осознать, соответствует ли выбор его желаниям, личностным ценностям, жизненным целям и смыслам. Если выбор профессии сделан правильно, то индивидуально-личностный потенциал раскрывается подобно расцветающему цветку и открываются реальные перспективы для планирования и построения карьеры. Таким образом, складываются все предпосылки для профессионального развития и самореализации личности. К сожалению, практика показывает, что современная молодежь испытывает серьезные затруднения в профессиональной социализации. Связано это с размытостью представлений о выбранной профессии и построении карьеры в ней [1, с.61]. Чаще всего молодые люди ассоциируют карьеру с должностью и статусом в обществе, что на самом деле не всегда отражает уровень профессионального самоопределения и самореализации их личности. Вышеизложенное определяет необходимость эффективной профориентации и формирования карьерных ориентаций молодых людей на этапе их обучения в вузе. В этом и заключается актуальность выбранной проблемы исследования. Материалы и методы Для решения задач исследования использован комплекс теоретических методов, адекватных природе и сущности построения карьеры и формирования карьерных ориентаций студентов на разных этапах обучения в вузе: 1) определение теоретико-методологической базы исследования карьерных ориентаций студентов предполагает обращение к методам теоретического анализа (феноменологический, логический, сравнительно-сопоставительный) и обобщения философских, социологических, психолого-педагогических исследований; 2) научное обоснование изучаемой проблемы основано на методах теоретического анализа, обобщения практического опыта, системно-структурном методе. Методологическую основу исследования составляют философские положения о диалектическом методе познания, сущности деятельности, участие в которой преобразует личность; теории системно-комплексного, личностно-деятельностного подхода; идеи формирования личности, становление профессионала; связи теории с практикой. Основные положения Важно понимать, что карьерные ориентации в структуре профессиональной направленности представляют собой динамическое образование. Они развиваются, трансформируются вместе с развитием личности студента. Возможность изучения этого процесса позволит прежде всего подобрать релевантные методы диагностики карьерных ориентаций студентов на разных этапах обучения в вузе. С другой стороны, исследование теории вопроса, этиологии ключевых понятий позволит нам выстроить четкую концепцию исследования и найти эффективные способы, позволяющие развивать карьерные ориентации студентов на разных этапах обучения в вузе. Таким образом, следуя логике организации научного исследования, целесообразно выделить существующие подходы к изучению карьерных ориентаций студентов, что позволит нам выстроить стратегию собственного исследования. По нашему мнению, формирование карьерных ориентаций напрямую коррелирует с профессиональной направленностью. Иначе говоря, они выступают в качестве значимого элемента в системе профессиональной направленности личности, процесса построения карьеры. Литературный обзор Ю.П. Хяюринен выделяет две базовые модели исследования карьеры: ŠŠ В первой модели изменения, которые происходят в обществе, и карьера взаимосвязаны. В ней психологические и социальные факторы взаимодействуют между собой. 60 Scientific and practical journal EJCRP&P № 1(2) 2023 ŠŠ Вторая модель акцентирует внимание на неизменности человеческой природы. Успех в карьере связывают с возрастными особенностями и различиями между индивидами. Исходя из данного подхода карьера, с одной стороны, рассматривается в качестве структурного компонента жизненного пути человека. С другой – выступает структурным элементом рынка труда [2]. С позиции рынка труда довольно популярны исследования карьеры в сфере психологии управления. Для работодателя важно создать благоприятные условия сотрудникам в продвижении их карьеры. Решение этих задач зачастую определяет необходимость разработки программ развития карьеры в компаниях. Такой подход довольно популярен в управленческой деятельности западных компаний [3]. Термин «карьера» в советское время воспринимался зачастую негативно, поскольку ассоциировался у многих с погоней за должностями [4]. Это объясняется существующей в то время общественной идеологией, ценностями общественно значимых целей в выполнении любой деятельности. Карьера связывалась с индивидуальностью, персонализированностью, что противоречило установленным канонам в обществе. Перемены в социально-политической и экономической жизни в конце ХХ столетия, распад СССР привели к пересмотру отношения и взгляда на термин «карьера» на территории постсоветского пространства. В российской науке основательно начал исследовать проблему карьеры Е.А. Климов, который рассматривал ее как часть профессионального самоопределения [5, c. 20–35]. Именно с началом появления его работ в российской и отечественной психологии сфор­ мировался подход, в русле которого карьера понимается как процесс, направленный на поиск путей реализации профессионального и личностного потенциала человека. Существенной особенностью процесса построения карьеры является приложение человеком собственных усилий, раскрытие своих способностей, умений, талантов, направленных на достижение опре­деленного статуса в обществе, успеха в профессиональной сфере. Так, по мнению Е.А. Могилевкина, Л.В. Куликова и др. карьера – это последовательность этапов развития человека в разных (основных) сферах жизни, которая помимо продвижения в профессиональ­ ной сфере способствует получению значимости для других и признания своей неповтори­ мости [6, 7]. Исходя из этого можно заключить, что карьера определяется не только успехом в профессио­ нальной деятельности, но и успехом в жизни вообще. Подтверждение этой мысли мы находим в трудах современных психологов [8]. Результаты и обсуждение В построении нашего исследования мы разделяем точку зрения таких ученых, как Д. Сьюпер [9] и Н.С. Пряжников, которые считают, что построение карьеры начинается и зависит от успешности профессионального самоопределения. Действительно, человеку в ходе его жизни приходится сталкиваться с выбором профессии после окончания школы, опреде­ ляться после окончания вуза, при переходе в другую сферу деятельности и т.д. Таким образом, карьеру мы понимаем, как процесс профессионального самоопределения личности, основанный на развитии Я-концепции и ценностного отношения к определенной профессиональной сфере. Проведенный анализ исследований карьеры показал, что они актуализированы в сфере управления и направлены на изучение связи между индивидуально-личностными характеристиками работника и построением карьеры; различных аспектов мотивации в построении карь­ еры; особенностях карьерного роста в различных профессиональных сферах и т.п. [10]. Если обратиться к анализу психологических исследований карьеры и карьерных ориентаций в Казахстане, то необходимо отметить их малочисленность. Существующие исследования в основном посвящены вопросам взаимосвязи карьерных ориентаций с различными аспектами трудовой деятельности в разных профессиональных сферах [11, 12]. Хотя необходимо отметить, что проблема профессионального самоопределения, вопросы профориентации набирают 61 Scientific and practical journal EJCRP&P № 1(2) 2023 актуальность и вызывают интерес у казахстанских исследователей, при раскрытии которых авторы обращаются и к вопросам развития карьеры [13]. Однако исследований, посвященных изучению развития карьеры и карьерных ориентаций, у студентов недостаточно. Среди наиболее ярких и значимых исследований по указанной проблематике можно выделить работы американского ученого Э. Шейна [14]. Он проводил много эмпирических исследований, результаты которых показали важность карьерных ориентаций как элемента профессиональной идентичности личности. В ходе эмпирических исследований им была установлена значимая взаимосвязь карьерных ориентаций с развитием карьеры, что позволило определить карьерные ориентации в качестве определяющего фактора оригинальности, индивидуальности процесса управления карьерой. По мнению Э. Шейна, карьерные ориентации тождественны карьерным установкам, в связи с чем начинают проявляться с началом приобретения профессионального опыта или профессиональной социализации [15]. По мнению Э. Шейна, в любой профессиональной деятельности существует несколько направлений продвижения личности, соответствующих ее карьерным ориентациям («якоря карь­еры»), которые, в свою очередь, связаны с представлением человека о себе. Исследование, проведенное ученым, показало, что личностные характеристики человека, демонстрируемые во время профессионального обучения, не являются определяющими факторами его будущей карь­еры. Это связано с тем, что в начале карьеры человек открывает для себя неожиданные соответствия и расхождения собственных ценностей, потребностей, способностей с требованиями организационной среды, в которой он находится при выполнении своей работы. Постепенное прояснение образа «я», сопряженного с собственными мотивами, потребностями, ценностями и талантами в профессиональной сфере, было названо процессом нахождения «якоря карьеры», который, стабилизируясь, начинает действовать в карьере как ведущая сила. «Якорь карьеры» – это «группа потребностей, ценностей и талантов, реализация которых в наибольшей степени значима для человека при осуществлении профессиональных выборов» [15, с. 23]. В начале Э. Шейн выделил «пять якорей» карьеры: менеджмент, профессиональная компетентность, стабильность, автономия/независимость, предпринимательство. В ходе последующих исследований, проведенных в 80-х гг. прошлого века, ученым выделено еще три карьерные ориентации: вызов, служение, интеграция стилей жизни. Карьерные ориентации, по мнению Н.И. Юртаевой [16], можно рассматривать в качестве регулятора поведения личности в социальной среде, благодаря которому выбирается оптимальный способ достижения успеха в карьере. Иначе говоря, карьерные ориентации выражают способ достижения успеха в построении карьеры и ее ценности. Заключение Таким образом, проведенный анализ исследований указанной проблемы показал, что чаще всего они сосредоточены на изучении различных аспектов развития карьеры в ходе трудовой профессиональной деятельности. Как за рубежом, так и в нашей стране интерес исследователей привлекают аспекты взаимосвязи карьеры с личностными характеристиками, мотивацией и др. Актуализируются исследования, посвященные различным аспектам профессионального самоопределения и его влияния на формирование успешной карьеры. Иначе говоря, большинство исследований реализованы в русле психологии менеджмента, организационной психологии и ориентированы на человека, который уже трудится (является сотрудником какой-либо компании). Однако мы считаем, что основы успешной карьеры закладываются еще на этапе обучения в вузе. Основу карьеры закладывают именно карьерные ориентации, которые включены в сис­ тему профессиональной направленности личности. В этой связи необходимо изучить особенности развития карьерных ориентаций на этапе обучения в вузе. Значимость этого, по нашему мнению, определяется тем, что период обучения в вузе важен не только для формирования 62 Scientific and practical journal EJCRP&P № 1(2) 2023 профессиональных компетенций, но и для определения целей, планов для успешного карьернопрофессионального развития. Конкретизация представлений о своем профессионально-личностном будущем в русле осваиваемой профессии происходит благодаря карьерным ориентациям. Следовательно, изучение личностных факторов, определяющих карьерные ориентации студентов на разных этапах обучения, и составляет перспективное направление нашего исследования. ЛИТЕРАТУРА 1 Харланова Т.Н. Развитие карьерных ориентаций студенческой молодежи в процессе профессиональной подготовки. Дисс. ... канд. психолог. н. – М., 2015. – 261 c. 2 Зеер Э.Ф. Психологические особенности управления карьерой / Э.Ф. Зеер, О.В. Таранова // Ежегодник Российского психологического общества. Материалы 3-го Всероссийского съезда психологов. – СПб., 2003. – Т. 3. 3 Молл Е.Г. Управление карьерой менеджера / Е.Г. Молл. – СПб.: Питер, 2012. – 198 с. 4. Кирт Н.Л. Динамика представлений о профессиональной карьере психологов образования в процессе профессионального самоопределения: дис. ... канд. психологических наук: 19.00.07 / Н.Л. Кирт. – Москва, 2000. – 158 c. 5 Климов Е.А. Введение в психологию труда / Е.А. Климов. – М.: Юнити. – 1998. – С. 8. 6 Могилевкин Е.А. Карьерный рост: диагностика, технологии, тренинг / Е.А. Могилевкин. – СПб.: Речь, 2007. – 336 с. 7 Куликов Л.В. Психология личности в трудах отечественных психологов: хрестоматия / Л.В. Куликов. – СПб.: Питер, 2008. – 460 с. 8 Пряжников Н.С. Мотивация трудовой деятельности / Н.С. Пряжников. – М.: Академия, 2012. – 368 с. 9 Super D.E. The psychology of career / D.E. Super. New York: Evanston, Harper & Row, 1957. 362 р. 10 Соловьева Е.М. Подходы к определению понятия «карьерные ориентации» / Е.М. Соловьева // Психология карьеры: материалы Всерос. научно-практ. конф. (заоч.), 2012. – С. 24–27. 11 Тажина Г.О., Таирова К.Т. Карьерные ориентации как предикторы организационной лояльности преподавателей университетов // Bullletin-psysoc.kaznu.kz/ URL: http://doi.org/10.26577/JPsS.2020.v73. i2.01 (дата обращения: 18.02.2023) 12 Лазаренко Д.В. Особенности мотивации и карьерных ориентаций государственных служащих // «Public administration and civil service» International scientific-analytical journal. URL: http:repository.apa.kz (дата обращения: 18.02.2023) 13 Сыздыкова К. Развитие системы профориентационной работы в школы как фактор расширения доступа к высшему техническому и профессиональному образованию // Программа для молодых исследователей в области публичной политики Фонда Сорос-Казахстан. – Алматы, 2018. URL: http:soros.kz (дата обращения: 20.02.2023) 14 Шейн Э. Организационная культура и лидерство: построение, эволюция, совершенствование / Э. Шейн – СПб.: Питер, 2002. – 336 с. 15 Schein E.H. Career anchors: the changing nature of work and careers / E.H. Schein, J. Maanen. San Francisco: Wiley, 2013. 88 p. 16 Юртаева Н.И. Управление карьерными ориентациями в профессиональном развитии студентов инженерного вуза. Вестник Казанского технологического университета. – 2012. – Т. 15. – 1: 300–306. – С. 37. REFERENCES 1 Harlanova T.N. (2015) Razvitie kar'ernyh orientacij studencheskoj molodezhi v processe professional'noj podgotovki. Diss. ... kand. psiholog. n. M., 261 p. (In Russian). 2 Zeer Je.F. (2003) Psihologicheskie osobennosti upravlenija kar'eroj / Je.F. Zeer, O.V. Taranova // Ezhegodnik Rossijskogo psihologicheskogo obshhestva. Materialy 3-go Vserossijskogo s#ezda psihologov. SPb. V. 3. (In Russian). 3 Moll E.G. (2012) Upravlenie kar'eroj menedzhera / E.G. Moll. SPb.: Piter, 198 p. (In Russian). 63 Scientific and practical journal EJCRP&P № 1(2) 2023 4. Kirt N.L. (2000) Dinamika predstavlenij o professional'noj kar'ere psihologov obrazovanija v processe professional'nogo samoopredelenija: dis. ... kand. psihologicheskih nauk: 19.00.07 / N.L. Kirt. Moskva, 158 p. (In Russian). 5 Klimov E.A. (1998) Vvedenie v psihologiju truda / E.A. Klimov. M.: Juniti. P. 8. (In Russian). 6 Mogilevkin E.A. (2007) Kar'ernyj rost: diagnostika, tehnologii, trening / E.A. Mogilevkin. SPb.: Rech', 336 p. (In Russian). 7 Kulikov L.V. (2008) Psihologija lichnosti v trudah otechestvennyh psihologov: hrestomatija / L.V. Kulikov. SPb.: Piter, 460 p. (In Russian). 8 Prjazhnikov N.S. (2012) Motivacija trudovoj dejatel'nosti / N.S. Prjazhnikov. M.: Akademija, 368 p. (In Russian). 9 Super D.E. (1957) The psychology of career / D.E. Super. New York: Evanston, Harper & Row, 362 p. (In English). 10 Solov'eva E.M. (2012) Podhody k opredeleniju ponjatija «kar'ernye orientacii» / E.M. Solov'eva // Psihologija kar'ery: materialy Vseros. nauchno-prakt. konf. (zaoch.) P. 24–27. (In Russian). 11 Tazhina G.O., Tairova K.T. Kar'ernye orientacii kak prediktory organizacionnoj lojal'nosti prepodavatelej universitetov // Bullletin-psysoc.kaznu.kz/ URL: http://doi.org/10.26577/JPsS.2020.v73.i2.01 (data obrashhenija: 18.02.2023). (In Russian). 12 Lazarenko D.V. Osobennosti motivacii i kar'ernyh orientacij gosudarstvennyh sluzhashhih // «Public administration and civil service» International scientific-analytical journal. URL: http:repository.apa.kz (data obrashhenija: 18.02.2023). (In Russian). 13 Syzdykova K. (2018) Razvitie sistemy proforientacionnoj raboty v shkoly kak faktor rasshirenija dostupa k vysshemu tehnicheskomu i professional'nomu obrazovaniju // Programma dlja molodyh issledovatelej v oblasti publichnoj politiki Fonda Soros-Kazahstan. Almaty. URL: http:soros.kz (data obrashhenija: 20.02.2023). (In Russian). 14 Shejn Je. (2002) Organizacionnaja kul'tura i liderstvo: postroenie, jevoljucija, sovershenstvovanie / Je. Shejn – SPb.: Piter, 336 p. (In Russian). 15 Schein E.H. (2013) Career anchors: the changing nature of work and careers / E.H. Schein, J. Maanen. San Francisco: Wiley, 88 p. (In English). 16 Jurtaeva N.I. (2012) Upravlenie kar'ernymi orientacijami v professional'nom razvitii studentov inzhenernogo vuza. Vestnik Kazanskogo tehnologicheskogo universiteta. V. 15. 1: 300–306. P. 37. (In Russian). Г.О. Абдуллаева,*¹ пед.ғ.к., қауымд.профессор. *e-mail: g.abdullayeva@turan-edu.kz ORCID ID: 0000-0002-8359-3117 И. Сүлейменов,¹ магистрант. e-mail: 22232256@turan-edu.kz ORCID ID: 0009-0006-8299-4070 1 «Тұран» университеті, Алматы қ., Қазақстан Студенттердің мансаптық бағдарлары: зерттеу тәсілдері Андатпа Мақалада студенттердің мансаптық бағдарларын зерттеудің өзектілігі мен қолданыстағы тәсілдері ашы­ лып, зерттеудің алдағы болашағы мен стратегиясы анықталады. Зерттеудің әдіснамалық негіздері көрсетілген, олардың арасында танымның диалектикалық әдісі туралы философиялық ережелер, қатысуы жеке тұлғаны өзгертетін іс-әрекеттің мәні; жүйелік-кешенді, тұлғалық-белсенділік тәсілінің теориялары; жеке тұлғаны қа­ лыптастыру идеялары, кәсіби маманның қалыптасуы; теорияның практикамен байланысы. Психологиядағы мансап пен мансаптық бағдарларды түсіну тәсілдеріне талдау жасалады, сонымен қатар мансаптық бағдарлар жеке тұлғаның кәсіби бағдарлау жүйесіне еніп, мансаптық құрылыстың негізін қалайтын авторлық ұстаным көрсетілген. Тірек сөздер: мансап, мансаптық бағдар, кәсіби өзін-өзі анықтау, кәсіби бағдар, ЖОО студенттері. 64 Scientific and practical journal EJCRP&P № 1(2) 2023 G.O. Abdullaeva,*¹ c.ped.sc., ass. professor *e-mail: g.abdullayeva@turan-edu.kz ORCID ID: 0000-0002-8359-3117 I. Suleimenov,¹ master student. e-mail: 22232256@turan-edu.kz ORCID ID: 0009-0006-8299-4070 1 Turan University, Almaty, Kazakhstan Career orientations of students: approaches to research Abstract The article reveals the relevance and existing approaches to the study of students orientations, identifies the prospects and strategy for further research. The methodological bases of research, among which philosophical positions about a dialectical method of knowledge, essence of activity, participation in which transforms a personality; theories of system-complex, personality-activity approach; ideas of formation of a personality, formation of the professional; connection of the theory with practice are reflected. The article presents the analysis of approaches to the understanding of career and career orientations in psychology, and also shows the author's position, which is expressed in the fact that career orientations are included in the system of professional orientation of the person and lay the foundation for career building. Key words: career, career orientations, professional self-determination, professional orientation, university students. 65 

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http://doi.org/10.3126/njr.v13i2.59964 REVIEW ARTICLE Intracardiac KCI Injection in Cesarean Scar Pregnancy: A Review Article Gupta A1, Thapa M2 1 Department of Intervention Radiology, Sri Ganga Ram Hospital, New Delhi, India Department of Intervention Radiology, Nepal Army Institute of Health Sciences, Kathmandu, Nepal 2 Received: September 25, 2023 Accepted: November 10, 2023 Published: November 24, 2023 Cite this paper: Gupta A, Thapa M. Intracardiac KCl injection in cesarean scar pregnancy: A Review Article. Nepalese Journal of Radiology 2023;13(2):3-8. http://doi.org/10.3126/njr.v13i2.59964 ABSTRACT Embryo implantation in a previous caesarean scar (CS) resulting in a caesarean scar pregnancy (CSP) is another rare but potentially catastrophic complication of a previous caesarean birth. CSP can be detected between days 33 and 94 of pregnancy. Methotrexate and potassium chloride (KCl) are the most common feticidal agents used. intracardiac KCl causes feticide and reduces the uteroplacental blood flow, thereby decreasing the rate of hemorrhage due to placenta previa, caesarian scar pregnancy and in cases of fibroids; it is easy to evacuate the macerated fetus. Intracardiac KCl causes feticide and reduces the uteroplacental blood flow, thereby decreasing the rate of hemorrhage and abortion time before termination of pregnancy (TOP) leading to an easy evacuation of the macerated fetus. In conclusion, feticide with intracardiac KCl is a safe procedure. Keywords: Cicatrix; Leiomyoma; Methotrexate; Potassium Chloride; Pregnancy INTRODUCTION many factors including clinical presentation, β-hCG levels, imaging features, and patient choice. In IR, the local treatment is widely used for termination of pregnancy using MTX as the most common feticidal agent or other agents such as potassium chloride, ethanol and hyperosmolar glucose.1,2,3,4,5,6,7 Cesarean scar pregnancy is defined as an ectopic pregnancy embedded in the myometrium of a previous Cesarean scar. The first case of CSP was reported in 1978 by Larsen and Solomon. The first detection of a 7-week cesarean scar pregnancy made in transvaginal US occurred in 1990. Studies indicate that CSP can be detected between days 33 and 94 of pregnancy. The incidence of CSP has been estimated to range from 1/1800-1/2500 of all CD performed and 1/531 for women who had at least one cesarean delivery. To date there have been only a few randomized studies on CSP and evidencebased management remains unclear. Until then, treatment should be individualized according to The technique of intracardiac KCl to induce fetal demise is well established.27 Intracardiac KCl is frequently used for fetal reduction in multiple pregnancies. Its use to induce fetal demise to reduce hemorrhage in medical termination of pregnancy is not very frequent, however, intracardiac KCl causes feticide and reduces the uteroplacental bl Correspondence to: Dr. Manish Thapa Department of Intervention Radiology Nepal Army Institute of Health Sciences Kathmandu, Nepal Email: emaildrmanish@gmail.com Licensed under CC BY 4.0 International License which permits use, distribution and reproduction in any medium, provided the original work is properly cited NJR VOL 13 No. 2 ISSUE 22 November, 2023 3 Gupta A et al. KCl flow, thereby decreasing the rate of hemorrhage due to placenta previa, caesarian scar pregnancy and in cases of fibroids; it is easy to evacuate the macerated fetus. Types of CSP There are two types of cesarean scar pregnancy proposed by Vial et al. CSP directed towards cervicoisthmic space or uterine cavity (type I, endogenic type) and CSP with a deep invasion of scar defect with progression towards the bladder and abdominal cavity (type II, exogenic type). The endogenic type of CSP could result in a viable pregnancy; yet with a high risk of bleeding at the placental site.13,14 METHODS We searched MEDLINE/PubMed electronic database using medical subheading search words such as ‘caesarean scar pregnancy’; ‘caesarean scar’; ‘caesarean scar local management’; ‘caesarean scar minimally invasive management’; ‘ectopic pregnancy’; ‘caesarean scar ectopic pregnancy’; ‘pregnancy, previous caesarean scar’; and ‘unusual ectopic pregnancies’ to obtain a comprehensive list of articles concerning this condition from January 1978 to March 2021. We also searched using the ‘Scholar Google’ search engine on the internet (www.scholar.google.com) for articles on this topic since 1987. The citations were cross-checked via related articles and links in PubMed. Additional articles were obtained from the cross-references of the relevant publications for bibliographical purposes. Presentation The symptoms can be seen from 5-6 weeks and as late as 16 weeks. Mild vaginal bleeding is the presenting symptom in 39% and approximately 16% complain of associated mild to moderate pain and 9% complain of only abdominal pain.15,16,17 Imaging Ultrasound It is the primary diagnostic tool for cesarean scar and pregnancy The advent of TVUS has made it possible to diagnose ectopic pregnancies at earlier gestational ages, reducing the risk of significant morbidity and mortality. The early diagnosis of CSP at approximately 6 weeks of gestation and as late as 16 weeks of gestation enables non-surgical treatment, resulting in short-term hospitalisation and preserving fertility. Early correct CSP diagnosis increases confidence and implementation of minimally invasive surgery.18 Predisposing factors, mechanism and pathophysiology The exact cause and mechanism is not well understood but it is possible that the conceptus penetrates the myometrium through a microscopic dehiscent tract of the cesarean scar or the gestational sac implantation occurs in a poorly healed cesarean section scar. Such a tract can also develop from the trauma of other uterine surgery, e.g. curettage, myomectomy, metroplasty, hysteroscopy and even manual removal of placenta. In CSP, the gestation sac is surrounded by myometrium and the fibrous tissue of the scar, quite separate from the endometrial cavity. Cesarean delivery is associated with increased risks of placenta previa, uterine rupture and placenta accreta. Among women with placenta previa and a previous cesarean section, the risk of placenta accreta was significantly higher when the placenta was implanted over the scar, supporting the theory that trophoblast adherence or invasion is enhanced when the scant decidualization of the lower uterine segment is further impaired by previous myometrial disruption.8,9,10,11,12 The following criteria have been followed for diagnosis of CSP:5,19,20,21 An empty uterine cavity and endocervical canal. Gestational sac located within the anterior myometrium of previous cesarean scar site in LUS. Color Doppler flow around the sac is characterized by a high velocity (peak velocity 0.20 cm/s) and low impedance (pulsatility index,1) blood flow; Negative ‘sliding organs sign’, that is inability to displace the gestational sac from its position at the level of the internal os using gentle pressure applied by the transvaginal probe. less than 5mm of myometrium or scar tissue NJR VOL 13 No. 2 ISSUE 22 November, 2023 4 Gupta A et al. KCl separating the sac from the bladder contamination through the needle tract during insertion, the initial 1 ml of aspirated heart blood was discarded. Thereafter, 2ml of 15% KCl was injected into the fetal heart under direct vision. A further 2ml was injected every 30 seconds until permanent fetal asystole was achieved, and the time was recorded. The fetal heart was observed on scan for a further 5 minutes of asystole with the needle in situ. To minimise any risk of maternal contamination with KCl, the needle was flushed with 5ml sterile water or saline before removal. Provided there were no contraindications to vaginal delivery, the women were given the choice to have labour induced or to await spontaneous onset of labour. Labour was induced if women did not go into spontaneous labour within 3 weeks.26 In most of the cases, less than 5mm thickness of myometrium between the gestational sac and bladder has been reported.22 It can also help to detect placenta accreta spectrum i.e. placenta or gestational sac embedded in the hysterotomy scar. Non-visualisation of myometrium between the urinary bladder and sac can also help in differentiation from cervicoisthmic pregnancy.8 In spontaneous abortion, the avascular gestational sac should be seen in the cervical canal on TVS, indicating detachment of sac from its implantation site, whereas in caesarean scar pregnancies, the gestational sac would appear well-perfused and would be located in the anterior uterine wall at the isthmus.23 Treatment Because of the rarity of the condition, the majority of CSPs are case reports or small case series reported in the literature, with no consensus on the preferred mode of treatment. Generally, termination of pregnancy (TOP) in the first trimester is strongly recommended, as there is a high risk of subsequent uterine rupture, massive bleeding and lifethreatening complications. This, in turn, may warrant life-saving hysterectomy and loss of future fertility. Magnetic resonance imaging MRI imaging can be an added advantage in equivocal cases for gestational sac location confirmation and surrounding organs. MRI and TVS are equally accurate in the diagnosis of CSP but scar implantation can be better evaluated with it. MRI T2-weighed sagittal section was used to identify the cesarean scar defect, trophoblastic layer, and myometrium separately. The MRI findings of cesarean scar pregnancy are the same as above mentioned sonographic findings concepts implantation on cesarean section scar, myometrial defect in association with empty endometrial cavity and placenta accreta spectrum.14,24,25 There are following types of CSP intervention Technique At least half an hour before the feticide procedure, all the patients were given pethidine 50 mg intravenously for sedation and an anti-emetic if necessary. After excluding allergies, a secondgeneration cephalosporin (mefoxin) 2gm was given intravenously as a prophylactic antibiotic. Lignocaine 2% was used to infiltrate the maternal abdominal skin at the site of entry into the fetal heart. A 20-gauge 15cm spinal needle was traversed through the predetermined tract, targeting the left ventricle or the most accessible chamber of the fetal heart. To reduce the risk of feto-maternal  KCl only  KCl + MTX  MTX only  Expectant Intracardiac potassium chloride (KCl) was first described as a method for inducing fetal demise in 1988 and has since grown in use to induce feticide before an abortion, by either intracardiac or umbilical vein administration. Post-KCl administration causes fetal asystole by disrupting the balance of intra- and extracellular potassium, which subsequently decreases myocyte action potential. As a result of this in-induced demise, the fetal tissue becomes macerated and softened, mimicking a spontaneous fetal demise, and thereby theoretically resulting in an easier surgical procedure.28,29 NJR VOL 13 No. 2 ISSUE 22 November, 2023 5 Gupta A et al. KCl There are very few studies and case reports where fetal demise is induced with intracardiac KCl before medical TOP to reduce the risk of hemorrhage either because of placenta previa or ectopic pregnancy at the scar site or due to large fibroids distorting the uterine anatomy. birth. The exponential rise in its incidence during the past 5-6 years may be a true increase in incidence because of the rising caesarean section rate worldwide or an apparent increase as a result of a more liberal use of TVS in early pregnancy. Little is known about its exact mechanism and natural history. According to Kaur et al., an average of 1.8cc of intracardiac KCl was required to achieve cardiac asystole which is less as compared to other studies. This is because the gestational age was less (10+4 weeks to 19 weeks) in our study as compared to a study by Sfakianaki et al. (range, 15.4-24.9 weeks) where on an average of 10 mL of strong KCl was required to reliably achieve fetal cardiac asystole.30,31 In conclusion, feticide with intracardiac KCl is a safe procedure and reduces the risk of hemorrhage and abortion time before TOP in scar pregnancy. CONFLICT OF INTEREST None SOURCES OF FUNDING Another study showed the average amount of potassium chloride required in their study was 4.7ml (range 2–10 ml), much less than the amounts reported by other authors. If the needle is correctly positioned in the left ventricle, a minimal amount of potassium chloride is required to achieve asystole as the potassium chloride is injected rapidly into the coronary circulation inducing immediate asystole.32 None REFERENCES The data shows that it is safe to use up to 20 mL of 15% KCl by the intracardiac route, but that on average only 10 mL of strong KCl is required to reliably achieve fetal cardiac asystole.33 The role of intracardiac KCl is to reduce the blood flow and thus the risk of hemorrhage or to reduce abortion time either in terms of conservative abortion or dilatation and curettage. According to Tufa et al., and Lohr et al., Inducing fetal demise with intracardiac KCl is associated with a decrease in D&E procedure duration or conservative management duration.34,35 Uterine artery embolization has also been used in previous studies to manage scar pregnancy but uterine artery embolization is a more invasive procedure with a longer duration than intracardiac KCl.36 1. Lai YM, Lee JD, Lee CL, Chen TC, Soong YK. An ectopic pregnancy embedded in the myometrium of a previous cesarean section scar. Acta Obstet Gynecol Scand 1995;74(7):573-6. https://doi.org/10.3109/00016349509024394 2. Larsen JV, Solomon MH. Pregnancy in a uterine scar sacculus-an unusual cause of postabortal haemorrhage. A case report. S Afr Med J 1978 28;53(4):142-3. Available from: https://pubmed.ncbi.nlm.nih.gov/653492/ [Accessed 5th November 2023] 3. Rempen A, Albert P. Diagnose und Therapie einer in der Sectionarbe implantierten Frühschwangerschaft [Diagnosis and therapy of an in the cesarean section scar implanted early pregnancy]. Z Geburtshilfe Perinatol 1990;194(1):46-8. Available from: https:// pubmed.ncbi.nlm.nih.gov/2180228/ [Accessed 6th November 2023] 4. Kwaśniewska A, Stupak A, Krzyżanowski A, Pietura R, Kotarski J. Cesarean scar pregnancy: uterine artery embolization combined with a hysterectomy at 13 weeks’ gestation–a case report and review of the literature. Ginekol Pol 2014;85(12):961-7. https://doi.org/10.17772/ gp/1890 5. Timor-Tritsch IE, Monteagudo A, Santos CONCLUSION Embryo implantation in the region of a previous caesarean section scar is a rare but potentially catastrophic complication of a previous caesarean NJR VOL 13 No. 2 ISSUE 22 November, 2023 6 Gupta A et al. KCl R, Tsymbal T, Pineda G, Arslan AA. The diagnosis, treatment, and follow-up of cesarean scar pregnancy. Am J Obstet Gynecol 2012;207(1):44-e1. https://doi.org/10.1016/j. ajog.2012.04.018 6. 7. 13. Vial Y, Petignat P, Hohlfeld P. Pregnancy in a Cesarean scar. Ultrasound Obstet Gynecol 2000;16(6):592-3. https://doi.org/10.1046/ j.1469-0705.2000.00300-2.x 14. Gonzalez N, Tulandi T. Cesarean scar pregnancy: a systematic review. J Minim Invasive Gynecol 2017;24(5):731-8. https:// doi.org/10.1016/j.jmig.2017.02.020 Shao MJ, Hu M, Hu MX. Conservative management of cesarean scar pregnancy by local injection of ethanol under hysteroscopic guidance. International Journal of Gynaecology and Obstetrics: Int J Gynaecol Obstet 2013;121(3):281-2. https://doi.org/10.1016/j. ijgo.2013.01.012 15. Seow K-M, Huang L-W, Lin YH, Yan-Sheng Lin M, Tsai Y-L, Hwang J-L. Caesarean scar pregnancy: issues in management. Ultrasound Obstet Gynecol 2004;23(3):247-53. https://doi. org/10.1002/uog.974 Peng P, Gui T, Liu X, Chen W, Liu Z. Comparative efficacy and safety of local and systemic methotrexate injection in cesarean scar pregnancy. Ther Clin Risk Manag 2015;11:137-42. https://doi.org/10.2147/ TCRM.S76050 8. Godin PA, Bassil S, Donnez J. An ectopic pregnancy developing in a previous caesarian section scar. Fertility and sterility 1997;67(2):398-400. https://doi.org/10.1016/ s0015-0282(97)81930-9 9. Fait G, Goyert G, Sundareson A, Pickens A Jr. Intramural pregnancy with fetal survival: case history and discussion of etiologic factors. Obstet Gynecol 1987;70(3):472-4. Available from: https://pubmed.ncbi.nlm.nih. gov/3627606/ [Accessed 6th November 2023] 16. Smith A, Ash A, Maxwell D. Sonographic diagnosis of cesarean scar pregnancy at 16 weeks. Journal of Clinical Ultrasound 2007;35(4):212-5. https://doi.org/10.1002/ jcu.20270 17. Rotas MA, Haberman S, Levgur M. Cesarean scar ectopic pregnancies: etiology, diagnosis and management. Obstet Gynecol 2006;107:1373-7. https://doi.org/10.1097/01. AOG.0000218690.24494.ce 18. Pędraszewski P, Wlaźlak E, Panek W, Surkont G. Cesarean scar pregnancy-a new challenge for obstetricians. J Ultrason 2018;18(72):56-62. https://doi.org/10.15557%2FJoU.2018.0009 19. Fylstra DL. Ectopic pregnancy within a cesarean scar: a review. Obstet Gynecol Surv 2002;57(8):537-43. https://doi. org/10.1097/00006254-200208000-00024 10. Coniglio C, Dickinson JE. Pregnancy following prior Caesarean scar pregnancy rupture: lessons for modern obstetric practice. Aust N Z Obstet Gynaecol 2004;44:162-6. https://doi. org/10.1111/j.1479-828X.2004.00176.x 20. Godin P-A, Bassil S, Donnez J. An ectopic pregnancy developing in a previous caesarean section scar. Fertil Steril 1997;67(2):398400. https://doi.org/10.1016/S00150282(97)81930-9 11. Miller DA, Chollet JA, Goodwin TM. Clinical risk factors for placenta previa-placenta accreta. American journal of obstetrics and gynecology 1997;177(1):210-4. https://doi. org/10.1016/S0002-9378(97)70463-0 21. Jurkovic D, Hillaby K, Woelfer B, Lawrence A, Salim R, Elson CJ. First- trimester diagnosis and management of pregnancies implanted into the lower uterine Caesarean section scar. Ultrasound Obstet Gynecol 2003;21(3):220-7. https://doi.org/10.1002/uog.56 12. Clark SL, KOONINGS PP, PHELAN JP. Placenta previa/accreta and prior cesarean section. Obstet Gynecol 1985;66(1):89-92. Available from: https://pubmed.ncbi.nlm.nih. gov/4011075/ [Accessed 8th November 2023] 22. Weimin W, Wenqing L. Effect of early NJR VOL 13 No. 2 ISSUE 22 November, 2023 7 Gupta A et al. KCl pregnancy on a previous lower segment cesarean section scar. Int J Gynecol Obstet 2002;77(3):201-7. https://doi.org/10.1016/ S0020-7292(02)00018-8 30. Kaur R, Goel B, Sehgal A, Goyal P, Mehra R. Feticide with intracardiac potassium chloride to reduce risk of hemorrhage in medical termination of pregnancy. J Gynaecol Womens Healthcare 1: 102 Abstract RESEARCH ARTICLE Open Access 2018;1(1). Available from: https://www.scholarena.com/article/ Feticide-with-Intracardiac-Potassium-Chloridto-Reduce-Risk-of-Hemorrhage-in-MedicalTermination-of-Pregnancy.pdf [Accessed 8th October 2023] 23. Tan G, Chong YS, Biswas A. Caesarean scar pregnancy: a diagnosis to consider carefully in patients with risk factors. Ann Acad Med Singapore 2005;34(2):216-9. https://doi. org/10.47102/annals-acadmedsg.V34N2p216 24. Osborn DA, Williams TR, Craig BM. Cesarean scar preg nancy: sonographic and magnetic resonance imaging find ings, complications, and treatment. J Ultrasound Med 2012;31(9):1449-1456. https://doi. org/10.7863/jum.2012.31.9.1449 31. Sfakianaki AK, Davis KJ, Copel JA, Stanwood NL, Lipkind HS. Potassium chloride-induced fetal demise: a retrospective cohort study of efficacy and safety. J Ultrasound Med 2014;33(2):337-41. https://doi.org/10.7863/ ultra.33.2.337 25. Timor-Tritsch IE, Monteagudo A. Unforeseen consequences of the increasing rate of cesarean deliveries: early placenta accreta and cesarean scar pregnancy. A review. Am J Obstet Gynecol 2012;207(1):14-29. https://doi.org/10.1016/j. ajog.2012.03.007 32. Pasquini L, Pontello V, Kumar S. Intracardiac injection of potassium chloride as method for feticide: experience from a single UK tertiary centre. BJOG 2008;115(4):528-31. https://doi. org/10.1111/j.1471-0528.2007.01639.x 26. Govender L, Moodley J. Late termination of pregnancy by intracardiac potassium chloride injection: 5 years’ experience at a tertiary referral centre. S Afr Med J 2013;103(1):47-51. https://doi.org/10.7196/SAMJ.6006 33. Bhide A, Sairam S, Hollis B, Thilaganathan B. Comparison of feticide carried out by cordocentesis versus cardiac puncture. Ultrasound Obstet Gynecol 2002;20(3):2302. https://doi.org/10.1046/j.14690705.2002.00797.x 27. Royal College of Obstetricians and Gynaecologists (1996) Termination of pregnancy for fetal abnormality in England, Wales and Scotland. London. RCOG Press. 34. Tufa TH, Prager S, Lavelanet AF, Kim C. Drugs used to induce fetal demise prior to abortion: a systematic review. Contracept X 2020;2:100046. https://doi.org/10.1016/j. conx.2020.100046 28. Westendorp AK, Miny P, Holzgreve W, De Wilde R, Aydinli K (1988) Selective fetocide by direct intracardiac injection of isotonic potassium chloride. Arch Gynecol Obstet 1988;244(1):59-62. https://doi.org/10.1007/ BF00931404 35. Sugawara J, Senoo M, Chisaka H, Yaegashi N, Okamura K (2005) Successful conservative treatment of a cesarean scar pregnancy with uterine artery embolisation. Tohoku J Exp Med 2069(3):261-5. https://doi.org/10.1620/ tjem.206.261 29. Singh S, Seligman NS, Jackson B, Berghella V. Fetal intracardiac potassium chloride injection to expedite second-trimester dilation and evacuation. Fetal Diagn Ther 2012;31(1):638. https://doi.org/10.1159/000333815 NJR VOL 13 No. 2 ISSUE 22 November, 2023 8 

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RESEARCH ARTICLE Motion prediction enables simulated MRimaging of freely moving model organisms Markus Reischl1☯, Mazin Jouda ID2☯, Neil MacKinnon2, Erwin Fuhrer ID2, Natalia Bakhtina ID2¤, Andreas Bartschat1, Ralf Mikut ID1, Jan G. Korvink ID2* 1 Institute for Automation and Applied Informatics, Karlsruhe Institute of Technology, Hermann-vonHelmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany, 2 Institute of Microstructure Technology, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯ These authors contributed equally to this work. ¤ Current address: ETH Zurich, Zurich, Switzerland * jan.korvink@kit.edu Abstract OPEN ACCESS Citation: Reischl M, Jouda M, MacKinnon N, Fuhrer E, Bakhtina N, Bartschat A, et al. (2019) Motion prediction enables simulated MR-imaging of freely moving model organisms. PLoS Comput Biol 15(12): e1006997. https://doi.org/10.1371/ journal.pcbi.1006997 Editor: Rahul Singh, San Francisco State University, UNITED STATES Received: March 30, 2019 Accepted: November 8, 2019 Published: December 19, 2019 Copyright: © 2019 Reischl et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: Support for the C. elegans Archive and for WormAtlas comes from NIH OD 010943 to D. H. Hall. MJ, NM, EF, NB and JGK acknowledge financial support from the European Research Council (ERC) under grant 290586 NMCEL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Magnetic resonance tomography typically applies the Fourier transform to k-space signals repeatedly acquired from a frequency encoded spatial region of interest, therefore requiring a stationary object during scanning. Any movement of the object results in phase errors in the recorded signal, leading to deformed images, phantoms, and artifacts, since the encoded information does not originate from the intended region of the object. However, if the type and magnitude of movement is known instantaneously, the scanner or the reconstruction algorithm could be adjusted to compensate for the movement, directly allowing high quality imaging with non-stationary objects. This would be an enormous boon to studies that tie cell metabolomics to spontaneous organism behaviour, eliminating the stress otherwise necessitated by restraining measures such as anesthesia or clamping. In the present theoretical study, we use a phantom of the animal model C. elegans to examine the feasibility to automatically predict its movement and position, and to evaluate the impact of movement prediction, within a sufficiently long time horizon, on image reconstruction. For this purpose, we use automated image processing to annotate body parts in freely moving C. elegans, and predict their path of movement. We further introduce an MRI simulation platform based on bright field videos of the moving worm, combined with a stack of high resolution transmission electron microscope (TEM) slice images as virtual high resolution phantoms. A phantom provides an indication of the spatial distribution of signal-generating nuclei on a particular imaging slice. We show that adjustment of the scanning to the predicted movements strongly reduces distortions in the resulting image, opening the door for implementation in a high-resolution NMR scanner. Author summary Magnetic resonance imaging (MRI) requires its subjects not to move, since movement will cause image artifacts. This is hard to achieve for adult humans, whom we can ask to PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1006997 December 19, 2019 1 / 16 Motion prediction enables simulated MR-imaging of freely moving model organisms Competing interests: The authors have declared that no competing interests exist. comply, but can currently only be achieved by sedation for other freely moving biological specimens. Because of the importance of non-invasive MRI as a technique to also capture metabolic information during activity, this is a huge deficiency of the methodology that is hampering progress. In our paper we ask the question whether it is possible to computationally combine optical information on specimen movement with MRI. Our approach is to predict the future movement and position of the specimen and thereby anticipate where it will be so as to specify correct MRI parameters. Our computer simulations show, for a freely moving worm, that a reasonable prediction is already possible for a short time window, and that we can control the amount of error of the resulting MRI image. Importantly, with the continuous speedup of computation, our simulations suggest that it is opportune now to implement such a system in hardware. Introduction A major challenge in biological science is to relate molecular regulation at the cellular level to response and behaviour at the organism level. Knowing this relationship lies at the foundation of every disease, and indeed also in understanding the healthy organism. An experiment establishing this relationship, as schematically shown in Fig 1, requires i) in vivo cellular-level detection of regulation-relevant molecules, such as metabolites and their production rates, ii) precise access to and application of biological perturbation mechanisms, and iii) an unburdened (naturally responding) organism. Clearly, even taken individually, these items are very hard to achieve in general, and correspond to major research areas in their own right. Fig 1. A freely moving organism is subject to a stimulus (mechanical, chemical, light, etc.) causing a response at all levels of detail (metabolomic, behaviour, etc.). The experiment designer relates the correlated output to a biological hypothesis and may adapt the stimulus. https://doi.org/10.1371/journal.pcbi.1006997.g001 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1006997 December 19, 2019 2 / 16 Motion prediction enables simulated MR-imaging of freely moving model organisms Small organisms such as C. elegans are biological model organisms useful for studying many human disorders, including neurodegenerative diseases [1, 2]. Model organisms have been the mainstay of biological sciences for decades, and thus a broad knowledge base already exists starting from the genome level, through developmental cycles, and up to behavioural response to applied stress. These platforms offer the opportunity to address the connection between molecular phenotype, which can be conveniently implemented due to the very rapid yet standardized life cycle of C. elegans, to behaviour. What remains is the technological challenge of satisfying the three requirements for robustly linking phenotype to behaviour. Nuclear magnetic resonance (NMR), which is a noninvasive and non-destructive technique, together with its imaging modality (MRI), is a strong candidate as the analytical method of choice, towards the ultimate goal of in vivo measurement of the molecular response. NMR is based on exciting the spin-active nuclei of the magnetized organism with radio frequency (RF) signals, and detecting their response via induced RF signals. Many nuclei are NMR sensitive, but in vivo molecular concentrations of metabolites are typically below millimolar levels, requiring high sensitivity to detect. NMR is a non-ionizing technique, making it superior to computed tomography (CT) based on X-rays. Although MRI microscopy currently only achieves spatial resolutions down to 4 μm, new techniques are partially overcoming these limits, such as the use of nitrogen vacancy centers in diamond, or the use of hyperpolarisation techniques. MRI is a Fourier imaging technique that uses magnetic field gradients to selectively excite parts of the object, and perform consecutive phase and frequency encoding of the excited spins. This so-called spatial encoding makes MRI a relatively slow imaging technique, as a single MRI image requires multiple consecutive signal acquisitions from the object that is being imaged. In conventional MRI, it is assumed that the organism (or object) being imaged is fixed in space, so that spatio-temporally varying magnetic fields are only due to the technical system. Any movement of the organism results in measurements from shifted spatio-temporal positions resulting in image artifacts and ensuing difficulty in data interpretation (see Fig 2). Motion-induced artifacts are the challenge since spatially localized spectroscopy (required for molecular profiling) necessarily requires repeated measurements to bring the signal level above the noise. In clinical applications of MRI, free body motion remains a challenge and there is a concerted effort underway to i) collect the MRI data faster than the motion; ii) collect the MRI data at moments when motion is minimal (triggering); iii) track patient motion and correct the MRI data during post processing; iv) track the motion and guide the spatial encoding to reflect the instantaneous geometrical configuration [3–6]; v) predict the motion and adjust the spatial encoding system in real-time, e.g., the prospective motion correction based on respiratory motion prediction [7]. Whilst these methods have been highly successful to control artefacts due to breathing, heartbeat, and low amplitude head movement, their assumptions for the kinematics of the underlying movement or the periodicity of motion are limiting. For example, the head is motion-captured in a model that assumes rigid six-degree-of-freedom body movement involving translations and rotations (x, y, z, θx, θy, θz) along the three orthogonal Cartesian axes. The direct translation of these techniques to MR measurements of small samples is not straightforward primarily because of the reduced size, more complex organism motion including writhing and wiggling, and rapid displacement across the detector’s sensitive region. Methods used to immobilise an organism can be considered to avoid these motion artifacts, such as clamping or freezing; however, such drastic measures typically introduce an undesired stress response into the molecular profile. The challenge of molecular measurement of non-stressed, small model organisms therefore still remains open. Given the advances in image processing, we believe there is an opportunity to address this challenge computationally. PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1006997 December 19, 2019 3 / 16 Motion prediction enables simulated MR-imaging of freely moving model organisms Fig 2. Concept of moving frame imaging: An object of interest is placed close to an NMR sensor (coil). For conventional cartesian acquisition of an MR image, N repetitions are required to fill the k-space matrix. The signal of one repetition is used to fill one k-space line. This acquisition scheme results in a total acquisition time of TR � N. The time scales of worm motion and repetition time TR are of the same magnitude, and with a standard Eulerian or fixed frame MR procedure, strong motion blurring occurs. To enable imaging free of blurring of a freely moving worm, the concept of a Lagrangian moving frame is required. https://doi.org/10.1371/journal.pcbi.1006997.g002 Advances in computer vision have revolutionized the speed and accuracy with which image analyses can be performed, and aim to reduce the need for expert knowledge e.g. in medical image analysis [8] and automated experiment handling [9]. These technologies are entering public awareness through the automation of highly complex processes, such as trajectory generation for self-driving vehicles (road, aerial) and surveillance for public safety. This is accomplished by real-time processing of dynamic images, in which accuracy and speed are of paramount importance. To name an example, advances in image and data processing algorithms are expected to make real-time dynamic spectrum imaging (achieving a hyperspectral imaging cube or hypercube) possible at all electromagnetic wavelengths. For instance, recording image and spectral data over 500 × 500 pixels, with a spectral resolution of 5nm over the visible spectrum, at 5 f s−1 has already been demonstrated [10]. Enormous data storage and processing power is required to perform such operations with sufficient spectral, spatial, and temporal resolution, and has motivated an increased effort in sparse imaging modalities. MRI also suffers from limitations in acquisition speed, which in this case are due to physical constraints such as the relaxation times. This renders MRI a relatively slow imaging technique PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1006997 December 19, 2019 4 / 16 Motion prediction enables simulated MR-imaging of freely moving model organisms compared to techniques that are based on the visual range of wavelengths. In MRI, data is acquired in ð~ k; tÞ-space (~ k being the spatial frequency), and the observer requires the transformed data in ð~ x ; tÞ space. Hence, a large number of acquired data is associated with each final image pixel or voxel, and motion or geometrical warping during imaging will introduce errors into the reconstruction algorithms. To obtain sufficient acquired signal power per voxel volume, object tracking must be implemented in order to ensure correct spatial and spectral co-localization over time. This is especially important in MRI microscopy, which depends on accumulated sampling for sufficient image resolution. With the novel capabilities of image and data processing algorithms, real-time image processing becomes feasible. This enables the extraction of information and motion prediction based on a conventional video stream which then can be used to steer the MR sequence in real-time. The prediction horizon will therefore be dependent on the signal acquisition time at a given frame rate and voxel spatial resolution. In this contribution we consider the preconditions for performing MRI experiments on unburdened small organisms. We will focus our attention on the nematode C. elegans, mainly because it fits in best with our own efforts towards in vivo metabolomic profiling, but we will address the underlying problem in more generality so that it is relevant also for other organisms. In this contribution we explore the possibility to completely remove the requirement of organism immobilisation, by providing the host observing technical system (e.g., a spectrometer or microscope) with a real time co-evolving Lagrangian coordinate system centered in the organism that provides the organism’s current center-of-gravity position and shape, paired with a robust prediction of the organism’s future position and deformed shape. There is a multitude of publications of so-called motion-trackers, which mainly quantify rigid body motions and ignore strain fields. Important ones are [11] which categorises behavior and morphology features out of C. elegans-videos by applying segmentation and tracking algorithms and [12], which applies methods of machine vision, data processing and tracking to evaluate drug assays and [13] to follow multiple worm within the same environment. Furthermore, motion decomposition methods using so-called Eigenworms apply methods from oscillation analysis to divide the worm motion into so called eigenmodes [14]. There are automated software-packages being able to quantitatively assess movement parameters (e.g. TrackA-Worm [15], Parallel Worm Tracker [16] etc.). However, there is a lack of an easy-to-use and sufficiently fast prediction algorithm for individually chosen positions within a single worm to be used to adjust the gradient system following a body part to be imaged. The prediction horizon available for the host system depends on the speed of image processing and latency of the capturing hardware (essentially the frame rate), as well as the smoothness of the organism’s motion. To gain insight into how movement prediction can enhance MRI signal detection, the paper introduces: • Data of a virtual phantom, combining high resolution electron microscope slice images [17] with conventional video-recordings of C. elegans moving in a Petri dish (Section Phantom Generation); • A new concept for location prediction in C. elegans, and detailed movement prediction, exploiting characteristic worm movements (Section MRI Simulation); • A computational platform, being able to evaluate the outcome of MR imaging with and without adapting the imaging parameters based on the movement prediction (Section MRI Simulation). PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1006997 December 19, 2019 5 / 16 Motion prediction enables simulated MR-imaging of freely moving model organisms • A measure for simulation success that compares prediction to the ‘true’ image through a similarity measure sxy, a technique first introduced in [18] and described in detail in the supporting information. Using this simulation model, we demonstrate the capabilities of the motion-prediction algorithm in MRI. Materials and methods Phantom generation The study is based on eight 10 s duration AVI-videos (sampling frequency 12 Hz) of C. elegans. The recording was done in a controlled environment with a fixed camera and constant illumination over time. The worm was enclosed in a technical setup including a microfluidic channel and a Petri dish of the host system, suitable for optical recording and other real-time measurements. Typically, if natural state studies are relevant, the organism can also be provided with an optically transparent nutritional substrate such as a gel containing E. coli bacteria. To simulate MR imaging of the moving worm, we artificially linked transmission electron microscope (TEM) images to the video of the worm, such that virtually scanning each location within the worm would deliver a simulated high-resolution MR-image. At each time-frame, the 1.2mm worm was segmented into 50 slices perpendicular to its center line. This resulted in a reasonable slice thickness of roughly 24μm. Subsequently, we assigned voxel MR signals adapted from the TEM images to each slice as follows: first, we removed the background from the TEM images and scaled them such that the dimensions of the body part in each slice were realistic. Second, we inverted the color map of the images, since the white regions of the TEM images corresponded to low density material, and therefore would appear dark in MR imaging due to the low proton signal intensity. This color inversion is just to make the sample images look like MR images. The reader should bear in mind that TEM and MRI use completely different imaging mechanisms, and therefore converting one to the other is not a straightforward task. In fact, having true MRI sample images is not essential to prove the efficiency of motion prediction in imaging enhancement, and indeed any arbitrary set of morphologically correct sample images would work. Finally, we reduced the number of pixels of the TEM images to 64 × 64, which corresponded to an MRI in-plane resolution of approximately 1.6 μm, and then assigned the voxel signals to the virtual MR slices. Although such a high volumetric resolution lies beyond the capabilities of currently available MRI scanners, it was chosen on purpose to allow more accurate assessment of the prediction algorithm. It is thus a good compromise between being close to a realistically achievable resolution, and being high enough for the structural similarity measure to work more accurately. Furthermore, considering the rapid advances in the field of MRI including higher fields, stronger gradients, and hyperpolarization techniques, the mentioned resolution of 1.6μm is believed to be achievable in the near future. Motion prediction New concept. In order to adjust the gradient system of the MRI, the worm needs to be detected and its future positions need to be predicted. Therefore, we introduced a new concept of real-time image processing of the worm, suitable for any video format, regardless of color model, and also independent of background structures. Fig 3a shows a block diagram of the basic steps of the concept. Starting with a raw image matrix Xraw, a robust estimation of the worm and its position Xworm within the video were determined. Based on the segmented worm, characteristic points such as center of gravity (COG) and position of the head region were determined. To introduce a coordinate system PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1006997 December 19, 2019 6 / 16 Motion prediction enables simulated MR-imaging of freely moving model organisms Fig 3. a: Concept for the prediction of defined locations within a worm, b: Parametrization of the worm, c-j: Snapshot of a sample video. c: grayscale image/video Xraw[k], d: Image with subtracted background, e: difference image, f: overlay with worm, g: skeletonization, h-j: Point of interest (POI, gray marker) and predictions for Δk = 30 (= 2.5 s) time samples (white marker). h-j: Time samples k = 50, k = 80, k = 110. Figures show the position every 30 samples and the prediction of the POI 30 samples ahead. Thus, the similarity of POI(80) vs. POIpred(80) and POI(110) vs. POIpred(110) shows the quality of the prediction. https://doi.org/10.1371/journal.pcbi.1006997.g003 along the center line of the worm, a skeletonization was applied, see Fig 3b. Assuming that body regions excluding the head would move along the center line, the worm velocity was used to predict positions xc,p of arbitrary selected points xc within the worm. A detailed description is given in the supporting information. Worm detection. This section briefly covers the worm detection and movement prediction. The preprocessing for the worm detection aims to reduce the computational complexity and consists of a grayscale conversion (standard Matlab conversion) of the videos as well as a decrease of the resolution by a factor of 9 (Fig 3c) (Decreasing the resolution proves real time processing to be possible / usage of low-end imaging hardware to be accurate (see Discussion)). For the segmentation of the worm, the background of the video is estimated and removed from the images (Fig 3d). The direction of motion is determined by relying on the position of the head and the COG. The COG is calculated based on the foreground pixels of the segmentation and is smoothed to obtain variations for future predictions (1st order low-pass filtering over time). The position of PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1006997 December 19, 2019 7 / 16 Motion prediction enables simulated MR-imaging of freely moving model organisms Fig 4. Evaluation (mean values and standard deviation) of the dataset given in section phantom generation. a: fixed location in the worm (s = 0.9), varying prediction horizons, b: fixed prediction horizon (Δk = 12, 1 second, varying locations, prediction horizon too high for s < 0.3). https://doi.org/10.1371/journal.pcbi.1006997.g004 the head is computed utilizing difference-images (Fig 3e) of the last 10 frames in the video. Fig 3f shows the detected head. Coordinate prediction. The position of the COG moves linearly and is predicted by linear extrapolation based on its past five positions. To predict the position of an arbitrary point of interest (POI) within the worm (which can easily be identified e.g. by a mouse click in the an NMR-simulation), we use the skeleton line as the center line of the segmentation and introduce a normalized coordinate s along it (head: s = 0, tail: s = 1, see Fig 3b and 3g). Assuming that each worm segment moves according to the current shape of the worm, following its predecessor segment with the velocity of the worm (The assumption is not perfectly valid, but it significantly simplifies processing and delivers reasonable results (see also Fig 4)), the velocity and the shape is used to predict the location sc of the POI after Δk time steps using sc = s − vΔk, (Fig 3h–3j) (If the prediction horizon is chosen too high, negative sc are avoided by setting the prediction to the topmost point (s = 0)). The prediction quality is evaluated using an Euclidean distance. Given the prediction horizon Δk, the predicted position based at time point k + Δk is calculated (x1,c,p[k + Δk], x2,c,p[k + Δk]) and the distance to the true position (x1,c[k + Δk], x2,c[k + Δk]) is measured. The average for all time samples is termed the mean prediction error. The dependency of the prediction accuracy to the prediction horizon, as well as to the predicted position of the worm, is given in Fig 4: The prediction error increases if the prediction horizon becomes larger. Regions near the head cannot be reliably predicted, since the head moves at a much higher frequency and in random patterns while scanning the surroundings and deciding on the moving direction. Furthermore, if the prediction horizon is set too high the predicted point lies outside the current shape of the worm (e.g. for Δk = 12 for all s < 0.3) (depending on the speed of the worm, however wild type worms mostly have the same velocity of approximately 0.13 mm s−1) and the topmost point (s = 0) is chosen as prediction, resulting in deviations). For all other regions, the uncertainty of the prediction stays roughly constant. Results MRI simulation Simulation-platform. The magnetic resonance imaging process is simulated using Matlab. The worm is assumed to reside within a strong and constant magnetic field B = (0, 0, Bz) of the MRI scanner. The software mimics a simplified yet acceptably accurate MR image PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1006997 December 19, 2019 8 / 16 Motion prediction enables simulated MR-imaging of freely moving model organisms acquisition process via a standard gradient-echo sequence [19] that takes place during worm motion as follows: • An image slice at a parametrised position s along the worm axis is defined as the image plane perpendicular to the worm axis. As previously mentioned, the worm is segmented into 50 slices whose voxels’ intensities are obtained from the TEM images. Thus s is the center of one of those slices. Ideally, to do MRI of a slice s, the center of the gradient system (the field of view FoV), GC, should be exactly at s over the entire signal acquisition process. This is unfortunately not the case as the prediction imposes some error resulting in GC being at another slice, at the desired slice but off-center, or both. In this case the MR excitation is done by calculating the signal intensity of each voxel in the FoV depending on the position of GC. If, for instance, GC is off-center, then some voxels of the true slice will be out of the FoV and the corresponding ones in the FoV will then be filled with zeros. The excitation step is followed by a phase encoding step, in which each voxel of the FoV is given a phase proportional to its position along one cross-sectional axis ξ. After a time delay TE/2, where TE (echo time) is the time from excitation to the center of the MR signal (echo), a frequency encoding gradient is applied, whereby each voxel of the FoV is assigned a frequency proportional to its position along an axis η perpendicular to the phase encoding axis (so that ξ � η = 0). The superposition of all voxel RF signals are simultaneously detected by a virtual coil. This so-called echo is recorded at instant TE, resulting in one line of k-space. The detection system is assumed to have a uniform spatial sensitivity. • After a time delay TR (repetition time), which is the time it takes to repeat the sequence in order to acquire a new line of the k-space, the algorithm calculates the anticipated new position and orientation of the selected slice (as the worm would have already moved to a new position). Now the slice at this new position is excited, phase encoded, and frequency encoded, resulting in a new line of k-space being filled. • The entire process is repeated until all the lines of k-space (in our case 64 lines) are complete. Once the imaging procedure is complete, the program reconstructs the anticipated MR image from the k-space data via Fourier transform. Algorithm 1 Gradient-echo MR imaging 1: procedure GRE(TE, TR) ⊳ Echo time and repetition time. 2: s = 0 ⊳ s parametrises slice position along worm axis. 3: while s 6¼ 1 do ⊳ cycle through the slices. 4: s s + δs ⊳ Next slice. 5: t = 0 ⊳ Initialize time counter. 6: N = 64 ⊳ Number of k-space lines. 7: k = −π ⊳ Set initial phase. 8: J = 1 ⊳ Set index. 9: while t 6¼ N � TR do 10: Pos. Pred. ⊳ Predict next position. 11: Mov. Grad. ⊳ Update imaging gradient. 12: Exc. ⊳ Excite slice. 13: t t + TE/2 ⊳ Advance time to half echo time. 14: Ph. Enc. (k + J � 2π/N) ⊳ Phase encode. 15: t t + TE/2 ⊳ Advance time to echo time. 16: Freq. Enc. ⊳ Frequency encode. 17: Echo ⊳ Signal acquisition. 18: t t + TR − TE ⊳ Advance time to next line. 19: J J + 1 20: end while PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1006997 December 19, 2019 9 / 16 Motion prediction enables simulated MR-imaging of freely moving model organisms 21: end while 22: end procedure The simulation is integrated into an interactive graphical user interface (GUI) to enable the user to set and change the imaging parameters easily and execute the processing without any programming skills (Fig 5). The simulated MRI signals can be chosen by the user and are either MR-like signals translated from real transmission electron microscopy (TEM) images, or any virtual MR-like images provided by the user. To start the simulation, an arbitrary slice of interest (with coordinate s) within the worm’s body is selected. For this point, the simulated MR image of a slice without movement is shown. In the display, the slice position s is denoted by a red line perpendicular to the center line of the worm, while the predicted position to which the virtual imaging gradient coordinate is set is denoted by a green line. Furthermore, the software allows the user to set the TR and TE imaging parameters, and to choose the prediction horizon (a value between 1 and 10 frames), which refers to the number of frames ahead for which the predicted worm position will be calculated. Our algorithm does not correct for the worm motion occurring during recording of a line of k-space. This is equivalent to the assumption that the MRI pulse sequence is based on very short echo times, which is possible, but taken at the expense of increased acquisition bandwidth and thus reduced signal-to-noise ratio (SNR). Simulation paradigms. We performed three MRI simulations using the optical video dataset from Section Phantom Generation to: Fig 5. Graphical user interface of a Matlab program that simulates the MR imaging experiment of a moving worm. The microscope movie of the worm is shown to the bottom left, and emulates real-time observation. The slice position is also shown. The top right shows the phantom MR image of the worm at the position of the slice. https://doi.org/10.1371/journal.pcbi.1006997.g005 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1006997 December 19, 2019 10 / 16 Motion prediction enables simulated MR-imaging of freely moving model organisms 1. confirm that the prediction enhances the MR imaging of the moving worm in general (Simulation 1); 2. measure the efficiency of the prediction algorithm as the desired resolution of the MR images increases in comparison to the resolution of the optical video used for the prediction (Simulation 2); and 3. measure the effect of an increased prediction horizon (the number of frames ahead for which the position is predicted, Simulation 3). In all the simulations, the repetition time (TR) was set to 83 ms, which corresponds to the frame rate of the optical video on which the prediction was based. We evaluate the simulation by quantifying the structural similarity sxy 2 f0; 1g 2 R between the true image and simulated image as introduced in [18]. Identical images return sxy = 1, whereas structural inequality delivers sxy = 0. Body position (Simulation 1). In Simulation 1, three slices (slice definition from Section Phantom Generation, first slice from the head (s = 0.02), second from the middle body (s = 0.5), third from the tail (s = 1) of the worm) are selected and an MR imaging simulation is performed (prediction horizon Δk = 1 (83 ms)). The results are illustrated in Fig 6a, each row shows the true slice on the left, the simulated MR image based on the prediction algorithm in the middle, and the simulated image when no position prediction is involved on the right. Clearly, the prediction algorithm significantly reduces the motion artifacts that would otherwise occur if the gradient system did not follow the worm as it moves. Moreover, we observe that prediction quality varies along the worm’s body. More specifically, the prediction performs better for the slices from the body and tail when compared with prediction of the head. This is axiomatic, since the worm rapidly moves its head laterally whilst scavenging for nutrition, thus the motional entropy of the head is higher (see also Fig 4). Fig 6b shows the simulation results of eight videos of different worms. The abscissa represents the slice position starting from the head (s = 0) to the tail (s = 1), while the ordinate shows the structural Fig 6. a: effect of position prediction on the quality of the MR images in the head (s = 0.02), body (s = 0.5) and tail (s = 1) regions. The prediction horizon in this case is 83 msec. b: dependence of the MR imaging quality (structural similarity) on the position of the slice. The figure displays the simulation results from eight videos of different worms. https://doi.org/10.1371/journal.pcbi.1006997.g006 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1006997 December 19, 2019 11 / 16 Motion prediction enables simulated MR-imaging of freely moving model organisms Fig 7. Effect of decreasing the ratio of the MR image resolution to the optical video resolution on the imaging quality for a prediction horizon of 1. The rows show the results for MR image:video resolution ratios of 4:1 and 2:1 respectively. Each row displays, from left to right, the reference slice, the MR image with position prediction, and the MR image without position prediction. https://doi.org/10.1371/journal.pcbi.1006997.g007 similarity between the prediction-based MR images and the original slices. To very good agreement with Figs 4 and 6b shows that the quality of the prediction-based MR images are higher for slices from the body and tail than the images taken near the head where the prediction uncertainty is usually higher. Image resolution (Simulation 2). In Simulation 2, a slice from the middle body section of the worm (s = 0.72) was selected and an MRI simulation was performed for different resolution ratios (= MR image: optical video). The idea here is to explore the effect of changing the MRI resolution (voxel size) on the quality of the prediction-based imaging. Imagine, for example, that one pixel of the optical video is 10 μm × 10 μm and the desired MRI isotropic resolution is also 10 μm × μm × 10 μm × 10 μm, then one pixel error in prediction results in one voxel error in the MRI k-space. On the other hand, if higher MRI resolution is wished, then one pixel error in prediction results in more than one voxel error in the MRI k-space. Decreasing the optical resolution can be used to speed up image processing, and thus decreasing the prediction horizon, if needed. The ratios of 4:1 and 2:1 are heuristically chosen, for which each pixel in the optical video respectively corresponds to 4 and 2 pixels in the MR image. Fig 7 demonstrates the results of this simulation—the rows correspond to the different resolution ratios while the columns depict the original slice, the image with prediction, and the image without prediction. The figure shows that the efficiency of the prediction declines as the desired resolution of the MR image increases (pixel size decreases), or alternatively, the optical resolution should be higher or at least equal to the desired MR resolution for a high quality prediction-based MR image. Indeed, the commercial optical imaging solutions can easily meet such demands of high resolution; however, upon implementation, any increase in number of pixels will be at the expense of the prolonged processing time needed for prediction. Nevertheless, this can be overcome by utilizing more powerful processors and by employing techniques such as parallel computing. PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1006997 December 19, 2019 12 / 16 Motion prediction enables simulated MR-imaging of freely moving model organisms Fig 8. Effect of enlarging the prediction horizon on the quality of the imaging (s = 0.52, see Fig 6). The images (1a-10a) show the reconstructed MR images based on the prediction algorithm for prediction horizons from 1 to 10 frames. The images (1b-10b) show the reconstructed MR images when no prediction is involved and when the initial position of the imaging gradient is set to the position of the slice after 1 (83 ms) to 10 (830 ms) frames respectively. (c) The similarity measure of the simulated image versus the prediction horizon. https://doi.org/10.1371/journal.pcbi.1006997.g008 Prediction horizon (Simulation 3). Simulation 3 varies prediction horizons for one slice (s = 0.52). Fig 8 illustrates the results of this simulation: Fig. (1a–10a) are the prediction-based simulated images for prediction horizons of Δk 1 to 10, respectively. In contrast, Fig. (1b–10b) display the results when prediction is not in action. Because only a few lines of the k-space are collected from the correct slice (depending on TR and the speed of the worm), the images in this figure exhibit a noticeable decrease in quality as the prediction horizon increases, leading to the conclusion that one should, whenever possible, minimize the prediction horizon. Of course, in an actual hardware realization of the system, the choice of the prediction horizon will be bounded by the speed of the image acquisition and processing units. The effect of increasing the prediction horizon is described quantitatively in Fig 8c by measuring the structural similarity between the original slice and the simulated image for both cases with and without prediction. The results confirm the variation in predictability along the length of the worm. The prediction horizon degradation is linear for the rear two-thirds of the worm, but falls off more rapidly for the head section, as expected from Fig 4. Moreover, a statistical assessment of the effect of the prediction horizon on the quality of the MR imaging was done: Regarding simulation results of four slices along the worm using the given eight videos it can be shown that the accuracy of prediction and thus the MRI quality decays with the increased prediction horizon but is in mean for all parameter combinations roughly three times better than without prediction. Discussion The results demonstrate that our prediction algorithm can markedly improve MR image quality of arbitrarily moving and deforming objects. The complete processing pipeline (including PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1006997 December 19, 2019 13 / 16 Motion prediction enables simulated MR-imaging of freely moving model organisms functions which will not be used in real-time processing, e.g. rotation, prediction of COG, bounding boxes etc.) takes 76 msec per frame on an average Laptop PC (core I7, fifth generation, 16 GB RAM) and has various possibilities for optimization (modern hardware, software parallelisation, hardware-filtering etc.). Decreasing the resolution of the optical video offers an additional option to reduce processing time up to a factor of 8 without significant loss of quality. Based on the current implementation, real-time processing is already possible for sample frequencies smaller than 13 Hz and application of the algorithm to a real MRI system is feasible. For the predictive information to be useful, the characteristic time (velocity) of sample motion tmotion and predictive accuracy needs to be determined. This can be done by recording a freely moving sample with the desired optical setup followed by an image analysis routine as described in this report. Models of the motion kinematics can be tested in order to maximize the prediction horizon given a user-specified predictive accuracy threshold. The threshold can be selected based on the expected error introduced within a single voxel with pre-selected dimensions. The predictive horizon together with the calculation time tpred then can be used to determine if the sample is ‘MR imageable’, i.e. by comparing the timescales to those required for MRI. For example, in this report it was observed that a prediction horizon of 83 ms requiring a calculation time of 76 ms yielded a predictive accuracy of approximately 57% as indicated by the structural similarity measure of the prediction-based MRI simulation, Fig 8c. On the other hand, a prediction horizon of 830 ms requiring a calculation time of 76 ms resulted in a prediction accuracy of approximately 30%. With the timescales of motion and predictive calculation defined, one must evaluate whether MRI is possible by comparing to the instrumental timescale (Only a standard cartesian MRI sequence is considered. The authors acknowledge the existence of more advanced sampling schemes, but these are outside the scope of the present discussion). The shortest relevant timescale is the time between spatial encoding steps, which in the case exemplified here is the repetition time TR (TR includes TE and the data acquisition time). It is during TR that prediction and hardware adjustments must be done prior to the subsequent spatial encoding step. In the MRI simulations described here, TR was 83 ms (including a TE of 4 ms) while tpred was 76 ms. Given the organism motion and hardware/experiment timescale regimes, one can estimate the potential for sample imaging with correction, summarized as follows: • tmotion < TR: the object is not MR imageable without motion artifacts. Conditions to slow the natural motion of the sample should be identified and implemented. • TR < tmotion, tpred: the object is MR imageable. Careful choice of TE and TR must be done so that the prediction calculation is complete before the next spatial encoding period. This places a restriction on the types of contrast that can be implemented. • tpred < TR < tmotion: the object is MR imageable. There is no restriction on the contrast weightings that can be implemented. To further improve the predictive quality, it is important to have kinematic models appropriate to the organism to be imaged. C. elegans is a convenient model for this reason given the advanced studies about behavioural phenotypes [11, 12, 20] and motion decomposition using so-called Eigenworms [14]. Extension of these models to organisms featuring similar motion characteristics should be straightforward (i.e. oscillation/undulation along the long axis— worms, snakes, swimming fish). As the kinematics becomes more complicated and/or sporadic, it will become necessary to introduce a method to guide the organism in order to introduce a predictive nature to its motion (food source, temperature gradient, etc.). Alternatively, PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1006997 December 19, 2019 14 / 16 Motion prediction enables simulated MR-imaging of freely moving model organisms shorter prediction horizons can be targeted together with faster MR imaging sequences such as the echo planar imaging (EPI), most likely at the expense of spatial resolution. To bring the presented method of in-situ real-time video capturing and motion prediction experiments for MR microscopy into daily routine, we so far lack available MR-compatible optical microscopes for high-field vertical bore magnets. However, there is strong progress to reach this technical integration in the near future. Initial results have already been presented in the early 2010s and a patent describing the adaptive objective to be integrated into an optical system has been granted in 2019 under the title “MR-compatible microscope, EP 2824471B1 (see e.g. https://worldwide.espacenet.com)”. Once the remaining hardware challenges are overcome the presented procedure for a tracking control loop will give the possibility to fully exploit and expedite these novel techniques for conducting MR microscopy of free-moving, undisturbed microscopic organisms. Supporting information S1 Text. This file describes the principal structure of the motion prediction algorithm. (PDF) S1 Fig. Structural similarity versus prediction horizon for slices (s = 0.1, s = 0.3, s = 0.5, and s = 0.9) for eight videos. (TIF) Acknowledgments We sincerely thank Dr. David H. Hall of Albert Einstein College of Medicine in New York for providing the high resolution images of C. elegans that we used as phantoms for high resolution NMR images. These lent our images more realism. The original images are from the MRC/LMB C. elegans Archive now curated by the Hall lab. These were generously donated by John White and Jonathan Hodgkin to the Hall lab. Author Contributions Conceptualization: Ralf Mikut, Jan G. Korvink. Data curation: Neil MacKinnon, Jan G. Korvink. Formal analysis: Mazin Jouda, Neil MacKinnon. Funding acquisition: Jan G. Korvink. Investigation: Markus Reischl, Mazin Jouda, Neil MacKinnon, Natalia Bakhtina, Andreas Bartschat. Methodology: Markus Reischl, Mazin Jouda, Neil MacKinnon, Erwin Fuhrer. Project administration: Neil MacKinnon, Ralf Mikut, Jan G. Korvink. Resources: Ralf Mikut. Software: Markus Reischl, Mazin Jouda, Natalia Bakhtina, Andreas Bartschat. Supervision: Ralf Mikut, Jan G. Korvink. Validation: Mazin Jouda, Natalia Bakhtina. Visualization: Markus Reischl, Mazin Jouda, Erwin Fuhrer, Andreas Bartschat. Writing – original draft: Markus Reischl, Ralf Mikut, Jan G. Korvink. PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1006997 December 19, 2019 15 / 16 Motion prediction enables simulated MR-imaging of freely moving model organisms Writing – review & editing: Markus Reischl, Mazin Jouda, Neil MacKinnon, Erwin Fuhrer, Ralf Mikut, Jan G. 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Handbook of MRI pulse sequences. Medical Physics. 2005; 32(5):1452–1452. https://doi.org/ 10.1118/1.1904597 20. Roussel N, Morton CA, Finger FP, Roysam B. A computational model for C. elegans locomotory behavior: application to multiworm tracking. IEEE Transactions on Biomedical Engineering. 2007; 54 (10):1786–1797. https://doi.org/10.1109/TBME.2007.894981 PMID: 17926677 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1006997 December 19, 2019 16 / 16 

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British Journal of Cancer (2000) 82(12), 1984–1990 © 2000 Cancer Research Campaign doi: 10.1054/ bjoc.2000.1163, available online at http://www.idealibrary.com on Enhancement of chemotherapy and radiotherapy of murine tumours by AQ4N, a bioreductively activated anti-tumour agent LH Patterson1, SR McKeown2, K Ruparelia1, JA Double3, MC Bibby3, S Cole4 and IJ Stratford4,5 1 School of Pharmacy and Pharmaceutical Sciences, De Montfort University, Leicester LE1 9BH, UK; 2School of Biomedical Sciences, University of Ulster, Jordanstown, Newtownabbey BT37 OQ, Northern Ireland; 3Clinical Oncology Unit, University of Bradford, Bradford BD7 1DP, UK; 4MRC Radiation and Genome Stability Unit, Harwell, Oxon OX11 ORD, UK; 5School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9PL, UK Summary AQ4 (1,4-Bis-{[2-(dimethylamino-N-oxide)ethyl]amino}5,8-dihydroxyanthracene-9, 10-dione) is a prodrug designed to be excluded from cell nuclei until bioreduced in hypoxic cells to AQ4, a DNA intercalator and topoisomerase II poison. Thus, AQ4N is a highly selective bioreductive drug that is activated in, and is preferentially toxic to, hypoxic cells in tumours. Five murine tumours (MAC16, MAC26, NT, SCCVII and RIF-1) have been used to investigate the anti-tumour effects of AQ4N. In only one tumour (MAC16) was AQ4N shown to be active as a single agent. However, when combined with methods to increase the hypoxic tumour fraction in RIF-1 (by physical clamping) and MAC26 tumours (using hydralazine) there was a substantial enhancement in anti-tumour effect. Notably, RIF-1 tumours treated with AQ4N (250 mg kg–1) followed 15 min later by physically occluding the blood supply to the tumour for 90 min, resulted in a 13-fold increase in growth delay. When combined with radiation or chemotherapy, AQ4N substantially increased the effectiveness of these modalities in a range of in vivo model systems. AQ4N potentiates the action of radiation in both a drug and radiation dose-dependent manner. Further the enhancement observed is schedule-independent with AQ4N giving similar effects when given at any time within 16 h before or after the radiation treatment. In combination with chemotherapy it is shown that AQ4N potentiates the activity of cyclophosphamide, cisplatin and thiotepa. Both the chemotherapeutic drugs and AQ4N are given at doses which individually are close to their estimated maximum tolerated dose (data not included) which provides indirect evidence that in the combination chemotherapy experiments there is some tumour selectivity in the enhanced action of the drugs. © 2000 Cancer Research Campaign Keywords: AQ4N; radiotherapy; bioreduction; cyclophosphamide; cisplatin; thiotepa Hypoxia occurs in regions of solid tumours as a result of poorly formed micro-vasculature coupled with the metabolic demand of tumour cells close to the available blood supply; this compromises the oxygen availability to more distant tumour cells (Brown and Giaccia, 1994). Hypoxic regions are known to be resistant to radiotherapy and conventional chemotherapy and this has led to much interest in the discovery of cytotoxins that kill hypoxic tumour cells (reviewed in Stratford and Workman, 1998). These agents are designed to be bioreduced selectively in cells low in oxygen to produce potent cytotoxins. Synthetic compounds with chemotherapeutic potential as bioreductive agents are usually based on nitroaromatic or quinone structures, e.g. nitroimidazoles and mitosenes (indoloquinones) (Workman and Stratford, 1993; Denny et al, 1996; Patterson and Raleigh, 1998). These agents are cytotoxic, on reduction, due to their ability to covalently bind to DNA that will not occur in oxygenated cells. A benzotriazene diN-oxide, tirapazamine, is currently the most promising bioreductive drug and is in phase II/III clinical trials. This agent can act as Received 1 October 1999 Revised 11 February 2000 Accepted 17 February 2000 Correspondence to: LH Patterson, The School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, UK 1984 a chemosensitizer and radiation enhancer due primarily to toxicity of the free radical species formed transiently during its reduction to the non-toxic reduction product SR4317. The toxic effect is probably caused by direct local damage to DNA that results in DNA strand-breaks and chromosome breaks that are difficult to repair (Brown and Wang, 1998). To date, therefore, bioreductive agents generate transient reactive species that are entirely dependent on hypoxia to generate the toxic species. In contrast, we report here preclinical data on a new class of bioreductive agent exemplified by AQ4N (1,4-bis{[2-(dimethylamino-N-oxide)ethyl]amino}5,8-dihydroxyanthracene-9, 10-dione). This agent is a prodrug designed to be excluded from cell nuclei (Patterson, 1993; Smith et al, 1997b) until metabolized in hypoxic cells to give AQ4, a stable, oxygeninsensitive metabolite (Smith et al, 1997a) (Figure 1). AQ4 is a DNA intercalator and potent inhibitor of DNA type II topoisomerase (Patterson, 1993; Patterson et al, 1994; Smith et al, 1997a). Previously we have shown that AQ4N is an effective enhancer of the anti-tumour effect of radiation in one murine tumour model, i.e. the T50/80 mammary carcinoma grown in BDF mice (McKeown et al, 1995, 1996). We now report on the efficacy of this drug in several murine models. These models were selected as they have previously been used successfully to show the efficacy of other bioreductive drugs when used in combination with radiation or chemotherapeutic agents (Stratford et al, 1989; Bremner et al, 1990; Bibby et al, 1989). Enhancement of chemotherapy and radiotherapy by AQ4N 1985 O OH O NH(CH2)2N(CH3)2 OH O NH(CH2)2N(CH3)2 +2e OH O NH(CH2)2N(CH3)2 AQ4N O OH O NH(CH2)2N(CH3)2 OH O NH(CH2)2N(CH3)2 +2e O OH NH(CH2)2N(CH3)2 AQM O AQ4 Figure 1 AQ4N and its reduction metabolities MATERIALS AND METHODS Materials AQ4N and 1,4-bis-{[2-(dimethylamino)ethyl]amino}5,8-dihydroxy-anthracene-9,10-dione (AQ4) were synthesized as described previously (Patterson et al, 1994). Cisplatin, cyclophosphamide, thiotepa and hydralazine were obtained from commercial sources. Materials for cell culture were from Flow Labs (Irvine, UK) and all other chemicals were used as purchased from Sigma (Poole, Dorset, UK). Murine tumour models MAC 16, MAC26, RIF-1, NT and SCCV11 tumour-bearing animals Pure strain NMRI male mice, aged 6–8 weeks were used for experiments with MAC16 and MAC26 tumours (Double and Ball, 1975; Bibby et al, 1987). Male C3H mice (8–12 weeks) were used for experiments using the RIF-1 and SCCVII tumours. NT tumours were grown in male CBA mice (Denekamp and Harris, 1975). RIF-1 and SCCVII tumours were maintained in mice by in vivo–in vitro passage as described previously by Twentyman et al (1980). Tumours were passaged using tumour brei from a donor mouse or by implantation of fragments. Tumours for experiments were implanted subcutaneously in the mid dorsal region of the back. All animal procedures were carried out under project licences issued by the Home Office, London, and UKCCCR guidelines (Workman et al, 1988) for the use of animals in experiments were adhered to throughout. Tumour systems and treatments Radiation treatments To allow local irradiation of tumours, unanaesthetized mice were gently restrained in polyvinyl jigs with lead shielding and a cut-away section over the tumour. Irradiations were done with 250 kV X-rays as described by Stratford et al (1989). In most experiments, single high doses of radiation were used (20 Gy for the SCCVII tumour; 25 Gy for the RIF-1 tumour, 12 Gy for the NT tumour). These doses were chosen to kill most of the oxic cells in these tumours, thereby creating tumours which contained predominantly radio-resistant hypoxic cells. This mode of treatment should reveal the cytotoxicity of bioreductive drugs selectively toxic to hypoxic cells when injected into mice after local irradiation of the tumours. Drug treatments AQ4N was usually given at its maximum tolerated single dose (MTD) which was 200–250 mg kg–1 (data not shown). However, in drug combination experiments, toxicity was observed with some chemotherapy agents, most notably cisplatin. The doses used were therefore at or close to the © 2000 Cancer Research Campaign MTD of the combination as determined from pilot experiments (data not shown). All drugs were dissolved in phosphate-buffered saline (PBS) and administered to mice by the intraperitoneal (i.p.) route in a 0.1 ml injection volume. Induction of hypoxia in tumours Two methods were employed to temporarily reduce the oxygen status of the tumours. The first of these was physically to occlude the blood supply to the tumours by applying a hinged, metal Dshaped clamp around the base of the tumour to occlude the vasculature for 90 min (Denekemp et al, 1983). The second was to treat mice with 10 mg kg–1 hydralazine. Both these methods can induce 100% radiobiological hypoxia in experimental tumours (Acker and Chaplin, 1987). In these experiments, mice were treated with AQ4N, then 15 min later, when it is presumed that tumour levels of drug will be high, the tumours were clamped or mice treated with hydralazine. Measurement of tumour response to treatment Tumour growth delay For NT tumours, treatment was initiated when the tumour reached 6.5–7.5 mm GMD (geometric mean of three orthogonal diameters). Tumours were measured three times weekly and the time taken to reach double its treatment volume was used as a measure of antitumour efficacy. For RIF-1 tumours, treatment was initiated when the tumours reached 100–200 mm3 and their growth followed until they had reached 4 × the treatment volume. MAC16 and MAC26 tumours were treated when the two longest orthogonal diameters were at least 4 and 5 mm. Tumour response was measured twice weekly by two-dimensional caliper measurements. For RIF-1, MAC16 and MAC26, tumour growth delay was calculated by subtracting the mean time to grow to 4 × treatment volume for control tumours from that obtained after drug exposure. In vivo–in vitro clonogenic assay C3H mice bearing SCCVII or RIF-1 tumours were treated upon reaching a volume of 100–200 mm3. After 24 h tumours were excised, minced and digested with an enzyme cocktail (6 mg DNase, 2 mg collagenase and 2 mg pronase per 10 ml PBS) to produce a single cell suspension. The cells were counted, diluted as necessary, plated and incubated at 37°C in 95% air/5% carbon dioxide for 12–14 days. Colonies were fixed, stained with 0.4% crystal violet in methanol and scored by eye. Surviving fraction (SF) was calculated as the number of colonies counted divided by the number of cells plated for a given treatment, divided by the same fraction determined for untreated control tumours. RESULTS Activity of AQ4N when combined with methods to induce hypoxia To investigate the effect of tumour hypoxia on AQ4N activity a physical clamp and a vascular ‘steal’ agent, hydralazine were used. Table 1 shows that AQ4N, clamping or hydralazine alone does not significantly delay tumour growth in RIF-1 or MAC26 tumours. However, making the tumours completely hypoxic for 90 min, using a physical clamp, after AQ4N administration does significantly delay tumour growth by 13-fold. Hydralazine, which British Journal of Cancer (2000) 82(12), 1984–1990 1986 LH Patterson et al Table 1 Activity of AQ4N in combination with hypoxia inducing protocols Treatment Tumour model Hypoxia AQ4N Clamp alone AQ4N + clamp AQ4N AQ4N + hydralazine Hydralazine Radation Radiation (25 Gy) Radiation (25 Gy) + AQ4N (AQ4N immediately after radiation) AQ4N Radiation (12Gy) AQ4N (AQ4N 30 min before radiation) + Radation (12 Gy) Growth delaya (days) n RIF-1 RIF-1 RIF-1 MAC26 MAC26 MAC26 0.6±0.5 1.1±0.9 14.1±1.7 0 5.5* 0 5 14 8 5 5 5 RIF-1 RIF-1 31.1±2.0 59.7±4.8 19 5 0.05±0.6 3.76±2.1 13.76±3.8** 8 8 8 NT NT NT Growth delay = time to reach 4 × tumour volume at treatment (treated group minus untreated group) for RIF -1 and MAC26; NT tumours were followed to 2 × treatment volume. AQ4N dosed i.p. MAC tumours: 50 mg kg–1; Hydralazine (10 mg kg–1) was given 10 min after AQ4N, RIF-1 and NT tumours: 200 mg kg–1; clamping for 90 min was carried out 15 min after AQ4N administration. See Materials and Methods for experimental details. Mann–Whitney U-test: * P < 0.05, **P < 0.01. Table 2 Activity of AQ4N in combination chemotherapy against murine tumours in vivo Treatment and Time schedule in minutes AQ4N Thiotepa AQ4N 1 min + thiotepa AQ4N CPM AQ4N 60 min + CPM AQ4N CPM AQ4N 60 min + CPM AQ4N Cisplatin AQ4N 30 min + cisplatin AQ4N Cisplatin AQ4N 30 min + cisplatin Drug dose (mg kg–1) 50 10 50/10 200 50 200/50 250 100 250/100 100 8 100/8 100 8 100/8 Tumour MAC16 MAC16 MAC1615.2** RIF-1 RIF-1 RIF-1 SCCVII SCCVII SCCVII RIF-1 RIF-1 RIf-1 SCCVII SCCVII SCCVII Growth delaya (days) 4.6 3.7 – 0.5 0.1 11.4*** – – – – – – – – – Surviving fractionsb – – – – – 1.4 0.0052 <0.00001 0.7 0.00023 <0.000001 0.77 0.0004 <0.000001 a Time to 4 × initial tumour volume (treated group minus untreated group). bSurviving fraction of clonogenic cells following treatment. All drugs were dosed i.p. See Materials and Methods for experimental details. CPM = cyclophosphamide. All results (n ≥ 5 per treatment group). Mann-Whitney U-test: *P < 0.05, **P < 0.01, ***P < 0.001. acts as a partial pharmacological clamp also, increases the antitumour effect of AQ4N in MAC26 tumours. Activity of AQ4N in combination chemotherapy Table 2 shows that when AQ4N (50 mg kg–1) or thiotepa (10 mg kg–1) alone were tested for anti-tumour efficacy against the MAC16 tumour only a small effect (approx. 4 days delay) on tumour growth was seen; combination of AQ4N and thiotepa at these concentrations significantly delayed tumour growth by over 15 days (P < 0.01). Table 2 also shows that against RIF-1 tumours, a combination of AQ4N (200 mg kg–1) and cyclophosphamide British Journal of Cancer (2000) 82(12), 1984–1990 (50 mg kg–1) significantly delays tumour growth whereas, as single agents, neither drug has a measurable effect. Similar results are seen with SCCVII where clonogenic cell survival was below the limit of detection following a combination of AQ4N (250 mg kg–1) and cyclophosphamide (100 mg kg–1). Scheduling of drug administration is important since tumour regression (in RIF-1) and clonogenic cell kill (in SCCVII) is greater when cyclophosphamide is administered 1 h prior to AQ4N compared to 1 h following AQ4N (results not shown). In both the RIF-1 and SCCVII models the antitumour effect of 8 mg kg–1 cisplatin was significantly enhanced by combination with 100 mg kg–1 AQ4N (Table 2). © 2000 Cancer Research Campaign Enhancement of chemotherapy and radiotherapy by AQ4N 1987 0.1 20 Gy only 0.01 Surviving fraction (SCCVII) Surviving fraction (SCCVII) 0.1 0.001 0.0001 0.00001 Limit of detection 0.000001 0 50 100 150 200 250 Dose of AQ4N (mg kg–1) after X-rays (20 Gy) Figure 2 The effect on surviving fraction of SCCVII tumours of various doses of AQ4N after 20 Gy of X-radiation. C3H mice bearing the SCCVII tumour were treated (five per group) with a range of AQ4N doses 30 min after exposure to 20 Gy X-radiation. Mice were sacrificed 24 h later, the tumour excised and the number of clonogenic cells determined as described in Materials and Methods. Results are given for individual tumours. Those which showed no clonogenic cells within the limits of detection of the assay are indicated with downward pointing arrows Surviving fraction (SCCVII) 10 0.01 20 Gy only 0.001 0.0001 0.00001 0.000001 –25 –20 –15 –10 –5 0 5 10 15 20 25 Time interval (h) between AQ4N (250 mg kg–1) and X-rays (20 Gy) Figure 4 The effect on surviving fraction of SCCVII tumours of dosing AQ4N at different times before or after X-radiation. C3H mice bearing the SCCVII tumour (at least three animals per treatment group) were irradiated with 20 Gy X-radiation. They were also administered with a single i.p. dose of AQ4N (250 mg kg–1) at known times intervals up to 24 h before (o) or after radiation (▼ ). Mice were sacrificed 24 h after each dosing interval, the tumour excised and the number of clonogenic cells determined as described in Materials and Methods. Results are given for individual tumours AQ4N only 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 0 5 10 15 20 25 30 X-rays dose (Gy) Figure 3 The effect on surviving fraction of SCCVII tumours of 250 mg kg–1 of AQ4N after various doses of X-radiation. C3H mice bearing the SCCVII tumour were treated with a range of X-radiation doses with (open circles) or without (closed squares) 250 mg kg–1 of AQ4N. Drug was administered 30 min after exposure to radiation. Mice were sacrificed 24 h later, the tumour excised and the number of clonogenic cells determined as described in Materials and Methods. Results are the means ± s.e.m. n = 5. Tumours which showed no clongenic cells within the limits of detection of the assay are indicated with downward pointing arrows AQ4N in combination with ionizing irradiation Previously we have shown (McKeown et al, 1995, 1996) that in the T50/80 tumour AQ4N enhances the effect of radiation. This was confirmed in the RIF-1 and NT tumour models (Table 1) and led to a more detailed study in the SCCVII model (Figures 2, 3 and 4). Figure 2 shows that AQ4N considerably enhances the clonogenic cell kill produced by a single dose of ionizing radiation (20 Gy) over a range of AQ4N concentrations (100–250 mg kg–1). In fact, AQ4N doses greater than 200 mg kg–1 in combination with ionizing radiation were so effective that the number of surviving clonogenic cells was below the limit of detection. At this dose of drug alone there was little, if any, measurable killing of tumour cells. We also investigated the effect of a range of radiation doses (0–30 Gy) immediately followed by a single dose of AQ4N (250 mg kg–1) in C3H mice bearing SCCVII tumours (Figure 3). There was a marked decrease in clonogenic cell survival as compared to irradiation alone especially at radiation doses above © 2000 Cancer Research Campaign 10 Gy. This will be because, at doses of 10 Gy and below, the response of the tumour will be dominated by the aerobic population of tumour cells, i.e. killing of the radiation-resistant hypoxic cells will only be revealed with single doses of radiation > 10 Gy. Since the level of tumour cell killing following 15 Gy plus AQ4N is only seen when radiation alone is given at 30 Gy, we can calculate a dose modification of 2.0 at this level of cell kill. The effect of administering AQ4N at various times before or after irradiation of SCCVII tumour-bearing mice was subsequently investigated (see Figure 4). This shows that i.p. injection of AQ4N (250 mg kg–1) up to 16 h before irradiation or up to 16 h following irradiation (20 Gy) produced a substantial increase in cell kill when these intervals were increased to 24 h the potentiating effect is lost. DISCUSSION The rationale underlying the development of AQ4N was to design a drug with potential for targeting DNA and DNA associated enzymes, but which was not active until metabolized in hypoxic tumour cells. The DNA affinity of several clinically useful agents, including the anthracyclines and mitoxantrone, is associated with a planar, electron-deficient chromophore bearing cationic amino side arm(s). It was proposed that an N-oxide of a tertiary aliphatic amine is electrically neutral and thus will exhibit a markedly decreased DNA binding affinity compared to the reduced cationic tertiary amine (Patterson, 1993). In fact previous studies have shown that AQ4 has excellent DNA binding affinity (Patterson et al, 1994) and is a DNA type II topoisomerase inhibitor (Smith et al, 1997a). In contrast AQ4N had a equilibrium binding to DNA which was too low to measure and AQ4N exhibited a 50-fold decrease in topoisomerase II inhibitory activity as compared to AQ4. AQ4N, in combination with hypoxia was a very poor cytotoxin against rodent and human cell lines whilst its reduction product, AQ4 is a potent cytotoxic agent (Smith et al, 1997a). This was confirmed in several other cell lines (Wilson et al, 1996). Previously using the T50/80 murine tumour model in vivo we have observed potent anti-tumour activity of AQ4N when used in British Journal of Cancer (2000) 82(12), 1984–1990 1988 LH Patterson et al combination with radiation (McKeown et al, 1995, 1996). For all bioreductive drugs to be useful clinically, tumour expression in vivo of appropriate reductases will be an important determinant of efficacy. Although not addressed in this study AQ4N is known to be reductively metabolized by haem containing enzymes including cytochrome P450s (CYPs). Specifically, AQ4N is a substrate for the CYP3A subfamily under hypoxic conditions (Raleigh et al, 1998) and CYP3A has been measured in RIF-1 and SCCVII tumours (Murray et al, 1998). CYP3A isoforms have been detected in a broad spectrum of human cancers including colon (Massaad et al, 1993; McKay et al, 1993) breast (Murray et al, 1993) and lung (Kivisto et al, 1995). Unlike tirapazamine it has not proved possible to metabolize AQ4N in cultured tumour cells, this is almost certainly due to the fact that cytochome P450s are known to be down-regulated ex vivo (Patterson et al, 1998). We have previously shown that the potential to metabolize AQ4N disappears within 24 h of excision in the T50/80 tumour (Hejmadi et al, 1996). The present studies were undertaken to further examine the potential of AQ4N to enhance radiation and chemotherapy in a number of in vivo murine tumours and hence support its use as a hypoxia activated bioreductive agent in the clinic. enhancement effects of other bioreductive agents, including tirapazamine (Kim and Brown, 1994; Siim et al, 1997) and E09 (Bibby et al, 1993) in these tumour types. MAC16 is an unusual model since it is poorly responsive to most conventional anticancer drugs (Bibby et al, 1988). This may be in part because MAC16 is a relatively slow growing tumour becoming necrotic as it grows suggesting the presence of a significant level of hypoxia. The significantly enhanced effect of thiotepa in combination with AQ4N suggests that this approach might have particular value in control of colon tumours. Standard chemotherapy drugs typically target the well-perfused and hence oxygenated tumour regions. The enhancement by AQ4N on the anti-tumour effects of these drugs may be explained by the existence in the hypoxic regions of AQ4 which would exert its cytotoxicity on cells as they attempt to re-enter the cell cycle, in response to the damage accrued by the well oxygenated cells. Recently, AQ4N over a range of concentrations has been shown to enhance the anti-tumour effects of cyclophosphamide in T50/80 implants. In particular, the anti-tumour effect of AQ4N (100 mg kg–1) combined with cyclophosphamide (100 mg kg–1) was shown to be equivalent to that produced by a single 200 mg kg–1 dose of cyclophosphamide alone, i.e. a 50% reduction in cyclophosphamide dose (McKeown et al, 1998). Enhancement of AQ4N metabolism in vivo by hypoxia Evidence that AQ4N is acting as a hypoxia-activated cytotoxin comes from the combination of this agent with clamping or hydralazine (Table 1). Clearly, the production of 100% hypoxia with a tumour clamp enhances the anti-tumour effect of AQ4N administered as a single agent and supports the contention that AQ4N can be bioreductively activated in vivo. Enhancement of the anti-tumour activity of bioreductive drugs with the vasoactive agent, hydralazine, has previously been shown with RSU 1069 (Acker and Chaplin, 1987; Stratford et al, 1997; Brown et al, 1987) and mitomycin-C (Adams et al, 1989; Quinn et al, 1992). It is known that the treatment of mice bearing the MAC26 colon tumour with hydralazine produces 80% shut down in functional tumour vasculature and greater than 60% reduction in tumour blood perfusion (Quinn et al, 1992). The present results show that hydralazine significantly enhances the anti-tumour efficacy of AQ4N again supporting the hypothesis that it is bioreductively activated in vivo despite poor activation in vitro. Our interpretation of the present data supports other published studies. Notably, AQ4N was active against the mouse mammary tumour MDAHMCa-4 when combined with the tumour blood flow inhibitor 5, 6dimethylxanthenone-4-acetic acid (DMXAA) (Wilson et al, 1996). In addition, the anti-tumour effect of AQ4N in vivo was potentiated in T50/80 tumours by combination with hypobaric hypoxia with a dose enhancement ratio of 5.1 (McKeown et al, 1995). Enhancement of chemotherapy by AQ4N From the results presented in Table 2 it can clearly be seen that AQ4N significantly enhances the effect of three standard chemotherapy agents (cisplatin, cyclophosphamide and thiotepa). This supports previous results that showed that AQ4N enhances the anti-tumour effect of cyclophosphamide and cisplatin in the T50/80 tumour (McKeown et al, 1998). The results with the RIF-1 and SCCVII model systems are consistent with the chemotherapeutic British Journal of Cancer (2000) 82(12), 1984–1990 Enhancement of radiation therapy by AQ4N Combination of AQ4N (200 mg kg–1) with ionizing radiation (25 Gy) slowed RIF-1 tumour growth by over 40% compared to radiation alone (Table 1). This encouraging result was explored more thoroughly using SCCVII implants, which showed that AQ4N, over a range of concentrations, greatly enhanced the positive effects of radiation (20 Gy). When AQ4N and radiation are administered at approximately the same time, a positive interaction can be explained on the basis that the two modalities are cytotoxic to different (i.e. oxic or hypoxic) cell populations. However, with AQ4N the observed enhancement is maintained even when drug is administered up to 16 h before radiation in the SCCVII tumour model (Figure 4); the stability and DNA affinity of AQ4 is likely to explain this phenomenon (Smith et al, 1997a; 1997b). Once produced, AQ4 will persist in the hypoxic cell fraction and kill any cell which attempts to re-enter the cell cycle following destruction of oxic cells by irradiation. When AQ4N was administered up to 16 h after irradiation, an enhancement was also observed. This suggests that formation of AQ4 in hypoxic cells that have not yet re-oxygenated, can still prevent them from repopulating the tumour. In T50/80 tumours a similar, although even longer, positive interaction time was observed with a 50% dosesparing effect reported for up to 4 days before and 6 h after irradiation (Hejmadi et al, 1996; McKeown et al, 1996). An enhanced anti-tumour activity post radiation was also observed in MDAHMca-4 tumours (Wilson et al, 1996). Overall this and previous studies are consistent with AQ4N acting as a bioreductive radiation enhancer. Differences in the time scales of the radiation enhancement by AQ4N in SCCVII compared to T50/80 tumours may be related to tumour clearance and/or host animal clearance of compound. The ability of cells to survive in the hypoxic compartment of tumours may also be a factor. Moore (1988) has reported that T50/80 tumours have a particularly high hypoxic fraction (60%) which suggests that T50/80 cells are capable of remaining viable, when hypoxic, for some considerable time; a © 2000 Cancer Research Campaign Enhancement of chemotherapy and radiotherapy by AQ4N 1989 factor which might also account for the very long times of interaction in this model previously observed (McKeown et al, 1995). In contrast, SCCVII tumours are significantly better oxygenated and have a faster doubling time than T50/80. This may explain the shorter time of maximal interaction, i.e. 16 h versus 96 h in the T50/80 tumor. The stable DNA-affinic agent AQ4, which is produced in the hypoxic cells, will compromise the cells in which it is generated for as long as they are viable. Once these cells die and become necrotic the cells, and the associated AQ4, are no longer capable of influencing tumour responses. This is likely to occur in fast growing tumours (e.g. SCCVII) more quickly than slow growing tumours especially those with a high proportion of hypoxic cells (e.g. T50/80). In conclusion AQ4N acts like a bioreductive drug when combined with hypoxia, chemotherapy or radiation treatment in vivo. The efficacy of the drug has now been shown against MAC16, MAC 26, NT, RIF-1 and SCCVII, T50/80 (McKeown et al, 1995, 1996) and MDAH-Mca-4 (Wilson et al, 1996). They all support the use of AQ4N as a bioreductive agent that has little or no intrinsic activity per se but is a very efficient enhancer of radiation as well as standard chemotherapy agents such as cyclophosphamide, cisplatin and thiotepa. An additional benefit of this bioreductively activated cytotoxin is the stable nature of the reduction product, AQ4, which may be capable of eliciting a bystander cell killing effect on proximate non-hypoxic tumour regions. In support of this confocal microscopy shows that viable cells preloaded with AQ4, the authentic reduction product of AQ4N liberate drug within 2 h despite the high DNA affinity of the latter (Smith et al, 1997b). REFERENCES Acker B and Chaplin D (1987) Potentiation of tumor-cytotoxicity of RSU-1069 by hydralazine: a new approach toward overcoming hypoxic radioresistance. Clin Invest Med 10: B114 Adams GE, Stratford IJ and Nethersell ABW (1989) Manipulation of the oxygenation of tumours: Activation of bioreductive drugs. Br J Radiol BIR Rep 19: 71–75 Bibby MC, Double JA, Ali SA, Fearon KCH, Brennan RA and Tisdale MJ (1987) Characterization of a transplantable adenocarcinoma of the mouse colon producing cachexia in recipient animals. J Natl Cancer Inst 78: 539–546 Bibby MC, Double JA and Loadman PM (1988) Unique chemosensitivity of MAC16 tumors to flavone acetic-acid (Lm975, Nsc-347512). Br J Cancer 58: 341–344 Bibby MC, Cronin BP and Phillips RM (1993) Evaluation of the cytotoxicity of the indoloquinone Eo9 in a human colon adenocarcinoma model. Intl J Oncol 3: 661–666 Bremner JCM, Startford IJ, Bowler J and Adams GE (1990) Bioreductive drugs and the selective induction of tumour hypoxia. Br J Cancer 61: 717–721 Brown JM and Giaccia AJ (1994) Tumor hypoxia – the picture has changed in the 1990s. Int J Radiat Biol 65: 95–102 Brown JM and Wang L.-H (1998) Tirapazamine: laboratory data relevant to clinical activity. Anti-Cancer Drug Design 13: 529–539 Brown JM, Zeman EM, Lemmon M and Hirst KV (1987) Selective antitumor therapy with a new class of bioreductive agents. Proc Am Assoc Cancer Res 28: 310 Denekamp J and Harris SR (1975) Tests of two electron affinic sensitizers in vivo using regrowth of an experimental carcinoma. Radiat Res 61: 191–197 Denekemp J, Hill S and Hobson B (1983) Vascular occlusion and tumour cell death. Eur J Cancer Clin Oncol 19: 271–279 Denny WA, Wilson WR and Hay MP (1996) Recent developments in the design of bioreductive drugs Br J Cancer 74: S32–S38 Double JA and Ball CR (1975) Chemotherapy of transplantable adenocarcinomas of the colon in mice. Cancer Chemother Rep 59: 1083–1089 Hejmadi MV, McKeown SR, Friery OP, McIntyre IA, Patterson LH and Hirst DG (1996) DNA damage following combination of radiation with the bioreductive drug AQ4N: Possible selective toxicity to oxic and hypoxic tumour cells. Br J Cancer 73: 499–505 © 2000 Cancer Research Campaign Kim IH and Brown JM (1994) Rexoygenation and rehypoxiation in the SCCVII mouse-tumor. Int J Radiat Oncol Biol Physics, 29: 493–497 Kivisto KT, Fritz P, Linder A, Friedel G, Beaune P and Kroemer HK (1995) Immunohistochemical localization of cytochrome-P450 3A in human pulmonary carcinomas and normal bronchial tissue. Histochem Cell Biol 103: 25–29 McKay JA, Murray GI, Weaver RJ, Ewen SWB, Melvin WT and Burke MD (1993) Xenobiotic-metabolizing enzyme expression in colonic neoplasia. Gut 34: 1234–1239 McKeown SR, Hejmadi MV, McIntyre IA, McAleer JJA and Patterson LH (1995) AQ4N – an alkylaminoanthraquinone N-oxide showing bioreductive potential and positive interaction with radiation in vivo. Br J Cancer 72: 76–81 McKeown SR, Friery OP, McIntyre IA, Hejmadi MV, Patterson LH and Hirst DG (1996) Evidence for a therapeutic gain when AQ4N or tirapazamine is combined with radiation. Br J Cancer 74: S39–S42 McKeown SR, Friery O, Gallagher R, Murray M, Clarke C, Patterson LH and Hirst DG (1998) Enhancement of cyclophosphamide anti-tumour activity with bioreductive drugs AQ4N and tirapazamine. Br J Cancer 78: 45 Massaad L, Dewaziers I, Ribrag V, Janot F, Morizet J, Beaune PH, Gouyette A and Chabot GG (1993) Screening of main drug-metabolizing-enzymes in mouse and human colon tumours. Bull Cancer 80: 397–407 Moore JV (1988) The dynamics of tumour cords in an irradiated mouse mammary carcinoma with a large hypoxic cell component. Jpn J Cancer Res (Gann) 79: 236–243 Murray GI, Weaver RJ, Paterson PJ, Ewen SWB, Melvin WT and Burke MD (1993) Expression of xenobiotic metabolizing enzymes in breast cancer. J Pathology 169: 347–353 Murray MM, Robson T, Gallagher R, Patterson LH, Hirst DG and McKeown SR (1998) Increased expression of cytochrome P450 isoforms in mouse tumours and their role in the enhanced activation of the bioreductive drug, AQ4N. 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Anti-Cancer Drug Design (in press) Quinn PKM, Bibby MC, Cox JA and Crawford SM (1992) The influence of hydralazine on the vasculature, blood perfusion and chemosensitivity of MAC tumors. Br J Cancer 66: 323–330 Raleigh SM, Wanogho E, McKeown SR, Burke MD and Patterson LH (1998) Involvement of human cytochromes P450 (CYP) in the reductive metabolism of AQ4N, a hypoxia activated anthraquinone di-N-Oxide prodrug. Int J Radiat Biol Chem Physics 42: 763–767 Siim BG, Menke DR, Dorie MJ and Brown JM (1997) Tirapazamine-induced cytotoxicity and DNA damage in transplanted tumors: relationship to tumor hypoxia. Cancer Res 57: 2922–2928 Smith PJ, Blunt NJ, Desnoyers R, Giles Y and Patterson LH (1997a) DNA topoisomerase II-dependent cytotoxicity of alkylaminoanthraquinones and their N-oxides. Cancer Chemother Pharmacol 39: 455–461 Smith PJ, Desnoyers R, Blunt N, Giles Y, Patterson LH and Watson JV (1997b) Flow cytometric analysis and confocal imaging of anticancer alkylaminoanthraquinones and their N-oxides in intact human cells using 647nm krypton laser excitation. Cytometry 27: 43–53 Stratford IJ and Workman P (1998) Bioreductive drugs into the next millenium. AntiCancer Drug Design 13: 519–528 Stratford IJ, Adams GE, Godden J, Howells N and Timpson N (1989) Induction of tumour hypoxia post-irradiation: A method for increasing the sensitizing efficiency of misonidazole and RSU 1069 in vivo. Int J Radiat Biol 55: 411–422 Stratford IJ, Godden J, Howells N, Embling P and Adams GE (1987) Manipulation of tumour oxygenation by hydralazine increases the potency of bioreductive radiosensitizers and enhances the effect of melphalan in experimental tumours. Radiat Res, 2: 737–742 British Journal of Cancer (2000) 82(12), 1984–1990 1990 LH Patterson et al Twentyman PR, Brown JM, Gray JW, Franko AJ, Scoles MA and Kallman RF (1980) A new mouse Tumor model system (RIF-1) for comparison of end-point studies. J Natl Cancer Inst 64: 595–604 Wilson WR, Denny WA, Pullen SM, Thompson KM, Li AE, Patterson LH and Lee HH (1996) Tertiary amine N-oxides as bioreductive drugs: DACA N-oxide, nitracrine N-oxide and AQ4N. Br J Cancer 74: S43–S47 British Journal of Cancer (2000) 82(12), 1984–1990 Workman P and Stratford IJ (1993) The experimental development of bioreductive drugs and their role in cancer therapy. Cancer Metastas Rev, 12: 73–82 Workman P, Balmain A, Hickman JA, McNally NJ, Mitchison NA, Pierrepoint CG, Raymond R, Rowlatt C, Stephens TC and Wallace J (1988) UKCCR guidelines for the welfare of animals in experimental neoplasia. Br J Cancer 58: 109 © 2000 Cancer Research Campaign 

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Close kin influence COVID-19 precautionary behaviors and vaccine acceptance of older individuals Bruno Arpino (  bruno.arpino@unifi.it ) University of Florence https://orcid.org/0000-0002-8374-3066 Valeria Bordone University of Vienna Giorgio Di Gessa University College London https://orcid.org/0000-0001-6154-1845 Article Keywords: Posted Date: June 2nd, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1699988/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/26 Abstract The family plays a central role in shaping health behaviors of its members through social control and support mechanisms. We investigate whether and to what extent close kin (i.e., partner and children) have mattered for older people in taking on precautionary behaviors (e.g., physical distancing) and vaccination during the COVID-19 pandemic in Europe. Drawing on data from the Survey of Health, Ageing and Retirement in Europe (SHARE), we combine its Corona Surveys (June-August 2020 and June-August 2021) with pre-COVID information (October 2019-March2020). We find that having close kin (especially a partner) is associated with a higher probability of both adopting precautionary behaviors and accepting a COVID-19 vaccine. Results are robust to controlling for other potential drivers of precautionary behaviors and vaccine acceptance, as well as to accounting for co-residence with kin. Our findings suggest that policy makers and practitioners may differently address kinless individuals when promoting public policy measures. Full Text In the early phases of the COVID-19 pandemic, individual precautionary behaviors were the only weapon to protect people from infection and reduce the spread of the virus in the community. Due to herd immunity remaining a distant target1 and given that COVID-19 vaccines neither permanently nor completely protect against infection2-4, precautionary health behaviors have remained crucial also after the launch of COVID-19 vaccination campaigns. Thus, governments across the globe have imposed or recommended behaviors such as physical distancing, mask-wearing, and frequent hand-washing. Although these have been presented as general guidelines for everyone, individuals at greater risk of developing a more severe case of COVID-19 when infected by the Coronavirus, such as older individuals, have been particularly encouraged to adopt precautionary health behaviors during all phases of the pandemic in order to limit admissions to Intensive Care Units and deaths from COVID-19-related conditions5-10. Monitoring and understanding compliance with COVID-19-preventive behaviors have thus become a prime target for research since the beginning of the pandemic11-18. More recently, the fight against the pandemic has entered a new stage with the approval by health authorities of effective COVID-19 vaccines. Although it is widely recognized that effective and equitable distribution of COVID-19 vaccines is a key policy priority19-20, ensuring their acceptance by the population is just as important. Thus, several studies have aimed at understanding the determinants of vaccine acceptance (i.e., actual vaccine intake and intention to be vaccinated)21-30. Despite the acknowledged importance of precautionary health behaviors and vaccines to limit the spread of the virus, compliance with guidelines and vaccine acceptance are anything but universal. Even among older adults, that are at the highest risk of COVID-19 complications, studies have shown that a large share of individuals does not follow the recommended precautionary behaviors11-12 and/or is unvaccinated and not willing to be vaccinated23-26. Socio-demographic characteristics of individuals such as gender Page 2/26 and education, and health conditions have been shown to be associated with both the adoption of precautionary health behaviors14-18 and vaccine acceptance27-30. For example, highly educated individuals and those in poorer health conditions were more likely to follow the guidelines and get vaccinated, while a gender “paradox” emerged31: women are more likely to adopt precautionary behaviors but less likely to accept COVID-19 vaccines. Other studies have considered the role of antiintellectualisms32, science skepticism33, information trust34 and misinformation35. We extend the existing work by analyzing whether having close kin (a partner and/or children) influences the adoption of precautionary health behaviors and vaccine acceptance. Indeed, social ties are known to influence health behaviors throughout the lifecourse36-38. In particular, partner and children tend to represent the most important social ties for older adults in terms of emotional closeness and intensity of support38-41. Therefore, numerous studies have investigated the role of having close kin (i.e., a partner and/or children) on health behaviors (see the reviews in [42-44]). The theoretical social-behavioral explanations of the importance of the family for health behaviors focus on the instrumental and emotional support that family members provide to each other complying with social norms of family obligations45-46. Family members complement thus the role of the health care system by providing material support, information, and motivation to prevent diseases and help adhering to medical treatments or recommendations47. The power of close kin to improve health is also explained by the social control function of family members, which exert pressures and control to inhibit unhealthy behaviors and to promote positive habits and lifestyles38,48,49. Social control affects health behaviors directly (through sanctions for deviant behaviors, regulation, and physical interventions) and indirectly (through internalization of norms of healthful behavior and facilitation of positive health behaviors)48. Partnership and parenthood, in particular, enhance a sense of obligation and greater self-regulation that discourage harmful behaviors and boost healthy ones48,50. Although under certain circumstances (e.g., family conflicts or multiple roles overload) kin may have a negative effect on health behaviors47,51, most studies have found positive effects38,43. Moreover, partnership tends to be more consistently beneficial for health and health behaviors as compared to parenthood48,52,53. The influence of family members on health behaviors is particularly strong when they live together48 and later in life54. In the context of the COVID-19 pandemic, the social control function of partners and children might have been particularly relevant to vehiculate information about the importance of adopting precautionary behaviors (e.g., wearing masks) and of vaccination. Similarly, children might have provided instrumental support to their older parents with (online and in-person) shopping in order to limit their in-person contacts. Thus, we should expect individuals with a partner and children to be more likely to adopt precautionary behaviors and (to be willing) to get vaccinated compared to their counterparts who lack these kin ties. Based on findings from the literature on family and health behaviors mentioned above, the effect should also be stronger for partnership than parenthood status. Page 3/26 We test the role of close kin in precautionary health behaviors and COVID-19 vaccine acceptance using large-scale representative data from the Survey of Health, Ageing and Retirement in Europe (SHARE), a survey on individuals aged 50 or more implemented in several European countries55. We combine data from the two SHARE Corona Surveys, administered in June-August 2020 and June-August 2021, with information from the latest pre-COVID wave (regular wave 8; October 2019-March 2020) from 27 countries. Our findings suggest that partnership and parenthood are positively associated with the likelihood of adopting precautionary health behaviors and to accept COVID-19 vaccine. The effect of having a partner is found to be stronger than that of having children. Our results urge policy makers and practitioners in the health sector to pay special attention to kinless individuals when designing interventions and recommendations related to precautionary health behaviors and vaccination. This research provides important insights to be better prepared for the next phases of the COVID-19 pandemic and in case of future pandemics. Results Having close kin and precautionary behaviors We first present the results based on the SHARE Corona Survey 1 (SCS1) which collected information on nine precautionary behaviors in June-August 2020. To ease interpretation of results, we present them graphically in Fig. 1 in terms of Average Marginal Effects (AMEs) with 95% confidence intervals obtained from fully-adjusted logistic regression models (see Methods for the socio-demographic and health variables we controlled for). The full table of regression estimates (log-odds) is reported in the Supplementary Materials (Table S.1). Results in Fig. 1 show that, overall, respondents who have close kin (partner or children) are more likely to adopt the suggested precautionary health behaviors against the spread of the virus compared to kinless older adults. As an example, compared to older people who do not have a partner, partnered older adults (independently of whether they have children or not) are about 6 percentage points more likely to use hand sanitizer or disinfection fluids more frequently than before the outbreak of the pandemic. The positive effect of kin is particularly evident for partnership: for most outcomes, having a partner and no children is more often associated with a higher probability of adopting precautionary behaviors than having children and no partner. In addition, the AMEs for those who have a partner and children are usually very similar and not statistically different to the AMEs for those who have a partner and no children. The only precautionary behavior where the combined effect of partnership and parenthood is both significantly and substantially higher than the effect of partnership alone is for reporting less shopping: partnered parents are about 5 percentage points more likely to report having left home for shopping less often or not at all since the outbreak of the pandemic than partnered respondents without children. Having close kin and vaccine acceptance Page 4/26 Next, we present results about vaccine acceptance based on SHARE Corona Survey 2 (SCS2; June-August 2021), the only SHARE survey where this information is available. Figure 2 presents estimated AMEs (with 95% confidence intervals) obtained from a fully-adjusted multinomial logistic regression (full regression estimates are available in Table S.2 of the Supplementary Materials). Figure 2 shows that the probability of being already vaccinated or planning to do so is about 5 percentage points higher for respondents who have a partner (independently of whether they have children or not). Correspondingly, older adults in a partnership are less likely of both being undecided about vaccination and of not intending to get vaccinated. Parenthood, instead, does not seem to play a role in vaccine acceptance. In fact, the AMEs of having children and no partner are very close to zero, and not statistically significant. In addition, the effect of partnership is neither substantially nor statistically modified by its combination with parenthood. Heterogeneity analyses Figures 3–8 present results obtained when interactions with gender, age groups, and country groups were considered to rule out the specificity of results for certain demographic or country groups (full regression estimates are available in Tables S.3-S.8 of the Supplementary Materials). Overall, results are very similar across gender (Figs. 3 and 4), age groups (Figs. 5 and 6) and country groups (Figs. 7 and 8), with statistically significant differences only observed in a bunch of cases, therefore confirming the importance of kinship (partnership, in particular) for precautionary behaviors and COVID-19 vaccine acceptance. Accounting for co-residence with kin The stronger effect on precautionary behaviors and vaccine acceptance found for partnership as compared to parenthood might be driven by typical living arrangements with different kin at older ages. In our sample, the vast majority (96.2%) of partnered older adults live with their partner. Instead, only 16.3% of older parents co-reside with at least one of their children. Thus, partners might be more likely to provide support and to exert control as compared to children simply because of the higher amount of time (and resources) shared. However, even analyses that account for living arrangements show that (co-residing) partners more clearly influence precautionary behaviors and vaccine acceptance compared to (coresiding) children (see Figure S.1 for precautionary behaviors and Figure S.2 for vaccine acceptance in the Supplementary Materials). Additional analyses As explained in the Methods section (see below), information on precautionary behaviors has been collected very differently in the second SHARE Corona Survey (SCS2) as compared to the first one. Nonetheless, analyses based on items in SCS2 yielded similar results to those based on items in SCS1: having close kin, and especially a partner, is associated with a higher probability of adopting precautionary behaviors (see Figures S.3 and S.4 in the Supplementary Material). Page 5/26 Finally, in additional preliminary analyses (available upon request) we also distinguished parents by their number of children but did not find this to matter. Also, given the slightly different sample size available for each outcome, we run the regression models selecting only observations available for all outcomes but results were barely affected. Discussion Precautionary behaviors have demonstrated efficacy at containing the spread of COVID-1956–58. Similarly, COVID-19 vaccines have been found to reduce the risk of infection, hospitalization, and death59–61. Thus, to slow the spread of the Coronavirus and limit its negative health consequences it is crucial to understand the factors associated with individuals’ adoption of precautionary behaviors and acceptance of COVID-19 vaccines. Our study focuses on the role of kin ties among older people, which the general (pre-COVID) literature on health behaviors found to be crucial for the adoption of healthy behaviors42–44,47−49,51−52. Our results show that having close kin is overall positively associated with older individuals’ likelihood of adopting precautionary behaviors and of being vaccinated or willing to get a COVID-19 vaccine. In particular, we find individuals in a partnership to be more likely to accept vaccine and to adopt (almost all) precautionary behaviors considered in this analysis. Results are robust to controlling for several other drivers of precautionary behaviors and vaccine acceptance (such as health and education), as well as to accounting for co-residence with kin. In addition, results are not specific for age, gender, or country groups. Most statistically significant associations are also substantially important. We find an adjusted difference in the probability of adoption of certain precautionary behavior (washing hand, using hand sanitizer, covering coughs and sneezes, reduced shopping) and of accepting COVID-19 vaccines of about 5 percentage points between partnered and unpartnered older adults, which is similar to the effect found in previous research for gender, health perception and chronic conditions11,15,18,30−31. Although our data do not include direct measures of social control, the positive effect of kin on older people’s adoption of precautionary behaviors and vaccine acceptance in the context of the COVID-19 pandemic is in line with predictions from the pre-COVID literature which finds ample evidence of positive effects of family social control on health behaviors, such as avoidance of alcohol and cigarette consumption38,62−64. Thus, it can be speculated that during a pandemic partners and children have an important role in encouraging and controlling the respect of public health measures and recommendations to reduce the risk of contagion and its negative health effects. Evidence in our study is also consistent with social support mechanisms identified in pre-COVID studies, showing that motivational and practical help from close kin may positively influence health behaviors38,54,65. In the context of a pandemic, partners and children may provide assistance and useful information to understand the importance of precautionary behaviors and vaccination. Practical help may also be a mechanism at work. Indeed, among the health behaviors analyzed, we find that having children is especially important for a specific outcome, i.e., limiting in-person shopping. Children, in this case, might Page 6/26 take the burden to go shop or order groceries online for their parents in order to reduce their risk of meeting strangers in a crowded indoor space, and therefore their risk of infection66–68. The generally stronger role that we find for partners compared to children in influencing precautionary behaviors and vaccine acceptance also fits with the predominant evidence in the general literature on family and health behaviors that reports larger associations with health behaviors of being in a partnership than of having children48,52−53. This is in part explained by the stronger and more effective social control received by partners38,63, and by their usually greater provision of emotional and practical support41,52,69. In addition, partners have been found to bilaterally influence each other’s behaviors, thus reinforcing the social support and control function of being in a partnership70. In addition, previous studies found that concerns about the possible consequences of COVID-19 for family members influences precautionary behavior and vaccine acceptance30. This mechanism might also contribute explaining the stronger effect we find for partnership than for parenthood: older individuals might be more concerned about reducing the risk of infecting their partner than their children, because partners are more likely to be older individuals with health pre-conditions. Our findings should be considered in light of some limitations. Our data could not account for the quality of relationships with partner and children for those individuals who have these ties. Previous research found that in case of conflicting relationships, family ties may also lead to health-compromising behaviors as coping mechanisms to deal with stress71. Also, the effectiveness of social control may vary with the type of behavior of the agent of the control72. Future research could examine more in detail possible heterogeneity in the role of kin ties in the context of the COVID-19 pandemic related to these and other factors (e.g., geographical distance to children and availability of friends). Also, an interesting avenue for future research is to examine the role of kin’s characteristics such as education and health. Finally, our results might be affected by differential response rates by family status during the pandemic. However, data quality controls showed that response rates remained satisfactory73. Despite these limitations, our findings shed some light on the complex role of kin ties during the COVID19 pandemic. It has been argued that family relationships (measured, for example, in terms of coresidence, frequent face-to-face contacts, etc.) may increase the chances of getting in contact with an infected person, thus constituting a risk to contract the virus. While it has been shown that conditional on having a (co-resident) family member infected the risk of getting the Coronavirus substantially increases74–75, the evidence on the (unconditional) risk of Coronavirus infection due to family ties per se is still scarce and, with few exceptions, is based on macro-level data. Also such macro-level analyses show mixed results76–82. A recent study83 based on part of the same individual-level data we used (SCS1) found that living with children was associated with a lower risk of Coronavirus infection for older women. Although it was not the focus on their study, the authors also found a similar effect for living with a partner for both men and women. These results are consistent with our findings of a positive association of close kin ties with precautionary behaviors and vaccine acceptance. Page 7/26 As Ross and colleagues52 wrote well before the onset of the COVID-19 pandemic, “a family is more than just a collection of people who might expose each other to infections and pollutants.” Thus, on the one hand, as all types of in-person contact, family contact can constitute a risk factor for Coronavirus infection. On the other hand, our study shows that partners, and to a lesser extent, children can also positively influence precautionary behaviors and vaccination. The overall effect of kin on risk of contagion and death is not easy to predict and it may vary with several factors, including extra-family (horizontal) relationships84 working status85 and age86 of family members. Our findings point to a potential positive role of kin in helping public health institutions to fight the pandemic and suggest that when analyzing the role of social relationships on COVID-19 outcomes rather than social network size per se one should account for (precautionary) behaviors and all types of contact (not limited to a specific type of ties, e.g., family) a person has. Understanding under which conditions social relationships may play a positive role in the context of a pandemic is of paramount importance and our study offers a new perspective and empirical evidence on this matter. Our findings that kin can have a positive influence on precautionary behaviors and vaccine acceptance suggest that policy makers and practitioners should focus especially on kinless individuals, especially those who are unpartnered, when designing measures to encourage the uptake and adherence to public health measures for COVID-19 prevention or in future pandemics. Methods Data The present study used data from the Survey of Health, Ageing and Retirement in Europe (SHARE)55. SHARE is a longitudinal survey on non-institutionalized individuals aged 50 + and their partners in 27 European countries and Israel. It is conducted biannually since 2004 and 9 waves of data have been collected till date. We use data from wave 8, which started in October 2019 but was suspended in all countries in March 2020 due to the COVID-19 outbreak. Regular data collection is based on computerassisted personal interviewing (CAPI), which provides pre-COVID information87. A special dataset, SHARE Corona Survey 188, was added to wave 8. This survey has been administered with CATI (computer assisted telephone interviewing) between June and August 2020 to collect information on individuals’ behaviors and conditions during the pandemic (SHARE Corona Survey 1; SCS1). We excluded observations from Portugal (because Portugal started the fieldwork of the regular wave 8 only a few weeks before the start of the first lockdown due to the pandemic), thus restricting the analyses to individuals from the 27 countries included in both regular and SCS1 data. We also use data from wave 9, i.e. SHARE Corona Survey 2 (SCS2)89 collected between June and August 2021. Our outcome variables (precautionary behaviors and vaccine acceptance) come from the two SHARE Corona Surveys; independent variables, instead, are measured from the pre-COVID wave 8 of SHARE because these variables not available in the Corona Surveys. We dropped individuals older than 85 because they constituted a small share of the sample (about 4%) with almost no variation for certain outcomes. Results were however not affected by this selection. After discarding observations with missing values on Page 8/26 the independent variables (640), our analytic samples comprise between 27,432 and 33,097 individuals depending on the outcome (the sample sizes differ because some outcomes do not apply to all respondents – those who declared who never left their home since the beginning of the pandemic – and because of missing values). Measures Using data from SCS1 we built nine outcome variables corresponding to nine different precautionary health behaviors. The questionnaire of the SCS1 is available at: http://www.share-project.org/datadocumentation/questionnaires/corona-questionnaire-1.html. All outcome variables are binary and coded so that 1 represents a precautionary behavior. More specifically the variables are constructed as follows (note that all of them refer to activities done or not since the outbreak of the pandemic and that italicized words refer to the names of the variables used in the models and reported in figures and tables). Washing hands equals one for respondents who report washing hands more than usual. Sanitizing hands equals one for respondents who report using special hand sanitizer or disinfection fluids more frequently than usual. Covering coughs and sneezes equals one for respondents who report paying special attention to covering cough and sneeze. Wearing masks equals one for respondents who report always wearing a face mask when in a public space. Keeping distance equals one for respondents who report always keeping distance from others in public. Less shopping equals one for respondents who report to have left their home for shopping less often or not at all since the outbreak of the pandemic. Less walks equals one for respondents who report to have left their home for going out for a walk less often or not at all since the outbreak of the pandemic. Less meetings equals one for respondents who report to have left their home for meeting with more than 5 people from outside their household less often or not at all since the outbreak of the pandemic. Less visits equals one for respondents who report to have left their home for visiting other family members less often or not at all since the outbreak of the pandemic. The SCS2 used a different questionnaire (available at: http://www.share-project.org/datadocumentation/questionnaires/corona-questionnaire-2.html), with questions which are not directly comparable with those in SCS1. Some of the questions about precautionary behaviors investigated in SCS1 were not kept in SCS2 (Washing hands; Sanitizing hands; Wearing masks; Less walks; Less visits); others changed the time reference (no longer ‘since the outbreak of the pandemic’ but either ‘in the three months preceding the survey’ or ‘compared to the first wave of the pandemic’); and there were some additional behaviors not included in SCS1 (such as going out to a restaurant). Therefore, these questions asked in SCS2 were only analyzed as robustness checks (and presented in Supplementary Files). Based on SCS2 items we built the following variables. Infrequent shopping equals one for respondents who report going out for shopping less often than once a week during the three months preceding the survey. Infrequent meetings equals one for respondents who report to have left their home for meeting with more than 5 people from outside their household less often than once a week during the three months preceding the survey. More covering of cough/sneeze equals one for respondents who report paying special attention to covering cough and sneeze more frequently. Keeping distance equals one for respondents who report to always pay special attention to keep distance from others in public during the Page 9/26 three months preceding the survey. Infrequent restaurants equals one for respondents who report going out to a restaurant less often than once a week during the three months preceding the survey. SCS2 additionally collected information in two consecutive steps on vaccination status and intent to get vaccinated. First, respondents were asked whether they had been vaccinated against COVID-19 at least once. Second, those who had not yet been vaccinated, were asked about their intention to do so, distinguishing whether they already had scheduled an appointment for vaccination, wanted to get vaccinated, did not want to get vaccinated, or were still undecided. We combined the information from these two questions and built a three-level categorical outcome variable: vaccinated or willing to get the vaccine (including vaccinated individuals and those who intend to get vaccinated); undecided; not willing to get the vaccine. The explanatory variable combined information on partnership and parenthood status: has a partner and children (respondents who are in a partnership and have at least one child); has a partner, no children (childless respondents with a partner); no partner, has children (unpartnered respondents, including widowed or divorced respondents, with at least one child); no partner, no children (respondents with no close kin – reference category in the models). In the main analyses we do not distinguish our main independent variable of interest according to living arrangements, i.e. we only account for having kin independently of whether respondents live with their partner or child(ren). In a robustness check we built a similar variable but considering kin availability only in case of co-residence (i.e. we dropped respondents who did not live with their partner or at least one child). The resulting variable had the following categories: has co-residing partner and children (respondents who live with their partner and with at least one child); has cores. partner, no children (childless respondents with a co-resident partner); no partner, has co-res. children (unpartnered respondents with at least one co-resident child); no partner, no children (respondents with no close kin – reference category). Control variables included the following: Age (in 5-year categories: 50–54 – reference category; 55–59; 60–64; 65–69; 70–74; 75–79; 80–85); Female (gender of respondent; male – reference category); Education (low – reference category; medium; high); Working status (retired – reference category; working; other); (Equivalized) Household income (continuous); Self-rated health (= 1 for respondents who rate their health as fair or poor; = 0 if health is rated as good, very good or excellent); Diagnosed illness (= 1 for respondents who self-reports of at least one doctor-diagnosed conditions including hypertension, diabetes, cancer, lung disease, heart disease, stroke and arthritis; = 0 otherwise); Gali (global activity limitations; = 1 for respondents whose activities are limited or severely limited because of health problems; = 0 otherwise); Respondent or close relatives tested positive (= 1 if the respondent or a close relative (partner, children, parents) has been tested for the Coronavirus and the result was positive; = 0 otherwise); Country of residence (reference category: Austria); Week of interview. As for education, the three groups are defined based on the International Standard Classification of Education (ISCED; http://www.uis.unesco.org/): low (ISCED 0–1, no or primary education, and ISCED 2, lower secondary education), medium (ISCED 3–4, higher secondary education), and high (ISCED 5–6, tertiary education). The independent variables have been measured using information from the regular SHARE wave 8 either Page 10/26 because they are time-invariant characteristics of because of unavailability of the information in the two SHARE Corona Surveys. Analyses Each of the precautionary health behavior described above represented a different binary outcome that we modeled using logistic regression. Thus, based on data from the SCS1 we estimated nine logistic regression models, one for each outcome. For vaccine acceptance, we use a multinomial logistic regression model. Although an ordering of the three categories of the outcome can be established (in terms of vaccine acceptance), a multinomial model allowed a higher degree of flexibility compared to an ordered logistic regression (i.e., it was possible to estimate separate effects of the independent variables for each category of the outcome). To ease interpretation of results, the main findings are reported in the main text graphically as Average Marginal Effects (AMEs) for the explanatory variable with 95% confidence intervals. Due to the categorical nature of our outcomes and explanatory variables, the AMEs are to be interpreted as the discrete effect of the independent variable (compared to the reference category – no partner, no children), i.e. as the difference between the predicted probabilities (in percentage points) across the groups being compared (e.g., has a partner and children vs no partner, no children). Full tables of regression estimates (estimated coefficients; log-odds) are reported in the Supplementary Materials. All control variables listed above have been included in all regression models. Among the additional analyses implemented, we considered heterogeneity analyses to rule out that the main findings only applied to certain demographic (gender and age) or country groups. More specifically, we have re-estimated the models that generated the main results by adding interactions between the explanatory variable and, in turn, gender, age (two groups: 50–64; 65+) and country groups. Countries have been grouped geographically: Northern/Central Europe (Denmark, Finland, France, Germany, Luxembourg, Netherlands, Sweden, Switzerland) and Southern/Eastern Europe (Bulgaria, Croatia, Cyprus, Czech Republic, Estonia, Greece, Hungary, Israel, Italy, Latvia, Lithuania, Malta, Poland, Romania, Slovakia, Slovenia, Spain). Although simple, this classification allows to capture considerable variation in family norms and similar grouping have been used in a previous study about the role of families on health and mortality90. Similarly to what we did for the main analyses, results are presented graphically, showing the estimated AMEs for the three categories of the explanatory variable corresponding to having close kin. However, this time we estimated separate AMEs for the two groups defined, in turn, by gender, age or country. In the few cases of a statistically significant difference (at the 5% level) between the AMEs this is indicated by an “x” used as a marker (the “x” is used for the highest AME among the two compared). Full tables of regression estimates (estimated coefficients; log-odds) are reported in the Supplementary Materials. Declarations Acknowledgments Page 11/26 This paper uses data from SHARE Wave 8 and 9 (DOIs: 10.6103/SHARE.w8.800, 10.6103/SHARE.w8ca.800, 10.6103/SHARE.w9ca800)), see Börsch-Supan et al. (2013) for methodological details. The SHARE data collection has been funded by the European Commission, DG RTD through FP5 (QLK6-CT-2001-00360), FP6 (SHARE-I3: RII-CT-2006-062193, COMPARE: CIT5-CT-2005028857, SHARELIFE: CIT4-CT-2006-028812), FP7 (SHARE-PREP: GA N°211909, SHARE-LEAP: GA N°227822, SHARE M4: GA N°261982, DASISH: GA N°283646) and Horizon 2020 (SHARE-DEV3: GA N°676536, SHARE-COHESION: GA N°870628, SERISS: GA N°654221, SSHOC: GA N°823782, SHARECOVID19: GA N°101015924) and by DG Employment, Social Affairs & Inclusion through VS 2015/0195, VS 2016/0135, VS 2018/0285, VS 2019/0332, and VS 2020/0313. 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The Lancet Healthy Longevity, 1(2), e80–e88. 86. Andrade, F. C. D., Quashie, N. T., & Schwartzman, L. F. (2022). Coresidence increases the risk of testing positive for COVID-19 among older Brazilians. BMC Geriatrics, 22(1), 105. Page 17/26 87. Börsch-Supan, A. (2022a). Survey of Health, Ageing and Retirement in Europe (SHARE) Wave 8. Release version: 8.0.0. SHARE-ERIC. Data set. DOI: 10.6103/SHARE.w8.800. 88. Börsch-Supan, A. (2022b). Survey of Health, Ageing and Retirement in Europe (SHARE) Wave 8. COVID-19 Survey 1. Release version: 8.0.0. SHARE-ERIC. Data set. DOI: 10.6103/SHARE.w8ca.800. 89. Börsch-Supan, A. (2022c). Survey of Health, Ageing and Retirement in Europe (SHARE) Wave 9. COVID-19 Survey 2. Release version: 8.0.0. SHARE-ERIC. Data set. DOI: 10.6103/SHARE.w9ca.800. 90. Zueras, P., Rutigliano, R., & Trias-Llimós, S. (2020). Marital status, living arrangements, and mortality in middle and older age in Europe. International journal of public health, 65(5), 627–636. Figures Figure 1 Having close kin (partner and children) and COVID-19 precautionary behaviors Page 18/26 Notes: The graph shows the effect of the explanatory variable (having kin) in the form of Average Marginal Effects (AMEs) with 95% confidence intervals from nine separate logistic regression models (one for each of the considered precautionary behaviors). Each AME compares the predicted probability of adopting a precautionary behavior for one of the three groups of older adults who have kin available (e.g., those who have both a partner and children) with the predicted probability of the outcome for the reference group (kinless, i.e. older adults who lack both partner and children). All control variables are included in the models. Full estimates are available in Table S.1 in the Supplementary Materials. Data are from SHARE Corona Survey 1 (June-August 2020). Figure 2 Having close kin (partner and children) and COVID-19 vaccine acceptance Notes: The graph shows results for the effect of the explanatory variable (having kin) in the form of Average Marginal Effects (AMEs) with 95% confidence intervals from a multinomial logistic regression model for the three-level categorical outcome vaccine acceptance. Each AME compares the predicted probability of a certain outcome category (e.g., being vaccinated or willing to get the vaccine) for one of Page 19/26 the three groups of older adults who have kin available (e.g., those who have both a partner and children) with the predicted probability for the reference group (kinless, i.e. older adults who lack both partner and children). All control variables are included in the models. Full estimates are available in Table S.2 in the Supplementary Materials. Data are from SHARE Corona Survey 2 (June-August 2021). Figure 3 Having close kin (partner and children) and COVID-19 precautionary behaviors by age group (65+ in blue; 50-64 in red) Notes: The graph shows the effect of the explanatory variable (having kin) in the form of Average Marginal Effects (AMEs) with 95% confidence intervals from nine separate logistic regression models (one for each of the considered precautionary behaviors). Each model includes an interaction between the explanatory variable and a dummy variable for age that distinguishes two groups. Thus, separate AMEs by age are obtained (65+ in blue; 50-64 in red). Each AME compares the predicted probability of adopting a precautionary behavior for each one of the three groups of older adults who have kin available (e.g., those who have both a partner and children) with that for the reference group (kinless, i.e. older Page 20/26 adults who lack both partner and children). Statistically significant differences (p<0.05) between the AMEs of the two considered age groups are indicated by an “x” in correspondence of the bigger AME. All control variables are included in the models. Full estimates are available in Table S.3 in the Supplementary Materials. Data are from SHARE Corona Survey 1 (June-August 2020). Figure 4 Having close kin (partner and children) and COVID-19 vaccine acceptance by age group (65+ in blue; 5064 in red) Notes: The graph shows results for the effect of the explanatory variable (having kin) in the form of Average Marginal Effects (AMEs) with 95% confidence intervals from a multinomial logistic regression model for the three-level categorical outcome vaccine acceptance. Each model includes an interaction between the explanatory variable and a dummy variable for age that distinguishes two groups. Thus, separate AMEs by age are obtained (65+ in blue; 50-64 in red). Each AME compares the predicted probability of a certain outcome category (e.g., being vaccinated or willing to get the vaccine) for each one of the three groups of older adults who have kin available (e.g., those who have both a partner and Page 21/26 children) with that for the reference group (kinless, i.e. older adults who lack both partner and children). Statistically significant differences (p<0.05) between the AMEs of the two considered age groups are indicated by an “x” in correspondence of the bigger AME. All control variables are included in the models. Full estimates are available in Table S.4 in the Supplementary Materials. Data are from SHARE Corona Survey 2 (June-August 2021). Figure 5 Having close kin (partner and children) and COVID-19 precautionary behaviors by gender (women in blue; men in red) Notes: The graph shows the effect of the explanatory variable (having kin) in the form of Average Marginal Effects (AMEs) with 95% confidence intervals from nine separate logistic regression models (one for each of the considered precautionary behaviors). Each model includes an interaction between the explanatory variable and a dummy variable for gender. Thus, separate AMEs by gender are obtained (women in blue; men in red). Each AME compares the predicted probability of adopting a precautionary behavior for each one of the three groups of older adults who have kin available (e.g., those who have Page 22/26 both a partner and children) with that for the reference group (kinless, i.e. older adults who lack both partner and children). Statistically significant differences (p<0.05) between the AMEs of the two genders are indicated by an “x” in correspondence of the bigger AME. All control variables are included in the models. Full estimates are available in Table S.5 in the Supplementary Materials. Data are from SHARE Corona Survey 1 (June-August 2020). Figure 6 Having close kin (partner and children) and COVID-19 vaccine acceptance by gender (women in blue; men in red) Notes: The graph shows results for the effect of the explanatory variable (having kin) in the form of Average Marginal Effects (AMEs) with 95% confidence intervals from a multinomial logistic regression model for the three-level categorical outcome vaccine acceptance. Each model includes an interaction between the explanatory variable and a dummy variable for gender. Thus, separate AMEs by gender are obtained (women in blue; men in red). Each AME compares the predicted probability of a certain outcome category (e.g., being vaccinated or willing to get the vaccine) for each one of the three groups of older Page 23/26 adults who have kin available (e.g., those who have both a partner and children) with that for the reference group (kinless, i.e. older adults who lack both partner and children). Statistically significant differences (p<0.05) between the AMEs of the two genders are indicated by an “x” in correspondence of the bigger AME. All control variables are included in the models. Full estimates are available in Table S.6 in the Supplementary Materials. Data are from SHARE Corona Survey 2 (June-August 2021). Figure 7 Having close kin (partner and children) and COVID-19 precautionary behaviors by country groups (SouthEast Europe in blue; North-West Europe in red) Notes: The graph shows the effect of the explanatory variable (having kin) in the form of Average Marginal Effects (AMEs) with 95% confidence intervals from nine separate logistic regression models (one for each of the considered precautionary behaviors). Each model includes an interaction between the explanatory variable and a dummy variable for country that distinguishes two groups (South-East Europe in blue; North-West Europe in red). Each AME compares the predicted probability of adopting a precautionary behavior for each one of the three groups of older adults who have kin available (e.g., those Page 24/26 who have both a partner and children) with that for the reference group (kinless, i.e. older adults who lack both partner and children). Statistically significant differences (p<0.05) between the AMEs of the two considered country groups are indicated by an “x” in correspondence of the bigger AME. All control variables are included in the models. Full estimates are available in Table S.7 in the Supplementary Materials. Data are from SHARE Corona Survey 1 (June-August 2020). Figure 8 Having close kin (partner and children) and COVID-19 vaccine acceptance by country groups (South-East Europe in blue; North-West Europe in red) Notes: The graph shows results for the effect of the explanatory variable (having kin) in the form of Average Marginal Effects (AMEs) with 95% confidence intervals from a multinomial logistic regression model for the three-level categorical outcome vaccine acceptance. Each model includes an interaction between the explanatory variable and a dummy variable for country that distinguishes two groups (South-East Europe in blue; North-West Europe in red). Each AME compares the predicted probability of a certain outcome category (e.g., being vaccinated or willing to get the vaccine) for each one of the three Page 25/26 groups of older adults who have kin available (e.g., those who have both a partner and children) with that for the reference group (kinless, i.e. older adults who lack both partner and children). Statistically significant differences (p<0.05) between the AMEs of the two considered country groups are indicated by an “x” in correspondence of the bigger AME. All control variables are included in the models. Full estimates are available in Table S.8 in the Supplementary Materials. Data are from SHARE Corona Survey 2 (June-August 2021). Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryMaterials.docx Page 26/26 

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Communicatio Socialis 34 (2001), Nr. 1: 99–106 Quelle: www.communicatio-socialis.de Frank Marcinkowski Autopoietische Systemvorstellungen in der Theorie der Massenmedien Vorschläge und Einwände Im Gefolge einiger Pionierarbeiten der frühen 90er Jahre (Spangenberg 1993, Marcinkowski 1993, Blöbaum 1994), in denen versucht worden war, Begriffe und Konzepte der neueren soziologischen Systemtheorie für die Theorie der Massenmedien fruchtbar zu machen, hat sich bis zum Ende des Jahrhunderts ein beachtlicher Fundus einschlägiger Diskussionsbeiträge angesammelt, unter denen die Reaktionen von Niklas Luhmann (1996; 1997, 1096-1109) besondere Aufmerksamkeit erregt haben. Ist bei Luhmann selbst durchaus Sympathie für die Idee der Konzeptualisierung von Massenmedien als autopoietischem Funktionssystem der modernen Gesellschaft erkennbar geworden, hat die - je nach Standort - gefeierte oder gefürchtete "Autopoietisierung" der Medien gerade innerhalb der Kommunikationswissenschaft nicht nur Epigonen, sondern auch vehemente Kritiker hervorgebracht und nicht zuletzt einer (Wieder-)Belebung handlungstheoretischer Konzepte der Journalismusforschung (Baum 1994, Neuherger 1996) Vorschub geleistet. Aber auch unter denen, die innerhalb des autopoietischen Paradigmas argumentieren, herrscht keineswegs Einigkeit in der Frage, wie das Mediensystem unter systemtheoretischen Vorzeichen im Detail beobachtet und beschrieben werden muss.l Die nachfolgenden Bemerkungen beziehen 1 Dem gewissermaßen vorgelagert ist die Frage, ob man das Konzept der Autopoiesis im fachfremdem - nämlich sozialwissenschaftliehen - Kontext überhaupt verwenden soll. Hierzu hat sich namentlich Humberto R. Maturana, der Erfinder des Begriffs, dezidiert geäußert. Maturana verwendet den Begriff der Autopoiesis ausschließlich zur Definition und Abgrenzung des Lebendigen. Soziologische Analogieschlüsse, wie sie Nildas Luhmann entwickelt hat, überzeugen ihn demgegenüber nicht. Maturana beharrt (als Nicht-Soziologe) vielmehr darauf, dass sich das Soziale durch andere Merkmale treffender charakterisieren lässt, als durch die Behauptung, alles Soziale ist im wesentlichen ein selbsterschaffendes Kommunikationsnetzwerk. Letztlich scheint ihm der Mensch, seine Einstellungen, Wünsche, Interessen und das daraus resultierende Handeln zur Charakterisierung des Sozialen unverzichtbar. Die Vorstellung einer Gemeinschaft (auch einer Theorie von Gemeinschaft). die das Individuum mundtot macht, ausschließt, negiert, scheint ihm vor dem Hintergrund autobiographischer Erfahrungen mit der Diktatur in seinem Heimatland unerträglich. Nüchtern betrachtet zielen diese Argumente allerdings auf den Entdeckungs- bzw. (potenziellen) Verwendungszusammenhang des Konzepts und scheinen mir insoweit im Begründungszusammenhang soziologischer Theorie von untergeordneter Bedeutung. https://doi.org/10.5771/0010-3497-2001-1-99, am 20.05.2024, 16:46:48 Open Access – - https://www.nomos-elibrary.de/agb 99 MARCINKOWSKI Stellung zu einigen der dabei sichtbar gewordenen Streitpunkte und sollen insoweit auch zur Präzisierung von Positionen beitragen, die der Verfasser in den vergangeneu Jahren wiederholt vorgetragen hat. Funktionsproblem Von grundlegender Bedeutung ist zunächst der Hinweis, das gesellschaftliche Funktionsproblem, mit dem auf die Ausdifferenzierung des Systems der Massenmedien reagiert wird, sei bisher noch nicht klar benannt. Träfe die Kritik zu, geriete die ganze Konstruktion ins Wanken. Tatsächlich lässt sich der Vorwurf bei genauem Hinsehen kaum halten. Sowohl Luhmann als auch Marcinkowski gehen bei ihren Überlegungen von der Riskanz und Anfälligkeit einer primär unter funktionalen Gesichtspunkten differenzierten Gesellschaft aus. Eine solche Formation, die ihren inneren Zusammenhalt nicht von einer hierarchischen Spitze aus beziehen kann, muss ständig um das Auseinanderdriften ihrer egoistischen Sonderperspektiven und Sondersprachen fürchten. Daran lässt sich die Überlegung knüpfen, dass gleichwohl in jedem Gesellschaftssystem Vorsorge dafür getroffen werden muss, dass sich gesellschaftliche Kommunikation auch thematisch auf sich selbst beziehen lässt, Gesellschaften also "imaginäre Konstruktionen der Einheit des Systems" (Luhmann 1997, 866) als Rahmenbedingungen der eigenen Kommunikation erzeugen und verwenden. Für diese Aufgabe, so lautet die Annahme, wird ein eigenständiges Funktionssystem ausdifferenziert, nämlich das System der Massenmedien. Sein Funktionsproblem ist die permanente Selbstbeobachtung und Anfertigung einer Selbstbeschreibung als (thematischer) Rahmen gesellschaftsintern möglicher Kommunikation. Sie macht unwahrscheinliche Kommunikation (und damit die Reproduktion der Gesellschaft) generell wahrscheinlicher, weil sie einen Entwurf von Welt allgemein zugänglich macht, der von jeder Kommunikation vorausgesetzt werden kann und nicht mitproduziert werden muss. Nun könnte man darüber streiten, ob eine solche Weltbeschreibung nicht auch in anderen Funktionsbereichen der Gesellschaft angefertigt wird und auf dieser Basis die Exklusivität des medialen Funktionsproblems bestreiten. Als Konkurrenten kämen in dem Falle etwa die Kunst oder die Wissenschaft in Frage und tatsächlich ist gar nicht auszuschließen, dass jede Gesellschaft eine Mehrzahl von Selbstbeschreibungen verfügbar hält. Die Besonderheit massenmedialer Konstruktion läge neben ihrer sachlichen Beliebigkeit und der Besonderheit der Themenbehandlung vor allem im ungeheuren Inklusionspotential der Massenmedien. Denn auf der Ebene der Beteiligung von Menschen an der Funktionserbringung https://doi.org/10.5771/0010-3497-2001-1-99, am 20.05.2024, 16:46:48 Open Access – - https://www.nomos-elibrary.de/agb 100 AUTOPOIETISCHE SYSTEMVORSTELLUNGEN IN DER THEORIE DER MASSENMEDIEN ist das Mediensystem das bei weitem inklusivste Sozialsystem moderner Gesellschaften und zweifellos inklusiver als Wissenschaft oder Kunst. Und schließlich kann die Argumentationskette noch einen Schritt weitergeführt werden: denn die Funktion der Massenmedien besteht nicht nur darin, dass sie eine Beschreibung der Gesellschaft verfügbar macht, sie besteht vor allem darin, dass sie diese Beschreibung beobachtbar macht. In den Medien kann man beobachten, wie man von anderen beobachtet wird. Medienrealität eröffnet damit allen Beobachtern in der Gesellschaft einen Zugang zum Modus der Beobachtung zweiter Ordnung und zur Reflexion. Reflexiv sind Systeme insoweit, als sie die Folgen ihres Operierens in der Umwelt und den Systemen ihrer Umwelt durch die Rückwirkungen auf sich selbst kontrollieren und als Konsequenz dieser Beobachtung die eigenen Möglichkeiten beschränken. Die Medienfunktion besteht folglich nicht darin, dass sie teilsystemische Abstimmung aktiv herstellt, wie die ältere Theorie dies noch fordert (,,Integrationsfunktion"), sondern darin, dass sie als Bedingung ihrer Möglichkeit fungiert, in dem sie den Aufbau von Reflexionskapazität bei den Teilen ermöglicht (vgl. dazu ausführlich Marcinkowski 2001). Systemgrenzen Ein zweiter Kritikpunkt knüpft unmittelbar an die Frage des Funktionsproblems an: die mangelnde Erkennbarkeit der Systemabgrenzung. Wenn man ein Funktionssystem als den Zusammenhang aller Kommunikationen bezeichnet, die sinnhaftauf ein spezifisches Bestandserfordernis der Gesellschaft bezogen sind, dann ist die Klärung der Funktion unabdingbare Voraussetzung der Grenzziehung. In dem Zusammenhang wird vermutet, gerade der Luhmannsche Entwurf würde zur näheren Bestimmung des Mediensystems zu stark auf die technischen Verbreitungsmedien zurückgreifen, die in ihm verwendet werden (vgl. etwa: Kohring 2000). Eine alternative Lesart besteht darin, die Medientechnik als eine Bedingung der Besonderung publizistischer Kommunikation zu verstehen, unter anderem, weil sie es erlauben, Themen nach eigenen Routinen auszuwählen und zu bearbeiten um sie anschließend einer spezifischen Karriere auszusetzen. Gerade weil technisch verbreitete Kommunikation nicht unmittelbar beantwortbar und insoweit keine Interaktion ist, 2 kann sie sich im Gegenteil aus den Interaktionskontexten des täglichen Le2 Wie in dem Zusammenhang die digitalen, "interaktiven" Online·Medien einzuschätzen sind, ist eine der vielen offenen Fragen, über die zu diskutieren wäre. https://doi.org/10.5771/0010-3497-2001-1-99, am 20.05.2024, 16:46:48 Open Access – - https://www.nomos-elibrary.de/agb 707 MARCINKOWSK/ bens lösen und eine besondere (und abgesonderte) Art der Codierung und Programmierung von Kommunikation praktizieren. Außerdem zwingt die Technik zu programmierter Selektion, denn durch die Entwicklung technischer Kommunikationsmittel potenzieren sich Kommunikationsmöglichkeiten und das führt zu einer noch schärferen Selektion dessen, was dann tatsächlich mitgeteilt werden kann, also zu eigenen Selektionskriterien. Und es ist diese Art der Codierung, die die Identität des Systems ausmacht, nicht etwa die Technik. Der Sinngehalt, den diese Form der Kommunikation mitführt, ist nicht der technische Akt der Publikation, sondern die Untersteilbarkeit des Bekanntseins bzw. das Bekanntsein des Bekanntseins von Themen und Beiträgen. Rein technisch gesehen erhöhen Verbreitungsmedien lediglich die soziale Reichweite von Kommunikation und steigern insoweit das Inklusionspotential des Systems. Zugleich wird aber Kommunikation anonymisiert. 3 Im Falle von Printmedien kann man nicht genau wissen, wer welche Texte wann gelesen hat, und im Hinblick auf elektronische Medien, wer wann was gesehen oder gehört hat. Das heißt aber, man kann zunächst gar nicht anders, als die Bekanntheit publizierter Sachverhalte zu unterstellen und dann beobachten, ob Kommunikation mit dieser Erwartung fortgesetzt werden kann. Und auf der anderen Seite kann man sich dem Druck kaum entziehen, über Themen zu reden, die allgemein als bekannt unterstellt werden. Man wird das auch dann versuchen, wenn man nur vage ahnt, wovon die Rede ist, und man wird versuchen, beim nächsten Mal besser vorbereitet zu sein. Um das auszudrücken, wurde auch vom symbolisch generalisierten Erfolgsmedium "Publizität" gesprochen. Exakt in diesem Sinne sind publizistische Systemgrenzen als Sinngrenzen etabliert. Autonomie vs. Abhängigkeit Schwer vermittelbar ist schließlich auch der Gedanke autonomer Verfügung über die eigenen Operationsgrundlagen in einem autopoietisch gedachten Mediensystem. Die klassische Theorie war es gewohnt, die Medien im Hinblick auf ihr Verhältnis zu den übergeordneten Bezugssytemen zu definieren, für das im Falle des (öffentlich-rechtlichen) Rundfunks an erster Stelle die Politik, im Falle des privatwirtschaftlich organisierten Printsektors zuvorderst die Ökonomie in Frage kamen. Auf der anderen Seite wurden dem gesamten Mediensystem, also auch seinen 3 Dies wiederum gilt für Online-Kommunikation in gleichem, wenn nicht in gesteigertem Maße. https://doi.org/10.5771/0010-3497-2001-1-99, am 20.05.2024, 16:46:48 Open Access – - https://www.nomos-elibrary.de/agb 102 AUTOPOIETISCHE SYSTEMVORSTELLUNGEN IN DER THEORIE DER MASSENMEDIEN kommerziellen Teilen, politische Funktionen zugeschrieben und mit der Zulassung privater Veranstalter wurden auch im Rundfunksystem Kriterien der Wirtschaftlichkeit implementiert. Das führte allerdings nicht zur Verunsicherung von derlei Dependenztheorien, sondern zu der heute weithin geteilten Annahme, der Rundfunk habe lediglich sein Referenzsystem gewechselt und werde nunmehr, genau wie die Printmedien auch, nicht mehr von der Politik, sondern von der Wirtschaft dominiert. Mit der Annahme einer "Kommerzialisierung" des gesamten Mediensystems wird diese Beobachtung auf den theoretischen Begriff gebracht. Systemisch gesprochen hätten wir es bei den Massenmedien insoweit lediglich mit einem Anwendungsfall von Wirtschaft zu tun. Demgegenüber hat eine autopoietische Medientheorie die These zu verteidigen, dass operativ geschlossene Kommunikationssysteme allenfalls wechselseitig resonanzfähig sind, aber keinesfalls extern beherrschbar. Unbefriedigend bleibt dabei zweifellos, dass mit Konzepten wie Irritation und Resonanz bisher lediglich hoch abstrakte Thesen über tatsächliche Wechselbeziehungen von publizistischem System und Umweltsystemen wie Wirtschaft oder Politik formulierbar sind, was die Erklärungs- und Prognosekraft des Ansatzes im Einzelfall erheblich einschränkt. Die relativ groben Analysen der Resonanzfähigkeit der Systeme füreinander verleiten dabei tendenziell zu reduktionistischen Annahmen über ihre wechselseitigen Einflusschancen, die dann nicht selten von drastischen Beispielen für Intersystemeffekte überrascht werden. Darüber hinaus wird die These von der operativen Schließung immer wieder in der Weise missverstanden, als verneine sie jegliche Kontaktmöglichkeit zwischen den Systemen. Dazu trägt auch der Umstand bei, dass das Konzept der strukturellen Kopplung von Systemen, mit dem an dieser Stelle gearbeitet werden müsste, nach wie vor als nicht hinreichend ausgearbeitet gilt, ein Mangel, der freilich nicht allein der systemischen Medientheorie anzulasten ist. Am intensivsten wurde bisher das Verhältnis von Mediensystem und Politik behandelt. Eine differenzierte Betrachtung der Publizistik/Politik-Beziehungen auf den Referenzebenen Interaktion, Organisation und Gesellschaft steht allerdings auch für diesen Fall aus. Erste Versuche bezogen sich vornehmlich auf die Makroebenen und nahmen insoweit die Frage einer spezifisch publizistischen Codierung in den Blick. Auch wenn hierüber keine Einigkeit unter den Beteiligten besteht, liegen doch immerhin diskutable Entwürfe vor. Danach orientieren die Massenmedien, wie alle anderen Funktionssysteme moderner Gesellschaft auch, ihre Umweltbeobachtung an einer exklusiven Sonderperspektive, nämlich an dem Gesichtspunkt ihrer unbedingten Präferenz für Information (Luhmann 1996, ebenso Blöbaum 1994) bzw. an der Akzeptanz gesellschaftshttps://doi.org/10.5771/0010-3497-2001-1-99, am 20.05.2024, 16:46:48 Open Access – - https://www.nomos-elibrary.de/agb 103 MARCINKOWSK/ weit unterstellbarer Bekanntheit von ausgewählten Sachverhalten als Themen publizistischer Kommunikation (Marcinkowski 1993). In beiden Fällen hat man es im Hinblick auf die Machtkommunikation von Politik mit deutlich unterscheidbaren Leitwerten zu tun. Auf der Ebene publizistischer Organisationen führt die Beobachtung systeminterner Differenzierung der Kriterienstruktur in Form dauerhaft verfestigter Programmbereiche zur Erklärung relativ enger Kopplungen von Massenmedien und Umweltsystemen, wobei Publizistik und Politik offensichtlich über den politischen Journalismus (bei Luhmann der Programmhereich "Nachrichten und Berichte") gekoppelt sind (vgl. Marcinkowski/Bruns 2000; Luhmann 1996, 53-82). Interaktionsbeziehungen zwischen Politikern und Journalisten wurden bisher nicht als Beispiel struktureller Kopplung angeführt. Offenbar sind Interaktionssysteme zu flüchtig, um dauerhafte, strukturell angelegte Beziehungen zu erklären. Der gelegentliche, meist buchstäblich "vorübergehende" Kontakt zwischen Journalisten und Politikern, so wichtig er im Einzelfall auch sein mag, scheint jedenfalls zu unbeständig, um Intersystembeziehungen darauf aufzubauen. Das hängt mit der hohen Störanfälligkeit des Anwesenheitsprinzips zusammen. Wenn Politiker verhindert sind oder Vertraulichkeit vereinbaren, können die Journalisten nur noch miteinander fachsimpeln. Die Bedeutung solcher Interaktionen, soweit sie denn zustande kommen, liegt darin, dass die Teilnehmer sich in der Interaktion aus den Rollenerwartungen lösen mögen, an die ihre Kommunikation innerhalb der Funktionssysteme gebunden wäre: Wenn der Journalist zusagt, nicht gleich zu veröffentlichen, mag er Mitteilungen erhalten, die der Position (Macht!) seines Gesprächspartners nicht unbedingt zuträglich sind. Das heißt, jenseits funktionaler Kommunikationsbezüge kann über andere Themen anders gesprochen werden. Damit baut die Interaktion freilich hohe Erwartungshaltungen auf, nämlich die, das genau das auch getan wird. Werden solche Erwartungen wiederholt enttäuscht, bricht die Interaktion auseinander. Insgesamt ist die Ablösung des Mediensystems von politischen Imperativen gerade vor dem Hintergrund medialer Expansion, Differenzierung und Komplexitätssteigerung auch empirisch relativ leichter vermittelbar. Schwieriger liegt der Fall mit der sogenannten "Kommerzialisierung", die einer medialen Verselbständigung so offensichtlich zu widersprechen scheint. Hier verführt die Bedeutung des Mediums Geld für Ausbildung und Bestand von Organisationen und die Bedeutung der Organisation für die Spezialisierung der Funktionssystems viele Beobachter dazu, alle zentralen Gesellschaftsbereiche (nach der CD U-Affäre unter Einschluss der Politik) als wirtschaftlich dominiert anzusehen. Unter systemtheoretischen Vorzeichen muss freilich zwischen dem "Erfolgsmedium" des https://doi.org/10.5771/0010-3497-2001-1-99, am 20.05.2024, 16:46:48 Open Access – - https://www.nomos-elibrary.de/agb 104 AUTOPOIETISCHE SYSTEMVORSTELLUNGEN IN DER THEORIE DER MASSENMEDIEN Funktionssystems und dem Medium Geld als Voraussetzung der Organisationsbildung im System unterschieden werden, um nicht weite Bereiche, darunter das System der Massenmedien umstandslos als Wirtschaft zu beschreiben. Das man eine Zeitung verkaufen muss, wenn man sie unterhalten will, ist selbstverständlich richtig und gilt im übrigen so lange, wie es überhaupt publizistische Medien gibt. Aber dieser Verkauf ist keine publizistische Operation, sondern eine ökonomische. So viel Differenzierungsvermögen muss man unterstellen können. Wer am Kiosk eine Zeitung aus dem Ständer nimmt und das passende Geldstück auf die Theke legt, der wird verstanden, auch wenn kein einziges Wort gefallen ist. Man kann insoweit von gelungener Kommunikation sprechen, aber einer Kommunikation, die ausschließlich der Autopoiesis der Wirtschaft nutzt und eben keine publizistische Kommunikation ist. Was die Zeitung mitteilt, was der Nutzer liest und welche Information er aus dem Gelesenen generiert, kann dem Kioskbesitzer im Prinzip egal sein, ebenso wie dem Verleger. Für den Leistungsbereich der Publizistik (Journalismus) ist die Information freilich wichtig, aber nicht unter ökonomischen Gesichtspunkten, sondern um zu sehen, woran man mit der nächsten Ausgabe erfolgreich anschließen kann. Das Medium Geld und der Zahlungsvorgang selbst liefert zu diesem Prozess jedenfalls keinen operativen Zugang. Natürlich mag es für Gründung und Unterhalt eines Mediums ökonomische Motive geben. "Neue Medien" entstehen, weil und solange man damit Geld verdienen kann. Aber dieses Motiv allein reicht keineswegs aus. Um gelungene oder gar erfolgreiche (folgenreiche) Kommunikation zu initiieren, sind journalistische Orientierungen unverzichtbar. Denn der erste Blick am Kiosk gilt nicht dem Preis einer Druckschrift. Man liest bestimmte Zeitungen und nicht andere aus Gründen, die die Kommunikation selbst mitliefert, nicht der Geld- bzw. Kostenmechanismus. Weder auf der Sender- noch auf der Empfängerseite dirigieren ökonomische Kriterien den Erfolg publizistischer Kommunikation. Ist der Journalismus insoweit autopoietisch? In der Form scheint mir die Frage falsch gestellt. Welche Einsichten resultieren, wenn man sich entschließt, Journalismus und Medien als autopoietisches Sozialsystem zu beschreiben? Die Frage gilt es zu beantworten. Und es ist dabei bewusst zu halten, dass und wie die Verhältnisse sich ändern, wenn man sie mit anderen Mitteln beobachtet. Es kann auch in der Medien- und Journalismustheorie nicht darum gehen, nicht-autopoietische Sichtweisen zu ersetzen oder gar zu eliminieren. Es geht um das Angebot einer "Sehhilfe", das nur dann angenommen zu werden verspricht, wenn es an undurchsichtigen Stellen mehr Klarheit zu schaffen vermag. Die bisherigen Erträge des systemtheoretischen Diskurses - so unvollständig sie noch lange Zeit bleiben werden - stimmen diesbezüglich optimistisch. https://doi.org/10.5771/0010-3497-2001-1-99, am 20.05.2024, 16:46:48 Open Access – - https://www.nomos-elibrary.de/agb 105 MARCINKOWSKI Literatur Baum, Achim, 1994: Journalistisches Handeln. Eine kommunikationstheoretisch begründete Kritik des Journalismus, Opladen. Blöbaum, Bemd, 1994: Journalismus als soziales System. Geschichte, Ausdifferenzierung und Verselbständigung. Opladen. Kohring, Matthias, 2000: Komplexität ernst nehmen. Grundlagen einer systemtheoretischen Joumalismustheorie, in: Löffelholz, Martin (Hrsg.), Theorien des Journalismus. Ein diskursives Handbuch, Opladen, S. 153-168. Luhmann, Niklas, 1996: Die Realität der Massenmedien. Opladen. Luhmann, Niklas, 1997: Die Gesellschaft der Gesellschaft, 2 Bde. Frankfurt/M. Marcinkowski, Frank 1993: Publizistik als autopoietisches System. Opladen Marcinkowski, Frank 2001: Massenmedien und die Integration differenzierter Gesellschaften: Was leistet Reflexion? In: Imhof, Kurt/Blum, Roger/Jarren, Otfried (Hrsg.): Medien und Integration. Mediensymposium Luzem, Bd. 7, Opladen (in Vorb.). Marcinkowski, Frank/Bruns, Thomas, 2000: Autopoiesis und strukturelle Kopplung. Inter-Relationen von Journalismus und Politik, in: Löffelholz, Martin (Hrsg.), Theorien des Journalismus. Ein diskursives Handbuch, Opladen, S. 209-223. Neuberger, Christoph, 1996: Journalismus als Problembearbeitung. Objektivität und Relevanz in der öffentlichen Kommunikation, Konstanz. Spangenberg, Peter, 1993: Stabilität und Entgrenzung von Wirklichkeiten. Systemtheoretische Überlegungen zu Funktion und Leistung der Massenmedien, in: Schmidt, Siegfried J. (Hrsg.): Literaturwissenschaft und Systemtheorie: Positionen, Kontroversen, Perspektiven, Opladen, S. 66-100. https://doi.org/10.5771/0010-3497-2001-1-99, am 20.05.2024, 16:46:48 Open Access – - https://www.nomos-elibrary.de/agb 

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www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 14), pp: 22825-22834 Research Paper Rottlerin as a novel chemotherapy agent for adrenocortical carcinoma Yi Zhu1,2, Minjie Wang3, Xu Zhao2,4, Lei Zhang2, Yigao Wu2, Bangqi Wang2, Weilie Hu2 1 Third Military Medical University, Chongqing, P.R. China 2 Guangzhou General Hospital of Guangzhou Military Command, Guangzhou, Guangdong, P.R. China 3 No. 422 Hospital of PLA, Zhanjiang, Guangdong, P.R. China 4 Southern Medical University, Guangzhou, Guangdong, P.R. China Correspondence to: Weilie Hu, email: huwl-mr@vip.sina.com Keywords: adrenocortical carcinoma, rottlerin, Wnt/β-catenin, anticancer agent Received: October 07, 2016    Accepted: January 24, 2017     Published: February 09, 2017 ABSTRACT Adrenocortical carcinoma (ACC) is a rare, but aggressive endocrine malignancy with a generally poor clinical outcome. There is no effective therapy for advanced and metastatic ACC. In our study, we found that an existing drug (rottlerin) exerted its tumour-suppressive function in ACC. Specifically, rottlerin inhibited cellular proliferation of ACC cell lines (NCI-H295R and SW-13) in a dose- and time-dependent manner. We also found that rottlerin induced cell apoptosis and promoted G0/G1 cell cycle arrest in ACC cell lines. The cellular migration and invasion of ACC cell lines were decreased after treatment with rottlerin. Further, the molecular expression of lipoprotein receptor related protein 6 (LRP6) and β-catenin were down-regulated in rottlerin-treated ACC cells, which indicated that Wnt/β-catenin signaling was involved in the tumour-suppressive function of rottlerin. To further confirm the anti-tumour function of rottlerin, a nude mouse ACC xenograft model was used. The xenograft growth curves and TUNEL assays demonstrated that rottlerin inhibited proliferation and induced apoptosis in the ACC xenograft model. Furthermore, we verified that rottlerin down-regulated the expression of LRP6 and β-catenin in vivo. The ACC cell line and xenograft mouse model data indicated that rottlerin significantly inhibited proliferation and induced apoptosis of ACC cells, likely via suppression of the Wnt/βcatenin signaling pathway. Our study indicated the potential therapeutic utility of rottlerin as a novel and potential chemotherapeutic agent for ACC. INTRODUCTION ACC. The combination of a cytotoxic drug and mitotane is recommended as first-line therapy in advanced ACCs, but the poor therapeutic efficiency (< 40%) and severe side effects limits clinical utility [4]. Therefore, new agents for ACC treatment are urgently required. Rottlerin(1-[6-[(3-acetyl-2,4,6-trihydroxy-5methylphenyl)methyl]-5,7-dihydroxy-2,2dimethyl-2H1-benzopyran-8yl]3-phenyl-2-Propen-1-one),also known as mallotoxin, is a natural plant polyphenol.Rottlerin is a traditional Indian medicine previously used as an antagonist of fertilization and treatment for cestode and trematode infections [5]. In recent years, rottlerin has been shown to regulate several cell processes, including cell circle regulation, cellular proliferation, apoptosis, Adrenocortical carcinoma (ACC) is a rare, but typically aggressive malignancy with an estimated annual incidence of 0.7–2.0 cases per million population. ACCs are highly aggressive with a poor prognosis and an overall survival rate at 5 years of only 25%–50% in most series [1, 2]. Until recently, radical surgical resection has been the only potentially curative option for ACC; however, approximately 40% of patients initially present with distant metastases. Even after seemingly complete removal of the tumor, recurrences occur in approximately 60%–80% of ACC patients [3]. Unfortunately, there is no effective therapy for patients with advanced or recurrent www.impactjournals.com/oncotarget 22825 Oncotarget autophagy, and migration [6]. Numerous studies have demonstrated that rottlerin plays an important role in antitumor activity in several cancers like prostate cancers and pancreatic cancer,and has great potential as a novel chemotherapeutic agent. Other studies have shown that rottlerin suppresses tumor cell survival in breast cancer, likely via suppression of the Wnt/β-catenin signal pathway [7]. Within the adrenal cortex, the Wnt/β-catenin cascade is involved in adrenal development and differentiation [8]. Indeed, abnormal functioning of the Wnt/β-catenin signal pathway may lead to adrenal tumourigenesis [9, 10]. In patients with ACC, β-catenin activation is associated with decreased overall survival. The Wnt/β-catenin pathway is expected to be an efficient targeting strategy for the treatment of ACC [11, 12]. Therefore, we sought to investigate the potential role and preliminary mechanisms of rottlerin in tumor cell proliferation, migration, and invasion in ACC cell lines and an ACC xenograft mouse model. In addition, we explored the potential therapeutic utility of rottlerin as a novel and potential chemotherapeutic agent for ACC. the rottlerin-treated group compared with the control and DMSO groups. After treatment with 2.5 μM rottlerin for 48 h, the percentage of apoptotic cells reached 13.0% in the NCI-H295R cells and 10.9% in the SW-13 cells. After treatment with 5 μM rottlerin for 48 h, the percentage of apoptotic cells reached 43.3% in the NCI-H295R cells and 38.3% in the SW-13 cells. The results suggest that rottlerin induces apoptosis in a dose-dependent manner. The results of AO/EB staining support these conclusions regarding morphology changes. The apoptosis rate increased in the rottlerin-treated group compared with the control and DMSO groups with AO/EB staining (Figure 2B, *p < 0.05). The apoptosis rate in the 5μM rottlerin-treated group was higher compared to the 2.5μM rottlerin-treated group (Figure 2B, *p < 0.05). Rottlerin caused cell cycle arrest of ACC cells in vitro Rottlerin inhibited cellular proliferation and may also be involved with cell cycle arrest. Therefore, we investigated the effect of rottlerin on the cell cycle of ACC cells using flow cytometry analyses. After treatment with 5 μM rottlerin for 48 h, G0/G1 cell cycle arrest occurred in ACC cells (Figure 1C). The percentage of cells in the G1 phase was 85.14% in NCI-H295R cells and 81.88% in SW-13 cells, which was significantly increased compared with the control group (Figure 1D, *p < 0.05). RESULTS Rottlerin inhibits ACC cell proliferation in vitro To investigate the effects of rottlerin on ACC, we first evaluated whether or not rottlerin decreases viability in ACC cell lines. NCI-H295R and SW-13 cells were treated with rottlerin at the indicated concentration for 24, 48, 72, 96, and 120 h. It was shown that rottlerin inhibited proliferation of ACC cells in a time- and dose-dependent manner (Figure 1A). At a concentration of 4 μM, rottlerin inhibited approximately 44.0% and 43.2% of NCI-H295R and SW-13 cells, respectively, after treatment for 48 h. In contrast, when exposed to 8μM rottlerin for 72 h, the inhibition ratio (1-viable cell%) was up to 70% in SW13 cells and 80% in NCI-H295R cells. The 5-day growth curves showed that the growth of rottlerin-treated cells was relatively slower than cells not treated with rottlerin in a dose-dependent manner (Figure 1B). The 48-h IC50 was 5.02 μM in NCI-H295R cells and 4.87 μM in SW-13 cells. Rottlerin inhibited cell invasion and migration of ACC cells Invasion and migration are related to the malignant features of ACCs [13]. To determine if rottlerin inhibits cell invasion and migration of ACC cells, we performed transwell invasion and migration assays. In the rottlerin treatment group, the invasion of ACC cells was impaired by rottlerin (5 μM); significantly fewer cells migrated to the lower side of the transwell chamber membrane than the control group (Figure 2C, *p < 0.05). The results of wound healing assay demonstrated that the migration rate of SW-13 cells was significantly reduced by rottlerin (5 μM) compared with the control group (Figure 3A). The 24 h migration rate was significantly decreased in SW-13 cells (Figure 3C, *p < 0.05). Rottlerin induced ACC cell apoptosis in vitro To further elucidate the mechanism by which rottlerin inhibits cellular growth in ACC cells, we determined whether or not rottlerin induces apoptosis in ACC cells. Flow cytometry was performed to show the apoptosis rate of ACC cells after treatment with rottlerin at the indicated concentration (5 μM [approximately the 48 h IC50] and 2.5 μM [approximately half of the 48 h IC50]). The percentage of apoptosis (early apoptosis plus late apoptosis) was identified in NCI-H295R and SW13 cells after rottlerin treatment (Figure 2A). The results showed an increase in the percentage of apoptotic cells in www.impactjournals.com/oncotarget Rottlerin inhibited tumor growth in vivo To investigate the effect of rottlerin on anti-tumor activities in vivo, we established a SW-13 xenograft tumor model in nude mice. With the administration of rottlerin for 4 weeks, tumor sizes and body weight were measured every 4 days. Assessment of tumor volume showed that the rottlerin-treated groups had delayed tumor growth compared to the control group and the group treated with DSMO (Figure 4A). There was no significant weight differences 22826 Oncotarget the control and DMSO-treated groups in vivo (Figure 5, *p < 0.05). Moreover, in the higher dose-treated group (rottlerin 4 mg/kg), the expression of β-catenin, LRP6, and p-LRP was lower than the lower dose-treated group (rottlerin 2 mg/kg). among the groups and there was no toxicity or side effects in the mice after 4 weeks of treatment (Figure 4B). Then, mice were sacrificed and the xenografts were extirpated. As shown in Figure 5, tumors were smaller in the experimental groups than the control and DSMO groups, and tumors were smaller in the rottlerin 4 mg/kg group than the rottlerin 2 mg/kg group. The mean volume of the tumors decreased from 3.23 cm3 (control) and 3.35 cm3 (DMSO) to 1.56 cm3 (rottlerin 4 mg/kg) and 1.94 cm3 (rottlerin 2 mg/kg); the difference was statistically significant (p < 0.05). TUNEL assays were performed to assess the anti-tumor activity of rottlerin in vivo (Figure 4C). The TUNEL assays indicated that the rottlerin-treated groups had higher apoptotic rates compared with the control and DMSO-treated groups. DISCUSSION There is no effective treatment for patients with locally advanced and metastatic ACC. Development of effective treatment or novel agents for patients with locally advanced and metastatic ACC is significant [14]. Rottlerin, which is extracted from Mallotus philippinensis, is a polyphenol that has been used as an ant-helminthic or a fertilization antagonist. Rottlerin has been used in humans for many years, has a good safety profile, and exhibits little toxicity, even after long-term exposure [5]. Recently, rottlerin has been shown to exhibit anti-neoplastic activity in several cancers, including breast, prostate, colorectal, and pancreatic cancers [7, 15, 16] by inhibiting various cellular pathways known to play important roles in carcinogenesis,like Wnt/β-catenin signaling pathway and mammalian target of rapamycin complex 1 (mTORC1) signaling.however, its antineoplastic activity in ACC has rarely been studied. Wnt/β-catenin signaling plays an important role in embryonic development and can lead to tumor formation when aberrantly activated [14, 17]. The Wnt pathway has been examined in relation to many cancers, such as leukemia, melanoma, lung cancer, and colorectal cancer Rottlerin suppressed the Wnt/β-catenin signaling pathway To determine if rottlerin suppresses the Wnt/βcatenin signaling pathway in ACC, and to elucidate the mechanism by which rottlerin mediates ACC cell apoptosis, some signaling molecules in the Wnt/β-catenin pathway were examined. Based on result of Western blotting, We can conclude that rottlerin reduced the expression of β-catenin, LRP6, and p-LRP in ACC cells in a dose-dependent manner (Figure 3B). Furthermore, we performed H&E (Figure 4D) and immunohistochemistry staining in xenograft tissues, and the results showed that rottlerin also down-regulated the expression of β-catenin, LRP6, and p-LRP compared with Figure 1: (A) Rottlerin both inhibited proliferation of ACC cells in a dose- and time-dependent manner. (B) Growth curve of NCI-H295R and SW-13 cells treated with rottlerin (0, 0.5, 1, 2, 4, and 8 μM) for 5 days. The growth of rottlerin–treated cells was relatively slower than untreated cells in a dose-dependent manner. (C) Rottlerin caused cell cycle arrest of ACC cells at the G0/G1 phase after treatment with 5 μM rottlerin for 48 h based on flow cytometric analysis. (D) Rottlerin caused cell cycle arrest of ACC cells at the G0/G1 phase compared with control (*p < 0.05). www.impactjournals.com/oncotarget 22827 Oncotarget [18]. Activation of the Wnt/β-catenin pathway occurs in ACC and is frequently caused by activating mutations of the gene encoding Wnt/β-catenin [18]. Therefore, blocking the Wnt/β-catenin pathway is expected to be an efficient targeting strategy for the treatment of ACCs [4]. Recent studies have reported that rottlerin blocks the Wnt/βcatenin signaling pathway in some cancers, including colorectal, prostate, and breast cancers [7]. In this study, we examined the effect of rottlerin on ACC cells. The results showed that rottlerin inhibited proliferation, induced apoptosis and G0/G1 cell cycle arrest, and decreased invasion and mobility of ACC cell lines. Furthermore, the results of animal experiments showed that rottlerin inhibited tumor growth in vivo, and there was no obvious toxic effect in nude mice. The results showed that rottlerin has anti-neoplastic activity in ACC, and could serve as a novel agent for the treatment of ACC. We then turned to study the mechanism underlying the antineoplastic activity of rotterlin against ACC. We detected the expression of several key components of the Wnt/β-catenin signaling pathway in ACC cell lines and xenografts of nude mice treated with rottlerin. Based on Western blot and immunohistochemistry staining, the expression of β-catenin, LRP6, and p-LRP6 was downregulated after treatment with rottlerin. We deduced that rottlerin plays a role as an anti-tumor agent in ACC related to Wnt/β-catenin signaling pathway,likely via suppression of the Wnt/β-catenin signaling pathway. β-catenin, which is encoded by the CTNNB1 gene, is a key component of this signaling pathway and has multiple functions, including mediation of cell adhesion and signal transduction [19, 20]. β-catenin combines with a variety of proteins to regulate cell proliferation and differentiation, which is critical for embryonic development and tumorigenesis [21]. In ACC, accumulation of β-catenin has frequently been noted, indicating activation of the Wnt signaling pathway. Mutations in β-catenin have been associated with a poor prognosis in patients with ACC, and higher-grade ACC is associated with higher β-catenin expression [22]. In our study,β-catenin was down-regulated by rottlerin in ACC cells, both in vitro and in vivo. LRP6, an indispensable co-receptor for the Wnt signaling pathway, is a member of the low-density lipoprotein receptor family, which interacts with the seven transmembrane receptor of the Frizzled (Fzd) Figure 2: (A) Flow cytometric analysis of annexin-V/PI staining of NCI-H295R and SW-13 cells. Cells were treated with rottlerin at the indicated concentration (2.5 μM or 5 μM) for 48 h. The dual parameter dot plots combining annexin-V-fluorescein isothiocyanate (FITC) and PI fluorescence show the viable cell population in the lower left quadrant (annexin-V2, PI2), apoptotic cells in the lower right quadrant (annexin-V+, PI2) and upper right quadrant (annexin V+, PI+), and necrotic cells in the upper left quadrant (annexin-V2, PI+). (B) Morphologic changes of ACC cells based on AO/EB staining. After treatment with rottlerin (2.5 μM or 5 μM) for 48 h, apoptosis was observed using a fluorescence microscope.In order to indicate ,the blue arrow point to a normal cell, the red arrow point to an early apoptotic cell, and the yellow arrow point to a late apoptotic cell. The rate of apoptosis in the rotterlin-treated group was higher compared with the control and DMSO groups (*p < 0.05). The apoptotic rate of cells in the higher concentration groups after treatment with 5 μM rottlerin for 48h was higher compared with the lower concentration groups (*p < 0.05). (C) The transwell invasion and migration assay showed that rottlerin inhibited cell invasion and migration of ACC cells (*p < 0.05). www.impactjournals.com/oncotarget 22828 Oncotarget family to activate the Wnt/β-catenin signaling pathway [23]. LRP6 is expressed in human cancer cell lines and up-regulated in human malignant tissues [24], and LRP6 silencing weakens Wnt/β-catenin signaling and inhibits cell proliferation and tumor growth in breast and prostate cancers [25]. LRP6 is readily expressed in ACC cells [26], but there has been limited research involving LRP6 in ACC. In our study, LRP6 and its phosphorylated form (p-LRP6) were down-regulated by rottlerin in ACC cells, both in vitro and in vivo. Blocking the Wnt signaling pathway is not the only mechanism underlying rottlerin anti-tumor activity. Some studies have indicated that rottlerin decrease carcinogenesis by impacting the mammalian target of rapamycin complex 1 (mTORC1) signaling [27]. Furthermore, rottlerin inhibits the markers of angiogenesis (COX-2, VEGF, VEGFR, and IL-8), and metastasis (MMP-2 and MMP-9), thus blocking production of tumorigenic mediators in the tumor microenvironment [28]. Rottlerin also inhibits epithelial-mesenchymal transition by up-regulating E-cadherin [29, 30]. According to these studies, we know that the anti-neoplastic activity of rottlerin is a multiple-signal pathway. The anti-tumor activity of rotterlin against ACC warrants further study. The recommended chemotherapy regimens for ACC are etoposide +doxorubicin+cisplatin+mitotane (EDP/M) or streptozotocin+mitotane (Sz/M). Whatever the chemotherapy regimen, mitotane is a key agent; however, the effective rate of treatment is only approximately 40%. Moreover, mitotane has intense side effects, thus limiting its clinical use [31, 32]. In this study, we attempted to identify a novel agent which suppresses ACC more efficiently and safer than traditional therapy. Based on our results, rottlerin is a novel and potential chemotherapeutic agent for ACC based on multiple signal pathway-related anti-neoplastic activity. Further studies should involve clinical trials better defining the anti-neoplastic activity of combination rottlerin and traditional chemotherapeutics or cytotoxic drugs. MATERIALS AND METHODS Cell lines and culture The ACC cell lines, NCI-H295R and SW-13, were purchased from the Cell Repository of the Chinese Academy of Science and originated from the American Type Culture Collection (ATCC; Manassas, VA, USA). The NCI-H295R cell line was cultured in DMEM (GIBCO, Grand Island, NY,USA), supplemented with 10% fetal bovine serum (MPBIO, Carlsbad, CA, USA) in a standard humidified incubator at 37°C in a 5% CO2 atmosphere. The SW-13 cell line was cultured in L15 media (GENOM, Shanghai, China) supplemented with 10% fetal bovine serum in a standard humidified incubator at 37°C in an atmosphere without CO2. Drugs and antibodies R00ottlerin was purchased from Sigma Aldrich (St. Louis, MO, USA). Rottlerin was dissolved in dimethyl Figure 3: (A) The wound healing assay showed that rottlerin reduced the cell migration rate of SW-13 cells compared with the control and DMSO groups. (B) WB assays of β-catenin, LRP6, and p-LRP were performed in NCI-H295R and SW-13 cells. The results showed that rottlerin down-regulated the expression of β-catenin, LRP6, and p-LRP in a dose-dependent manner. (C) The 24 h migration of SW-13 decreased after rottlerin treatment compared with the control and DMSO-treated groups (*p < 0.05). www.impactjournals.com/oncotarget 22829 Oncotarget Apoptosis analyses by flow cytometry sulfoxide (DMSO; GIBCO) at 10mM and stored in dark at 4°C. The final concentrations of rottlerin used for different experiments were prepared by diluting the stock solution with media solution. The antibodies used for Western blotting and immunohistochemistry staining were as follows: anti-LRP6 and anti-p-LRP6 antibodies were purchased from Affinity Bioscience (Cincinnati, OH, USA); and anti-β-catenin antibody was purchased from ABCAM (San Francisco, CA, USA). NCI-H295R and SW-13 cells were cultured in 6-well plates and treated with rottlerin in the dark (concentration : 5 μM and 2.5 μM ). The control group was not treated with rottlerin and a DMSO treated-group was established as well. After incubation for 48 h, cells were harvested, washed with PBS twice, resuspended in binding buffer, stained with an annexin-V/PI solution (Dojindo) at room temperature, then analyzed using a FACScan system (BD Biosciences, San Jose, CA, USA). Cell viability Acridine orange/ethidium bromide (AO/EB) staining NCI-H295R and SW-13 cells were plated at a density of 2000 cells/well in flat bottom 96-well plates (100μl media per well). After 24 h, cells were treated with rottlerin at various concentrations (0, 0.5, 1, 2, 4, and 8 μM) in the dark. Cell viability was measured after 24, 48, 72, 96, and 120 h using the Cell Counting Kit 8 (Dojindo, Osaka, Japan) with a microplate reader at 450 nm. All experimental concentrations were assessed in triplicate.The half maximal inhibitory concentration (IC50) was calculated with GraphPad Prism 5. Morphologic assessment of apoptotic cells was performed using the AO/EB staining method. NCIH295R and SW-13 cells were cultured in 6-well plates (5 × 104 cells/ml). After a 24-h incubation, the cells were treated with rottlerin in the dark (concentration = 5 μM and 2.5 μM) for 48 h. After washing twice with PBS, the cells were stained with 1ml of AO/EB (Sigma Aldrich) Figure 4: (A) Tumors in the rottlerin-treated groups were smaller than the control and DMSO-treated groups. (B) There was no significant difference in weight among the groups (*p > 0.05), and the rottlerin-treated groups showed delayed tumor growth compared to the control and DSMO-treated groups (*p < 0.05); there was no significant difference in tumor volume between the control and DSMO-treated groups (**p > 0.05). Tumors in the rottlerin 4 mg/kg group grow more slowly compared with the rottlerin 2 mg/kg group (*p < 0.05). (C) Tunnel assays indicated that rottlerin induced apoptosis in vivo. The tumor sections were observed under a light microscope. The apoptotic cells appeared brown in color, when the normal cells appeared blue. The apoptotic rates of the groups showed that the rottlerin-treated groups had higher apoptotic rates than the control and DMSO-treated groups (*p < 0.05). The apoptotic rate in the rottlerin 4mg/kg group was higher than the rottlerin 2 mg/kg group (*p < 0.05). (D) Hematoxylin and eosin (H&E) staining of tumor tissue. www.impactjournals.com/oncotarget 22830 Oncotarget for 3min and imaged under an inverted fluorescence microscope.Five hundred cells were counted under a microscope (magnification = 200×). The apoptosis rate was calculated using the following formula: apoptosis rate = (early apoptotic cells + late apoptotic cells)/the total number of cells.The nuclei of early apoptotic cells had a bright green fluorescence, the late apoptotic cell nuclei had a bright orange fluorescence, and the nuclei of normal cells had a ground glass-like dim green fluorescence. pre-treated with the respective concentrations of rottlerin (5 μM) for 24 h. The cells (1 × 105) in 0.2 ml of culture medium without fetal bovine serum were added to the upper chamber. The lower chamber was filled with 0.6 ml of complete medium containing 30% fetal bovine serum. After a 24 h incubation,cells remaining on the upper side of the transwell membrane were removed using a cotton swab.The membrane was washed with ice cold PBS twice.Cells that invaded the underside were fixed with 4% formaldehyde, stained with crystal violet, and counted in five randomly selected fields under a microscope (magnification of 200×). We also measured the migration rate using a wound healing assay.SW-13 cells were plated in 6-well plates (1 × 106cells/ml); after attachment the cells were scratched using a sterile pipette tip.The experimental group was treated with 5μM rottlerin. The cells were photographed at various time points (0, 12, and 24 h; magnification = 100×). The migration rate at 24 h was calculated using the following formula:migration rate = (1-the width of the cell wound at 24 h/the width of the cell wound at 0 h)%. Cell cycle analyses by flow cytometry NCI-H295R and SW-13 cells were plated in 6-well plates. After a 24 h incubation, the cells were treated with rottlerin in the dark (concentration = 5 μM and 2.5 μM) for 48 h. The cells were collected, washed twice in cold PBS, mixed in 300 ml of 1×binding buffer, and incubated at room temperature for 15 min with propidium iodide (PI), NP-40, and RNaseA (Beyotime, Shanghai, China). The cell cycle was analyzed by flow cytometry and the percentage of cells in the different phases was calculated using ModFit LT software (Verity Software House). Animal experiments Invasion and migration assay Animal experiments were performed using 4-weekold male nude mice (athymic, BALB/C nu/nu), which were purchased from the Provincial Animal Center (Guangdong, China). The animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals and approved by the Animal Cell invasion and migration were assessed using a Transwell chamber (GreinerBio, Frickenhausen, Germany) containing a polycarbonated filter with 8lM pores coated with Matrigel (BD Biosciences, BD Biosciences,San Jose, CA, USA).The cells in the experimental group were Figure 5: The expression of β-catenin, LRP6, and p-LRP by immunohistochemistry staining in xenograft tissues. The positive expressing proteins appeared brown in color.The results showed that rottlerin down-regulated the expression of β-catenin, LRP6, and p-LRP compared with the control and DMSO-treated groups (*p < 0.05) in vivo. The suppression was stronger in the higher dosetreated group (rottlerin 4 mg/kg) than the lower dose-treated group (rottlerin 2 mg/kg; **p < 0.05). www.impactjournals.com/oncotarget 22831 Oncotarget Investigation Committee of the General Hospital of Guangzhou Military Command. Mice were housed in a standard animal laboratory with free access to water and food. The mice were kept under constant environmental conditions with a 12 h light-dark cycle. SW-13 cells were (3 × 106 cells) suspended in 200 μl of serum-free culture medium and inoculated subcutaneously in the flank region of nude mice. Tumors were permitted to grow to approximately 4mm, and mice were randomized into 4 treatment groups (control, DMSO, rottlerin 4 mg/kg, and rottlerin 2 mg/kg) with 5 mice per group. All the mice were injected intraperitoneally daily for 4 weeks. Rottlerin was dissolved in DMSO, and diluted with normal saline (NS). The control group was injected with 0.5 ml of NS and the DMSO group was injected with 0.45 ml of NS mixed with 50 μl of DSMO. The rottlerin groups were injected with rottlerin (4 mg or 2 mg per kilogram). The mouse weights and tumor volumes were monitored every 4 days. Using Vernier calipers, the tumor volume was calculated according to the following formula: A × B × B/2, where A is the length of the tumor and B is the width. After 4 weeks, the mice were euthanized and the xenografts were removed from the animals. Tumor tissues were fixed in 10% formalin solution and paraffin-embedded. The sample sections were prepared for hematoxylin and eosin (HE) staining, immunohistochemistry staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. and ethanol. The slides were rinsed twice with PBS and treated for 15 min at 37°C with proteinase K (15 mg/ml in 10 mM Tris/HCl [pH 7.4–8.0]). Endogenous peroxidases were blocked using 3% hydrogen peroxide in methanol at room temperature for 10 min. The tissue sections were then analyzed with an in situ Cell Death Detection KitPOD (Roche, Basel, Switzerland) in accordance with the manufacturer’s instructions. The reaction was visualized using microscopy. Cell numbers of five randomly selected fields(400×) were counted under a microscope and analyzed with Imaga-ProPlus 6.0 software. Western blotting NCI-H295R and SW-13 cells were treated with rottlerin in the dark (concentrations = 0, 2.5, 5, and 10 μM) for 48 h. The cells were washed twice with PBS solution, then lysed with RIPA Lysis Buffer (Beyotime, Shanghai, China) and a protease inhibitor (Thermo Scientific, Waltham, MA, USA). Protein concentrations were determined with a Pierce BCA Protein Assay Kit (Beyotime). Equivalent amounts of total protein (60 μg) were boiled and electrophoretically separated on a 10% polyacrylamide gel at 80 volts. The proteins were transferred to a polyvinylidene difluoride membrane. The membranes were blocked for 60 min with a 5% milk solution prepared in PBS. The membranes were incubated overnight at 4°C with 1:500 (anti-p-LRP6 and anti-LRP6) or 1:3000 (antiβ-catenin) dilutions of the primary antibodies. The membranes were washed three times for 5 min each with Tween 20 (1:1000 dilution in PBS) and incubated for 45 min with the appropriate peroxidase-conjugated secondary antibody (1:5000 dilution). The membranes were washed with Tween 20-PBS three times for 10 min each and were developed using an Odyssey two-color infrared laser imaging system. The signal generated by β-actin was used as an internal control. Immunohistochemistry staining Tumor tissues sections from paraffin-embedded tumors were de-paraffinized and rehydrated using xylene and ethanol, and immersed in 3% hydrogen peroxide solution for 10 min to block endogenous peroxidase. Sections were boiled for 30 min in 10 mM citrate buffer solution (pH 6.0) for antigen retrieval. Slides were incubated for 45 min with 5% bovine serum albumin and incubated overnight at 4°C with anti-LRP6, antip-LRP6, and anti-β-catenin antibodies. The specimens were incubated for 45 min at 37°C with the appropriate peroxidase-conjugated secondary antibody and visualized using the DAB Detection Kit (BOSTER, Shanghai, China) following the manufacturer’s instructions. All sections with immunohistochemical staining were observed and the pictures of 6 randomly selected but homogeneous in staining and cell numbers fields (400×) were photographed by an Olympus microscope (IX-70 OLYMPUS, Japan) under high power view. The integrated optical density (IOD) in each image was measured with the same setting for all the slides, and the results were analyzed with Image-ProPlus 6.0 software[33]. Statistics Statistical analyses were performed using SPSS17.0 software and all the data from at least three experiments are presented as the mean ± S.D. The statistical difference between the means was analyzed with a Student’s t-test or one-way ANOVA. Compared with the control, a p < 0.05 was considered to be statistically significant. ACKNOWLEDGMENTS We thank Dr. WANG Jie and Dr. XIA Bin for providing instructions for experiments, and Dr. WANG Wei for providing advise in experimental design. CONFLICTS OF INTEREST TUNEL assays The authors declare that there are no conflicts of interest that could be perceived as prejudicing the impartiality of the research reported. Tumor tissue sections from paraffin-embedded tumors were de-paraffinized and rehydrated using xylene www.impactjournals.com/oncotarget 22832 Oncotarget FUNDING This research did not receive any specific grant from any funding agency in the public, commercial, or not-forprofit sectors. 10. Authors’ contributions Conceived and designed the experiments: Weilie Hu and Yi Zhu. Performed the experiments: Yi Zhu, Minjie Wang,Xu Zhao, Lei Zhang.Analyzed the data: Yi Zhu , Minjie Wang,Yigao Wu and Bangqi Wang. Contributed reagents/materials/analysis tools: Yi Zhu, Minjie Wang, and Xu Zhao. Wrote the paper: Yi Zhu. 11. 12. REFERENCES 1. Ayala-Ramirez M, Jasim S, Feng L, Ejaz S, Deniz F, Busaidy N, Waguespack SG, Naing A, Sircar K, Wood CG, Pagliaro L, Jimenez C, Vassilopoulou-Sellin R, et al. Adrenocortical carcinoma:clinical outcomes and prognosis of 330 patients at a tertiary care center. Eur J Endocrinol. 2013; 169:891–899. 13. 14. 2. Bilimoria KY, Shen WT, Elaraj D, Bentrem DJ, Winchester DJ, Kebebew E, Sturgeon C. Adrenocortical carcinoma in the United States:Treatment utilization and prognostic factors. Cancer. 2008; 113:3130–3136. 3. Fassnacht M, Kroiss M, Allolio B. Update in Adrenocortical Carcinoma. J Clin Endocrinol Metab. 2013; 98:4551–4564. 15. 4. 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ISSN: 2597-8012 JURNAL MEDIKA UDAYANA, VOL. 10 NO.9,SEPTEMBER, 2021 JMU Jurnal medika udayana Diterima: 2021-01-20 Revisi: 2021-02-02 Accepted: 15-09-2021 HUBUNGAN KARAKTERISTIK, KEPUASAN KERJA, MOTIVASI PERAWAT DAN DOKTER DENGAN KOMITMEN PENCAPAIAN STANDAR PELAYANAN MINIMAL DI INSTALASI GAWAT DARURAT RUMAH SAKIT UMUM DAERAH KABUPATEN BADUNG MANGUSADA Ni Made Widya Juliati, Dyah Pradnyaparamita Duarsa, Putu Cintya Denny Yuliyatni Departemen Ilmu Kesehatan Masyarakat, Fakultas Kedokteran Universitas Udayana Email : madejuliatiwidya@gmail.com ABSTRAK Latar Belakang : Rendahnya angka pencapaian standar pelayanan minimal di RSUD Badung dan tidak tercapainya target standard pelayanan minimal (SPM) yang telah ditetapkan menunjukkan beberapa permasalahan. Motivasi, kinerja dan kepuasan kerja yang rendah dapat mempengaruhi target tercapainya komitmen dalam pencapaian standar pelayanan minimal yang ditetapkan di IGD. Tujuan : Tujuan penelitian ini untuk mengetahui hubungan karakteristik, kepuasan kerja, motivasi perawat dan dokter dengan komitmen pencapaian standard pelayanan minimal di IGD. Metode: Penelitian ini menggunakan metode cross-sectional dengan 59 sampel yaitu seluruh tenaga kesehatan di IGD, dengan instrument penelitian berupa kuesioner dengan metode angket. Selanjutnya dilakukan analisis univariat, bivariat dengan chi-square menggunakan nilai P dengan tingkat kesalahan 5% atau sebesar 0,05, analisis multivariat dengan regresi logistik yang disertakan dalam uji multivariat dengan nilai p<0,25. Hasil: Analisis univariat pada variabel komitmen pencapaian SPM ditemukan 61% responden tidak mempunyai komitmen Pada analisis bivariat tenaga kesehatan yang mempunyai komitmen lebih banyak dijumpai pada tenaga kesehatan umur 36-45 tahun, jenis kelamin laki – laki, pendidikan sarjana, masa kerja lebih dari 5 tahun, status perkawinan menikah, dan penghasilan > 2,5 juta. Variabel yang paling dominan adalah Jenis kelamin dengan nilai OR 0,187 (95%IK = 0,043 - 0,805). Simpulan: Secara umum, dengan rendahnya komitmen pencapaian SPM khususnya pada tenaga kesehatan berjenis kelamin perempuan, perlu menjadi perhatian pihak rumah sakit. Manajemen rumah sakit diharapkan dapat menyeimbangkan antara jenis kelamin laki-laki dan perempuan dalam rekrutmen tenaga kesehatan serta memeberikan kompensasi rutin apabila target tercapai, memperjelas aturan dan kebijakan serta mensosialisaikannya. Kata kunci: Kepuasan Kerja, Motivasi, Komitmen, Komitmen Standar Pelayanan Minimal ABSTRACT Background : The low level of achievement of minimum service standards at Badung Regional Hospital and not achieving the minimum service standard targets that have been set indicate several problems. Motivation, performance and low job satisfaction can affect the target of achieving commitment in achieving the minimum service standards set in the IGD. https://ojs.unud.ac.id/index.php/eum doi:10.24843.MU.2020.V10.i9.P01 1 HUBUNGAN KARAKTERISTIK, KEPUASAN KERJA, MOTIVASI PERAWAT…, Ni Made Widya Juliati, Dyah Pradnyaparamita Duarsa, Putu Cintya Denny Yuliyatni Objectives: The purpose of this study was to determine the relationship between characteristics, job satisfaction, motivation of nurses and doctors with a commitment to achieving minimum service standards in the emergency department Methods: This study used a cross-sectional method with 59 samples, namely all health workers in the ER, with a questionnaire as the research instrument. After the data was collected, editing, coding, processing, and cleaning were carried out, then univariate, bivariate analysis with chisquare analysis was carried out and multivariate analysis along with other control variables using logistic regression. Results: Univariate analysis on the variable commitment to achieving MSS found that 61% of respondents had no commitment. In the bivariate analysis there was a significant relationship between age, sex, education, years of service, income, job satisfaction, compensation, work load, attitude to work procedures and supervision with a commitment to achieving minimum service standards. The most dominant variable was gender with an OR value of 0.187 (95% CI = 0.043 - 0.805). Conclusion: Most respondents fall into the category of not having a commitment to achieving minimum service standards. The dominant variable is gender, female gender is less committed than male gender. Hospitals are expected to balance the sexes of men and women in the recruitment of health workers. Build a regular communication platform, provide regular compensation when targets are achieved, clarify rules and policies and disseminate them. Keywords: Job Satisfaction, Motivation, Commitment, Commitment to Minimum Service Standar PENDAHULUAN Pelayanan kesehatan yang baik akan meningkatkan efektifitas mutu pelayanan kesehatan. Penerapan pelayanan kesehatan dengan penerapan keselamatan pasien dapat meminimalkan tingkat kesalahan dan berdampak pada penurunan biaya yang harus dibayar oleh pasien akibat masa rawat yang lama¹. Instalasi gawat darurat merupakan salah satu jenis pelayanan rumah sakit yang ditujukan untuk memberikan pelayanan kesehatan secepatnya pada kasus gawat darurat untuk mengurangi risiko kematian atau cacat. Instalasi Gawat Darurat adalah ujung tombak pelayanan kesehatan di rumah sakit³. Hasil laporan monitoring pelayanan IGD dari sembilan indikator standar pelayanan minimal yang telah ditetapkan oleh rumah sakit, terdapat tiga indikator yang belum tercapai. Jumlah kematian pasien selama tahun 2014 (satu tahun) ≥ 24 jam sebanyak 26 orang dengan nilai 0,026 pada tahun 2015 sebanyak 65 orang dengan nilai 0,065, sedangkan standar yang ditetapkan oleh rumah sakit sebesar 0,003. Jumlah pasien dirujuk pada tahun 2014 sebanyak 450 orang (4,5%), sedangkan tahun 2015 sebanyak 686 orang (19%), jumlah tersebut tidak mencapai target karena standar yang ditetapkan ≤ 3% per tahun. Jumlah tenaga perawat yang bersertifikat https://ojs.unud.ac.id/index.php/eum doi:10.24843.MU.2020.V10.i9.P01 ATLS/BTLS/ACLS/PPGD yang masih berlaku pada tahun 2014 untuk tenaga dokter dan perawat 16 orang (36%) sedangkan tahun 2015 sebanyak18 orang (37%), sedangkan jumlah tenaga dokter dan perawat yang belum mempunyai atau setifikat ATLS/BTLS/ACLS/PPGD yang tidak berlaku tahun 2014 sebanyak 29 (64%), sedangkan tahun 2015 sebanyak 31 orang (63%), indikator ini tidak mencapai target karena standar nilai yang ditetapkan rumah sakit sebesar 100% untuk tenaga Penelitian ini dilakukan untuk mengetahui hubungan karakteristik, kepuasan kerja, dan motivasi dengan komitmen dalam pencapaian standar pelayanan minimal di IGD RSUD Kabupaten Badung Mangusada. METODE Penelitian ini merupakan penelitian kuantitatif analitik, menggunakan rancangan potong-lintang. Penelitian dilakukan pada bulan Januari 2020 di IGD RSUD Kabupaten Badung Mangusada. Jumlah sampel sebanyak 59 orang, dengan kiteria insklusi semua dokter dan perawat yang bekerja di IGD dengan status pegawai Negeri Sipil dan Tenaga Kontrak dan kriteria eksklusi Dokter dan perawat magang dari instansi pendidikan ataupun instansi lain. Penelitian ini 2 HUBUNGAN KARAKTERISTIK, KEPUASAN KERJA, MOTIVASI PERAWAT…, menggunakan kuesioner yang sebelumnya telah dilakukan uji validitas dan reliabelitas. Penelitian ini telah mendapatkan kelaikan etik dari Komite Etik Penelitian Fakultas Kedokteran Universitas Udayana/Rumah Sakit Umum Pusat Sanglah Denpasar dengan Nomor: 2954/UN/14.2.2.VII.14/LP/ 2019 tertanggal 6 Desember. HASIL Hasil Analisis Univariat Diketahui dari tabel 1 sebagian besar responden dengan kelompok umur 36-45 tahun (86,4%), jenis kelamin perempuan (72,9%), pendidikan tingkat sarjana (89,8%), masa kerja > 5 tahun (86,4%), dengan status menikah (96,6%), penghasilan lebih dari 2,5 juta (83,1%) mempunyai komitmen. Tabel 1. Karakteristik Tenaga Kesehatan di IGD RSUD Mangusada Variabel Frekuen Persentase (n=59) si (f) (%) Umur 20-35 tahun 36-45 tahun Jenis kelamin Perempuan Laki-laki Pendidikan Diploma Sarjana Masa kerja < 5 Tahun ≥ 5 Tahun Status BlmMenikah Menikah Penghasilan < 2,5 Juta >2,5 Juta 8 51 13,6 86,4 43 16 72,9 27,1 6 53 10,2 89,8 8 51 13,6 86,4 2 57 3,4 96,06 10 49 16,9 83,1 Tabel 2. Komitmen, Kepuasan Kerja, Motivasi di IGD RSUD Mangusada Variabel (n=59) Komitmen Punya Tidak Kep. Kerja Puas Tidak puas Motivasi Rendah Tinggi Kompensasi Rendah Tinggi Kondisi kerja Tdk nyaman Nyaman Beban kerja Rendah Tinggi P.prosedur kerja Tidak sesuai Sesuai Supervisi Tidak Ada Frekuensi (f) Persentase (%) 36 23 61 39 53 6 89,8 10,2 24 35 40,7 59,3 8 51 13,6 86,4 16 43 27,1 72,9 15 44 25,4 74,6 9 50 15,3 84,7 6 53 10,2 89,8 Hasil Analisis Bivariat Tenaga kesehatan yang mempunyai komitmen lebih banyak dijumpai pada tenaga kesehatan umur 36-45 tahun (45,1%), jenis kelamin laki – laki (56,33%), pendidikan sarjana ( 56,6%), masa kerja lebih dari 5 tahun (45,1%), status perkawinan menikah (40,4%), dan penghasilan > 2,5 juta (44,9%), disajikan pada tabel 3. Secara umum tenaga kesehatan di RSUD Mangusada Kabupaten Badung kurang mempunyai komitmen (61%). Hasil penilaian kepuasan kerja, sebagian besar merasa sangat puas dalam bekerja (89,8%). Responden memiliki motivasi kerja yang tinggi (59,3%). https://ojs.unud.ac.id/index.php/eum doi:10.24843.MU.2020.V10.i9.P01 3 Ni Made Widya Juliati, Dyah Pradnyaparamita Duarsa, Putu Cintya Denny Yuliyatni Tabel 3. Hubungan Karakteristik dengan Komitmen Pencapaian SPM di IGD RSUD Mangusada Var. Bebas Umur 20-35 36-45 JK P L Pddkn D1/3 S1 Masa kerja < 5 Th ≥ 5 Th Status Blm Mnikah Mnikah Penghasi lan <2,5 Jt ≥2,5 Jt Komitmen Tidak Mempun Nila Mempun yai ip yai n(%) n(%) Variabel Bebas PR 8(100) 28(54,9) 0 23(45,1) 0,0 15 1,8 2 29(67,4) 7(43,8) 14(32,6) 9(56,33) 0,09 7 1,5 4 6(100) 30(56,6) 0(0) 23(43,4) 0,03 9 1,7 6 8(100) 28(54,9) 0(0) 23(45,1) 0,01 5 1,8 2 2(100) 34(59,6) 0(0) 23(40,4) 0,25 0 1,6 7 9(90) 27(55,1) \1(10) 22(44,9) 0,03 9 1,6 3 Tenaga kesehatan yang mempunyai komitmen lebih banyak ditemukan pada tenaga kesehatan yang mempunyai kepuasan kerja yang baik (41,5%), Motivasi tinggi (97,1%), kompensasi baik (43,1%), kondisi kerja nyaman (41,9%), beban kerja rendah (53,3%), penyikapan prosedur kerja sesuai (44%), dilakukannya supervise (43,4%). https://ojs.unud.ac.id/index.php/eum doi:10.24843.MU.2020.V10.i9.P01 Tabel 4. Hubungan Kepuasan Kerja dan Motivasi dengan Komitmen Pencapaian SPM di IGD RSUD Mangusada Kep. kerja Tidak puas Puas Motivasi Rendah Tinggi Kompen sasi Rendah Baik Kondisi kerja Tidak nyaman Nyaman Beban kerja Rendah Tinggi P.prosed ur kerja Tidak sesuai Sesuai Supervis i Tidak Ada Komitmen Tidak Mempu Mempu nyai nyai n(%) n(%) Nil ai p P R 5(83,3) 31(58,6) 1(16,7) 22(41,5) 0,2 37 1,4 2 4(16,7) 1(2,9) 20(83,3) 34(97,1) 0,0 61 7,2 9 7(87,5) 29(56,9) 1(12,5) 22(43,1) 0,0 99 1,5 3 11(68,8) 25(58,1) 5(31,3) 18(41,9) 0,4 58 1,1 8 7(46,7) 29(65,9) 8(53,3) 15(34,1) 0,1 87 1,4 1 8(88,9) 28(56) 1(11,1) 22(44) 0,0 63 1,5 8 6(100) 30(56,6) 0(0) 23(43,4) 0,0 39 1,7 6 4 HUBUNGAN KARAKTERISTIK, KEPUASAN KERJA, MOTIVASI PERAWAT…, Hasil Analisis Multivariat Tabel 5 menunjukkan hanya jenis kelamin yang berhubungan dengan komitmen dengan nilai p kurang dari 0,05. Jenis kelamin memiliki nilai p =0,017, dengan nilai OR 7,833 dan 95% IK sebesar 1,440 – 42,603. Tabel 5. Analisis Uji Multivariat Hubungan Karakteristik, Kepuasan Kerja, Motivasi Perawat dan Dokter dengan Komitmen Pencapaian Standar Pelayanan Minimal di IGD RSUD Mangusada Variabel OR Nilai p 95% IK (n =59) Umur 1512788 0,999 0,000 - (-) 272,803 JK 7,833 0,017 1,440 – 42,603 Tk.pendid -ikan 27173,4 1,000 0,000 – (-) 47 Status P.kawinan 13,432 1,000 0,000 – (-) Penghasilan 1,843 0,676 0,105 – 32,412 Kepuasan kerja 0,000 0,999 0,000 – (-) Kompens asi 1,843 0,676 0,105 – P. 32,412 prosedur kerja 5,147 0,266 0,286 – 92, 528 supervisi 7987233 1,000 0,000 – (-) 001115, 020 PEMBAHASAN Hasil analisis komitmen pencapaian standar pelayanan minimal diperoleh (61%) responden tidak mempunyai komitmen. Menurut Sopiah, komitmen merupakan dimana orang-orang dalam organisasi tetap tinggal oleh karena membutuhkan gaji atau karyawan tersebut tidak diterima kerja di tempat lain atau bertahannya karyawan di dalam suatu organisasi disebabkan karena organisasi tersebut memberikan banyak keuntungan yang tidak didapatkan di organisasi yang lain⁸. https://ojs.unud.ac.id/index.php/eum doi:10.24843.MU.2020.V10.i9.P01 Jenis kelamin memberikan pengaruh tidak langsung kepada kinerja individu, karena ada perbedaan bawaan antara pria dan wanita⁷. Perubahan peran pada perempuan di masa kini dimana perempuan juga memiliki kebebasan dan keinginan untuk pengembangan peran, tetapi faktor status perkawianan dimana wanita menikah mempunyai tanggung jawab lebih pada rumah tangga, sedangkan laki – laki cenderung lebih agresif dan lebih besar kemungkinan dalam memiliki harapan untuk sukses¹º. SIMPULAN Sebagian besar responden masuk katagori tidak mempunyai komitmen pencapaian standar pelayanan minimal. Variabel yang berhubungan dengan komitmen pencapaian standard pelayanan minimal adalah jenis kelamin. Variabel yang tidak berhubungan dengan komitmen pencapaian standard pelayanan minimal antara lain: karakteristik (umur, pendidikan, masa kerja, status perkawinan, penghasilan), kepuasan kerja, motivasi terdiri dari lima sub variabel diantaranya: kompensasi, kondisi kerja, beban kerja, penyikapan prosedur kerja dan supervisi. SARAN 1. Bagi Manajemen Rumah Sakit a. Rumah sakit diharapkan dapat menyeimbangkan tenaga kesehatan antara jenis kelamin laki-laki dan perempuan dalam rekrutmen karyawan. b. Target indikator yang ditentukan rumah sakit dalam pencapaian SPM perlu dikaji lagi. Apabila target indikator tercapai tenaga kesehatan perlu diberikan reward. 2. Untuk Peneliti Selanjutnya Mengingat beberapa kelemahan dari penelitian ini, diharapkan dijadikan sebagai data dasar untuk mengadakan penelitian yang lebih kompleks dan spesifik menggunakan desain penelitian kualitatif agar mengeksplorasi lebih dalam komitmen pencapaian standard pelayanan minimal dengan jenis kelamin. 5 Ni Made Widya Juliati, Dyah Pradnyaparamita Duarsa, Putu Cintya Denny Yuliyatni DAFTAR PUSTAKA 1. 2. 3. 4. 5. 6. 7. 8. Gerri, Kaufan & Dorothy, M.C. The Effect of Organisational Culture on Patient SafetyResearch Databese, The university of York. The Univercity of York. 2013. https://doi.org/10.7748/ns2013.06.27.43.50. e7280 Departemen Kesehatan RI. Pedoman Insiden Keselamatan Pasien Rumah Sakit (Patient safety). Jakarta. 2008. Scharein, P &Trendelenburg., M. Critical Incidents in a Tertiary Care Clinic for Internal Medicine. BMC Research Notes. 6 (1). 2013. 1 . https://doi.org/10.1186/17560500-6-276 RSUD Kabupaten Mangusada. Laporan Standar Pelayananminimal Rumah Sakit Umum Daerah Kabupatrn Mangusada. 2015. Didik Widiyanti. Pengaruh Disiplin Kerja, Motivasi Kerja, Gaya Kepemimpinan Kerja Terhadap Kualitas Kinerja Karyawan Dinas Kesehatan Kabupaten Sragen, Tesis. Surakarta: Fakultas Ekonomi & Bisnis Program Study Ekonomi Manajemen Universitas Muhamadiyah Surakarta. 2015. Angraeni, S. Analisis Faktor-Faktor yang Berhubungan dengan Kepuasan Kerja Perawat Pelaksana di Ruang Rawat Inap RS. MH. Thamrin Jakarta Pusat. Tesis Program Pasca sarjana FIK-UI Jakarta. 2011. Chan, Sow Hup. Organizational identification and commitment of members of human development organization, Jurnal of Management Development. 2006. Sopiah. Prilaku Organisasi. Yogyakarta. ANDI. 2008. https://ojs.unud.ac.id/index.php/eum doi:10.24843.MU.2020.V10.i9.P01 9. 10. 11. 12. 13. 14. 15. 16. Wijono, S. Psikologi Industri & Organisasi dalam suatu Bidang Gerak Psikologi Sumber Daya Manusia. Jakarta. Kencana. 2010. Chan, Sow Hup. Organizationalidentification and commitment of members of human development organization, Jurnal of Management Development. 2006. Wibowo. Manajemen Kinerja. Jakarta: Rajawali Pers. 2011. Siangian, Sondang. Manajemen Sumber Daya Manusia. Jakarta. Bumi Aksara. 2006. Robbins, S.P. Alih Bahasa Pujaatmaka, H &Molan, B. Perilaku Organisasi: Konsep Kontroversi, Aplikasi. Jakarta. PT Prelindo. 2006. Smith,S. Reducing Medical Errors andIncreasing Patient Safety in Health Care. Proreques Dissertasions Publishing. Utica Collage. 2016. https://doi.org/10183250. Luthans. Alih Bahasa Yuwono, dkk. Perilaku Organisasi. Yogyakarta. ANDI. 2006. Ilyas Y. Perencanaan SDM Rumah Sakit. Teori. Metoda dan Formula. Jawa Barat: Fakultas Kesehatan Masyarakat UI. 2004. 6 

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MATCHING FUND SEKTOR PARIWISATA DESA MINGGIRSARI DALAM PERSPEKTIF HUKUM PARIWISATA Ongky Ramadhan Baseno Fakultas Hukum Universitas 17 Agustus 1945 Surabaya Jl. Semolowaru 45, Surabaya, 60118 E-mail: ongkyramadhan26@gmail.com Abstrak Indonesia adalah negara yang berpotensi untuk menjadi tujuan pariwisata di dunia. Pariwisata adalah suatu kegiatan yang menyediakan jasa akomodasi, transportasi, makanan, rekreasi, serta jasa-jasa lainnya. Perdagangan jasa pariwisata melibatkan beberapa aspek, yaitu aspek ekonomi, budaya, sosial, agama, lingkungan, keamanan dan aspek lainnya. Dari berbagai aspek tersebut, aspek ekonomilah yang mendapat perhatian paling besar dalam pembangunan pariwisata sehingga pariwisata dikatakan sebagai suatu industri. Berdasarkan pada ketentuan latar belakang masalah tersebut di atas maka dapat ditentukan rumusan masalah, Bagaimana pengelolaan desa wisata Minggirsari yang sesuai dengan prinsip hukum kepariwisataan. Jenis penelitian yang digunakan dalam penulisan paper ini adalah metode penelitian hukum normatifPariwisata adalah suatu kegiatan yang secara langsung menyentuh dan melibatkan masyarakat, sehingga membawa berbagai dampak terhadap masyarakat setempat. Bahkan pariwisata dikatakan mempunyai energi dobrak yang luar biasa, yang mampu membuat masyarakat setempat mengalami metamorphose dalam berbagai aspeknya. Pendahuluan 1. Latar Belakang Masalah Indonesia adalah negara yang berpotensi untuk menjadi tujuan pariwisata di dunia. Pariwisata adalah suatu kegiatan yang menyediakan jasa akomodasi, transportasi, makanan, rekreasi, serta jasa-jasa lainnya. Perdagangan jasa pariwisata melibatkan beberapa aspek, yaitu aspek ekonomi, budaya, sosial, agama, lingkungan, keamanan dan aspek lainnya. Dari berbagai aspek tersebut, aspek ekonomilah yang mendapat perhatian paling besar dalam pembangunan pariwisata sehingga pariwisata dikatakan sebagai suatu industry.1 Peranan sektor pariwisata nasional semakin penting sejalan dengan perkembangan dan kontribusi yang diberikan sektor pariwisata melalui penerimaan devisa, pendapatan daerah, 1 I Putu Gelgel, Industri Pariwisata Indonesia Dalam Globalisasi Perdagangan Jasa (GATS-WTO) ImplikasiHukum dan Antisipasinya, Refika Aditama, Bandung, 2009, h. 22. pengembangan wilayah, maupun dalam penyerapan investasi dan tenaga kerja serta pengembangan usaha yang tersebar di berbagai pelosok wilayah di Indonesia. Menurut Buku Saku Kementerian Pariwisata (2016), kontribusi sektor pariwisata terhadap Produk Domestik Bruto (PDB) nasional pada tahun 2014 telah mencapai 9 % atau sebesar Rp 946,09 triliun. Sementara devisa dari sektor pariwisata pada tahun 2014 telah mencapai Rp 120 triliun dan kontribusi terhadap kesempatan kerja sebesar 11 juta orang. Melalui mekanisme tarikan dan dorongan terhadap sektor ekonomi lain yang terkait dengan sektor pariwisata, seperti hotel dan restoran, angkutan, industri kerajinan dan lainlain. Melalui multiplier effect-nya, pariwisata dapat dan mampu mempercepat pertumbuhan ekonomi dan penciptaan lapangan kerja. Itulah mengapa, percepatan pertumbuhan ekonomi dan penciptaan lapangan kerja yang lebih luas dapat dilakukan dengan mempromosikan pengembangan pariwisata.2 Matching Fund adalah bentuk nyata dukungan dari Kementerian Pendidikan dan Kebudayaan (Kemendikbud) Republik Indonesia untuk penciptaan kolaborasi dan sinergi strategis antara Insan Dikti (lembaga perguruan tinggi) dengan pihak Industri. Lulusan pendidikan tinggi mendapat pekerjaan yang layak. Matching Fund menjadi salah satu nilai tambah terbentuknya kolaborasi antara dua pihak melalui platform Kedaireka. Dukungan Matching Fund ini diprioritaskan bagi kolaborasi yang berkontribusi terhadap pencapaian 8 (delapan) Indikator Kinerja Utama Perguruan Tinggi yang telah ditetapkan oleh Kemendikbud: • Lulusan pendidikan tinggi mendapat pekerjaan yang layak • Mahasiswa mendapat pengalaman di luar kampus • Dosen berkegiatan di luar kampus • Praktisi mengajar di dalam kampus • Hasil kerja dosen berguna bagi masyarakat dan diakui internasional • Program studi kampus bekerja sama dengan mitra kelas dunia • Kelas bersifat kolaboratif dan partisipatif • Program studi berstandar internasional Kaitannya di dalam hal ini ialah mahasiswa diberi tugas atau program kerja di dalam pelaksanaan Kuliah Kerja Nyata (KKN) untuk meningkatkan sektor ekonomi yang berada pada Desa Minggirsari. Mengingat adanya Pandemi Covid-19 juga tentunya berdampak pada sektor ekonomi khususnya di bidang pariwisata Desa Minggirsari. 2. Rumusan Masalah 2 Isharyanto dkk., Hukum Kepariwisataan Dan Negara Kesejahteraan (Antara Kebijakan Dan Pluralisme Lokal), Halaman Moeka Publishing, Bogor, 2019, h. 3 Berdasarkan pada ketentuan latar belakang masalah tersebut di atas maka dapat ditentukan rumusan masalah sebagaimana berikut: Bagaimana pengelolaan desa wisata Minggirsari yang sesuai dengan prinsip hukum kepariwisataan? 3. Metode Penelitian Jenis penelitian yang digunakan dalam penulisan paper ini adalah metode penelitian hukum normatif (normative legal research), dimana untuk menemukan peraturan-peraturan yang berlaku, prinsip-prinsip, dan disertai pula dengan doktrin-doktrin, guna menjawab isu hukum yang dihadapi.3 Pembahasan Alasan sektor pariwisata dipacu untuk dijadikan komoditi andalan di samping migas sebagai komoditi pendukung kelangsungan pembangunan nasional , antara lain adalah:4 a. Pola perjalanan wisata dunia yang terus menerus meningkat dari tahun ketahun; b. Pariwisata tidak begitu terpengaruh gejolak ekonomi dunia, di samping pertumbuhannya yang lebih cepat daripada pertumbuhan ekonomi dunia; c. Meningkatkan kegiatan ekonomi daerah dan pengaruh ganda dari pengembangan pariwisata tampak lebih nyata; d. Komoditi pariwisata tidak mengenal proteksi atau quota seperti komoditi lainnya; e. Potensi pariwisata Indonesia yang tersebar diseluruh wilayah Indonesia yang beranekaragam macamnya tak akan habis terjual; f. Pariwisata sudah menjadi kebutuhan hidup manusia pada umumnya. Dalam living culture, unsur-unsur yang bisa dijadikan sebagai daya tarik antara lain tradisi suatu suku bangsa tertentu, upacara dan ritual keagamaan, seni pertunjukan, dan sebagainya. Sedangkan dalam cultural heritage, daya tarik yang ditawarkan dapat berupa benda-benda peninggalan sejarah dan purbakala, lansekap budaya, dan sebagainya. Wisata berbasis budaya adalah salah satu jenis kegiatan pariwisata yang menggunakan kebudayaan sebagai objeknya. Pariwisata jenis ini dibedakan dari minat-minat khusus lain, seperti wisata alam, dan wisata petualangan. Pariwisata budaya sebagai salah satu produk pariwisata merupakan jenis pariwisata yang disebabkan adanya daya tarik dari seni budaya suatu daerah. Pariwisata budaya pada intinya merupakan jenis pariwisata yang menawarkan kebudayaan yang berupa atraksi budaya baik yang bersifat tangibel atau konkret maupun intangibel atau abstrak, juga yang 3 4 Peter Mahmud Marzuki, Penelitian Hukum, Jakarta, Kencana Pernada Media Group, 2010, h. 35. Hari Karyana, Kepariwisataan, Penerbit PT. Gramedia Widiasarana Indonesia, Jakarta, 1997, h. 7 bersifat living culture (budaya yang masih berlanjut) dan cultural heritage (warisan budaya masa lalu), sebagai daya tarik utama untuk menarik kunjungan wisatawan. Pariwisata secara teori dapat ditinjau dari berbagai fenomena yang mempengaruhi berkembangnya kegiatan pariwisata. Menurut Hari Karyana ada 5 (lima) fenomena yang berpengaruh terhadap penyelenggaraan pariwisata yaitu fenomena sosial, fenomena ekonomi, fenomena psikologi, fenomena geografi dan fenomena budaya.5 Keinginan pemerintah untuk mengembangkan pariwisata di Indonesia pada dasarnya disebabkan oleh beberapa faktor. Pertama adalah semakin berkurangnya peranan minyak sebagai penghasil devisa jika dibandingkan dengan waktu yang lalu; kedua merosotnya nilai ekspor kita dari sektor non minyak; ketiga prospek pariwisata yang tetap memperlihatkan kecenderungan meningkat secara konsisten; dan keempat besarnya potensi yang kita miliki bagi pengembangan pariwisata di Indonesia, khususnya pada Desa Minggirsari. Pariwisata adalah suatu kegiatan yang secara langsung menyentuh dan melibatkan masyarakat, sehingga membawa berbagai dampak terhadap masyarakat setempat. Bahkan pariwisata dikatakan mempunyai energi dobrak yang luar biasa, yang mampu membuat masyarakat setempat mengalami metamorphose dalam berbagai aspeknya.6 Pengembangan pariwisata di Indonesia sebenarnya sangat potensial yang terkait dengan daya tarik wisata berbasis budaya. Terkait dengan hal itu salah satu sistem yang dikehendaki oleh Program Pembangunan Nasional (Propenas) dibidang pariwisata adalah pembangunan pariwisata yang berbasis komunitas budaya karena disadari bahwa memang keanekaragaman budaya bangsa Indonesia merupakan kekayaan yang tidak ternilai harganya dan jumlahnya sangat melimpah dari Sabang sampai Merauke. Dalam ketentuan Undang-Undang Nomor 10 Tahun 2009 dapat disimpulkan pada bagian pasal-pasal undang-undang sebagai berikut : a. Pasal 2 , asas-asas yang menggambarkan bahwa UU No. 10 Tahun 2009 melibatkan masyarakat dalam pengembangan pariwisata yaitu asas manfaat, asas kekeluargaan, asas adil dan merata, asas partisipatif, asas demokrasi. b. Pasal 3 yang mengatur fungsi kepariwisataan. c. Pasal 4 yang mengatur mengenai tujuan penyelenggaraan kepariwisataan. d. Pasal 5, prinsip penyelenggaraan kepariwisataan huruf c, d, e. 5 6 Ibid. I Gde Pitana dan Putu G. Gayatri , Sosiologi Pariwisata, Penerbit Andi, Yogyakarta, 2005, h. 109. e. Pasal 19 yang secara tegas menyebutkan peran serta masyarakat dalam penyelenggaraan pariwisata: Ayat (1) Setiap orang berhak memperoleh kesempatan memenuhi kebutuhan wisata; melakukan usaha pariwisata;, menjadi pekerja/buruh pariwisata;. Berperan dalam proses pembangunan kepariwisataan. Ayat (2)Setiap orang dan/atau masyarakat di dalam dan disekitar destinasi pariwisata mempunyai hak prioritas untuk menjadi pekerja/buruh; hak konsinyasi ; dan hak pengelolaan. Pengembangan pariwisata berbasis masyarakat merupakan sebuah pendekatan yang melibatkan dan meletakkan masyarakat sebagai pelaku penting dalam konteks paradigma baru pembangunan yaitu pembangunan berkelanjutan (suistainable development paradigm). Pariwisata berbasis masyarakat merupakan peluang untuk menggerakkan segenap potensi dan dinamika masyarakat guna mengimbangi peran pelaku usaha pariwisata berskala besar. Pariwisata berbasis masyarakat tidak berarti merupakan upaya kecil dan local semata , tetapi perlu diletakkan dalam konteks kerjasama masyarakat secara global. Dalam konsep pariwisata berbasis masyarakat terkandung di dalamnya konsep pemberdayaan masyarakat. Upaya pemberdayaan masyarakat pada hakekatnya selalu dihubungkan dengan karakteristik seseorang sebagai suatu komunitas yang mempunyai ciri, latar belakang dan pemberdayaan masyarakat , yang terpenting adalah dimulai dengan bagaimana menciptakan kondisi suasana atau iklim yang meningkatkan potensi masyarakat untuk berkembang. Di dalam pelaksanaan KKN pada Desa Minggirsari terdapat beberapa kendala antara lain yakni: • Bantuan marketing dalam pembuatan sosial media agar wisatawan lebih mudah mengetahui wisata alam yg berada di desa minggir sari. • Memperbaiki rute jalan ke wisata alam agar lebih baik dan mudah untuk di akses. • Pendampingan dan pelatihan untuk menjaga situs bersejarah di Desa Plunturan • Tidak terdapat tempat pemandian atau toilet di daerah sumber air • Tidak ada tempat parkir yang luas untuk wisatawan • Kurang banyaknya tempat sampah • Kurang petunjuk arah untuk menuju ke tempat wisata Hal tersebut yang memuat program kerja sebagaimana KKN yang dilaksanakan pada Desa Minggirsari Blitar. Namun dengan bantuan para warga sekitar pelaksanaan Kuliah Kerja Nyata (KKN) Tematik Matching Fund pada Desa Minggirsari Blitar dapat terselenggara dengan baik dan memberikan dampak yang positif bagi masyarakat sekitar Penutup 1. Kesimpulan Berdasarkan pada ketentuan tersebut di atas maka, pelaksanaan Kuliah Kerja Nyata (KKN) Tematik Matching Fund pada Desa Minggirsari Blitar dapat terselenggara dengan baik dan memberikan dampak yang positif bagi masyarakat sekitar. Disamping itu diharapkan program kerja yang dilakukan mahasiswa ini akan menimbulkan dampak yang bagus baik dilingkup sektor pariwisata, ekonomi, maupun hukum. Diharapkan Desa Minggirsari kedepannya dapat mengembangkan beberapa sektor khusunya pada sektor pariwisata, mengingat para masyarakat warga Desa Minggirsari sangat antusias terhadap hal seperti ini. 2. Ucapan Terimakasih Penulis mengucapkan terimakasih yang sebesar-besarnya kepada beberapa pihak yang membantu untuk kelancaran pelaksanaan Kuliah Kerja Nyata (KKN) ini. Lebih lanjut saya ucapkan terimakasih yang sebesar-besarnya kepada dosen pembimbing serta seluruh perangkat dan warga Desa Minggirsari. DAFTAR PUSTAKA Hari Karyana, Kepariwisataan, Penerbit PT. Gramedia Widiasarana Indonesia, Jakarta, 1997 I Gde Pitana dan Putu G. Gayatri , Sosiologi Pariwisata, Penerbit Andi, Yogyakarta, 2005 I Putu Gelgel, Industri Pariwisata Indonesia Dalam Globalisasi Perdagangan Jasa (GATSWTO) ImplikasiHukum dan Antisipasinya, Refika Aditama, Bandung, 2009 Isharyanto dkk., Hukum Kepariwisataan Dan Negara Kesejahteraan (Antara Kebijakan Dan Pluralisme Lokal), Halaman Moeka Publishing, Bogor, 2019 Peter Mahmud Marzuki, Penelitian Hukum, Jakarta, Kencana Pernada Media Group, 2010 

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“Talqin va tadqiqotlar” Respublika ilmiy-uslubiy jurnali №7 FIZIKA FANI RIVOJLANISH TARIXINI O’RGANISHNING DIDAKTIK JIHATLARI Muydinova Madina Alisherovna Andijon davlat pedagogika instituti katta o’qituvchisi Abdunabiyeva Maftunaxon Solijon qizi Andijon davlat pedagogika instituti Ta’limda axborot texnologiyalari mutaxassisligi magistranti Qosimjonova Xamidaxon Botirjon qizi Andijon davlat universiteti Fizika-matematika fakulteti talabasi Annotatsiya: Ushbu maqolada fizika fanini rivojlanish tarixi o’rganish bosqichlari, vazifalari, rivojlanishidagi fanlar bilan bog’liqligi va rivojlanish tarixining davrlari haqida keng yoritilgan. Bundan tashqari, o’quv darslarida fizika fani tarixini o’qitishning ahamiyati va samaradorligi tahlil etilgan. Kalit so’zlar: rivojlanish tarixi, analitik ma’lumot, tarixiy fakt, tabiatshunoslik, jamiyat qonunlari, astornomiya, ishlab chiqarish, ilmiy kashfiyot. Insoniyat o’zi yashayotgan olam to’g’risidagi bilimlarni to’satdan va tayyor tugallangan holda ololmaydi. Oddiy, misol: Nyutonning qonunlari degan nom bilan ataluvchi hozirgi maktab o’quvchisiga ham tanish bo’lgan harakat qonunlarini tushunish minglab yillarni talab qilgan. Yana ko’p yuz yillar davomida bu qonunlarning har doim ham har qanday holatlar uchun ham qo’llab bo’lmasliginii tushunildi[1]. Ya’ni juda katta tezlikdagi va juda kichik o’lchamdagi zarrachalar harakatiga bu qonunlarni qo’llab bo’lmasligi aniqlandi. Insoniyat bilmaslikdan bilishga bo’lgan uzoq va mashaqqatli masofani bosib o’tdi. Bunda u unchalik aniq va to’liq bo’lmagan bilimlarni aniq va to’liq tushunchalar bilan almashtirib bordi. Hech bir ish yo’q joydan boshlanmaydi, balki har bir yangi avlod o’zidan avvalgi o’tganlar to’xtagan joydan ishni boshlaydi va o’zi amalga oshirgan ishlarni keyingi keluvchilarga topshiradi. Bu yerda I. Nyutonning quyidagi so’zlarini eslash muxum ahamiyat kasb etadi. “Agar men boshqalarga nisbatan uzoqni ko’ra olgan bo’lsam, buning sababi, men buyuk kishilarning yelkalarida turganligim uchundir”[2]. Yevklid va Arximedlarsiz Nyuton, Nyutonsiz Eynshteyn yoki Bor, Xorazmiy va Forobiysiz Beruniy yoki Ibn Sino, Umar Xayyom va Umar Chag’moniysiz Ulug’bek, bularsiz Qozizoda Rumiy (zamonasining Aflotuni) yoki Ali Qushchi bo’lmas edi. Ya’ni, har bir avlod o’zidan avval o’tganlar merosini o’rganib, ular qoldirgan ishlar zaminida fan va texnikaning kelgusi rivojini amalga oshiradi. 172 “Talqin va tadqiqotlar” Respublika ilmiy-uslubiy jurnali №7 Har qanday bilimning rivojlanish jarayonini o’rganish alohida bir fan - tarix fanining asosiy masalasini belgilaydi. Shu jumladan fizika fani rivojlanish tarixi, fan tarixining bir bo’lagini tashkil qilib, u fizika fanining rivojlanishi jarayonini o’rganadi. Har qanday fanning asosiy vazifasi shu fan shug’ullanayotgan sohada amalda bo’lgan qonunlarni ochishdan iborat bo’lganligi kabi fizika tarixi fanining ham vazifasi fan rivojlanishini boshqaruvchi qonunlarni yaratishdan iborat. Yuzaki qaraganda bunday qonunlarning bo’lishi mumkin emasdek ko’rinadi. Oldindan Farobiy, Beruniy, Ibn Sino, Arximed, Nyutonlarning dunyoga kelishini bilib bo’lmaydi, olimlar fikri va tafakkurini boshqarib bo’lmaydi. Tashqaridan bir ko’rishda fan tarixi buyuk daholarning nazorat qilib bo’lmaydigan faoliyatlari natijasiga o’xshab ketadi. Albatta ular faoliyatini tabiatda yuz beruvchi biror harakat qonunlariga o’xshash qolipdagi qonunlarga o’xshatib bo’lmaydi[3]. Mubolag’asiz aytish mumkinki, fan bu insoning murakkab tafakurri va ijodiy faloiyat mahsulidir. Biroq fan rivoji muhum o’rin tutuvchi ma’lum bir tarixiy sharoitlarda amalga oshadi. Bu narsani esa, albatta, ilmiy tahlil qilish mumkin. Tarixan ma’lumki ilm-fan rivoji iqtisodiy rivojlanish bosqichlariga bog’liq. Qaysi joyda ishlab chiqarish, sanoat, madaniyat rivojlanishi yuqori bo’lsa o’sha joyda fan rivojlanishi ham yuqori darajada bo’ladi. Hozirgi kunda bu yanada yaqqol namayon bo’lib bormoqda.Chunki, ilm fan hozirgi davr rivojida juda katta ijtimoiy mablag’ni talab qiladi. Fizika fanini rivojlanish tarixi o’rganish bir qator bosqichlarda amalga oshiriladi: –ma’lumotlar yig’ish –analitik ma’lumotlar orasidagi bog’lanish va sabablar - analitik natijalarni umumlashtirish va fandagi asosiy qonunlarni ochish. Fanning rivojlanish tarixi bu umumiy fizikaviy bilimlarning ajralmas bir qismdir, chunki fizika fanning o’rganish bilan uning tarixi ham o’rganib boriladi, bu fanning qonun qoidalarning ochilishi, ushbu qonun ochishda olimlarning tutgan o’rnidir[4]. Fizika tarixini bilish uchun asosan shu qonunlarning yozilishini, asl nusxasini o’rganish uchun asil ma’nosini o’rganishda muhim o’rin egallaydi. Har qanday fanning tarixini o’rganishda, bu fan aslida qanaqangi fan, bu fan o’z taraqqiyotida umumbashariy rivojlanishda jamiyat taraqqiyotida qanaqangi o’rin tutadi va qanday metodlar bilan o’rganilishi kerak. Shu nuqtai nazardan qaragan fizika tarixi o’rganish, jamiyat taqqiyotining bosqichini o’rganishda tabiatshunoslik fanning yetakchilari bilan bir qatorda tutgan o’rni muhimdir. Chunki fizika atrofimizni o’rab olgan tabiat qonunlarni o’rganish bilan bog’liqlikligi tufayli, uni o’rganish tarixi ham juda qadimiy bo’lib, hozirgi kungacha 2000 yildan ortiq vaqtni o’z ichiga oladi[5]. 173 “Talqin va tadqiqotlar” Respublika ilmiy-uslubiy jurnali №7 Fizika fani o’tgan XX asr davomida shunchalik o’zgarib ketdiki, fizika fani o’rganishda uning oddiy fizik xodisalardan boshlab uning hozirga kundagi murakkab taraqqiyotda davom etayogan taraqqiyotini inson ongiga singdirib borish uchun ham fizikani fan tarixi bilan biog’lab o’rganish muximdir. Fizika tarixi fani fizikaviy bilimlarning ajralmas bir qismdir. Shu sababli fizika fanini o’rganish fizika o’qituvchilaridan fizikani chuqur o’rganish, uni analiz qilish, qonunlarining bir biriga bog’liqligini ochib berish, o’quvchilarga uni bir-biri bilan uzviy bog’liqligini ko’rsatib berish, qonunlarni moxiyatini tushuntirish, fanning jamiyat taraqqiyotida tutgan o’rnini ochib berilishidan iboratdir. Fizikaning rivojlanish tarixini o’rganishda quyidagi vazifalarni amalga oshiriladi: - Tarixiy faktlarni topish va oydinlashtirish; - Tarixiy faktik materiallarni analiz qilish, uning moxiyatini ochish; - Fizikaviy umumiy qonunlarni o’zaro bog’liqligini ochish. Dunyo taraqqiyotidagi barcha fanlar tarixi asosiy imkoniyat yo’nalishidagi vazifani hal qiladi: 1. Tabiat qonunlari tarixi; 2. Jamiyat qonunlari tarixi. Tarixiy fanlar shuni ko’rsatadiki, fizika fani o’z rivojlanishida boshqa fanlar bilan birinchi navbatda tabiatshunoslik fani tarixi bilan uzviy bog’liqdir, fizika fani esa bu fanlarning rivojlanishida o’zining munosib xissasini qo’shadi. Shuning uchun ham ko’pgina fanlar rivojlantirgan olimlarni ham ko’p qirrali deb qarashimiz mumkin. Bular fizika, astronomiya, ximiya, biologiya, matematika va h.k. fanlarning rivojlanishiga kattagina hissa qo’shgan olimlar Abu Rayxon Beruniy, Mirzo Ulug’bek, Ibn Sino, R. Boel, E. Mariott, G.Kavendesh va boshqalar shular jumlasidandir. Fanlar tarixini o’rganar ekanmiz qadimda bu fanlarning barchasi falsafiy qarashlardan kelib chiqqanini ko’rishimiz mumkin. Qadimda olimlarning “Donolik ishqibozlari” deyilgan[6]. So’ngra fanlar o’rganilib qonuniyatlarning ochilishi natijasida barchasi alohida fan sifatida shakllana bordi. Fizika fani ham o’z rivojlanishida ko’pgina fanlar bilan bog’liqdir. Masalan fizika fani o’z rivojlanish tarixida matematika fani bilan uzviy bog’liqdir, chunki matematikasiz fizik qonunlari tushuntirish mumkin emas. Matematikani ilmiy metod sifatidagi ahamiyati fizikani o’qitishda juda keng va yaxshi o’z aksini topdi. Ular matematika formulalarida ifodalanadi, bular esa fizik qonunlardan xulosaalar chiqarishda ayrim holatlarni isbotlashda va fizik masalalarni echishda ishlatiladi. Fan tarixida minglab yillar davomida astronomlar osmon xodisalari to’g’risida faqat ko’zga ko’rinadigan yorug’lik vostasida olinadigan informastiya bilan cheklanib keladilar. Ular bu xodisalarni elektromagnit 174 “Talqin va tadqiqotlar” Respublika ilmiy-uslubiy jurnali №7 nurlanishlarning keng spektridagi torgina tirqishi orqali o’rganar edilar deb aytish mumkin. 1965- yillar radiofizikasining rivojlanishi tufayli radioastronomiya vujudga keldi va bizning koinot haqidagi tasavvurlarimiz xaddan tashqari kengayib ketdi. So’ngi yillarda muxim o’zgarishlar bo’ldi. Astronomiyadagi astrofizika ko’proq ahamiyat kasb eta boshladi va ishlab chiqarishni rivojlantirishda, ishlab chiqarish jamiyatning moddiy farovonligining o’sishiga olib keladi[7]. Demak, fanning rivojlanishi ishlab chiqarishni boshqarar ekan, ishshlab chiqarish fizikaning yangi qonunlar ochishiga har doim ta’zyiq o’tkazib turadi. Masalan, Gyugensning mayatnik nazariyasi – mexanik soatning ixtiro qilishga olib keldi. Termodinamikadan S.Karno stikli bug’ mashinsining ixtiro etinlishiga asos bo’ldi. A.S.Popov tomonidan radioning kashf etilishi radiofizikaning rivojlanishiga olib keldi va hakozolar. Shuningdek fizika fanining rivojlanish tarixi nazar solsak, fanning taraqqiyoti bevosita ishlab chiqarishni rivojlantirish tarixi bilan bog’liqligini ko’ramiz. Chunki fizik fanining yutuqlari bevosita texnika va texnalogiyalarda qo’llanib, u esa o’z navbatida ishlab chiqarish kularini rivojlantirishda muxim o’rni egallaydi. Fizika fanining rivojlanishi jamiyatning ijtimoiy tuzilishiga ham bog’liqdir. Chunki fanning rivojlanishida jamiyatning iqtisodiy siyosiy tuzilishi ham muhim rol o’ynaydi. Masalan: Quldorlik tuzumi davridan keyingi ilk feodalizm davrida fan taassurotlar bilan rivojlanmadi, chunki o’sha davrdagi quldor ham, feodal ham ishlab chiqarishni rivojlantirishi bilan qiziqmas edi. Fizika faqat ishlab chiqarish bilan bog’liq bo’libgina qolmay, u barcha tabiiy fanlar bilan ham bog’liqdir. Buni qanning tarixi o’rganishimizda ko’rib boramiz. Fizika fani rivojlanish tarixini quyidagi davrlari uning rivojlanishiga qarab o’rganib boriladi. 1. Qadimgi zamon fizikasi(fizik tushunchalarning shakillanish davri) a) antik davr fizikasi (eramizdan oldingi III asrga qadar bo’lgan davr) b) O’rta asr fizikasi (III-X asr) v) Geliostentrik sistema uchun kurash(X-XVI asr ) 2. Klassik fizikaning asosiy yo’nalishlar shakllanisi davri a) Ilmiy inqilobning tugallanishi (XVIII asr). b) Fizikaning asosiy yo’nalishlarining rivojlanishi (XIX asr). 3. Fizikada ilmiy inqilobning asosiy yo’nalishlari (zamonaviy fizika shakllanisl davri) a) Elektron nazariya va elektrodinamikaning rivojlanishi. b) Eynshteyning nisbiylik nazariyasi v) Atam va yadro fizikasining rivojlanishi. g) Fizikaviy inqilobning birinchi bosqichi Rezerford-Bor nazariyalar, kvant mexanikaning rivojlanishi. d) yadro fizikasining rivojlanishi 1918 – 1938 yillar. 175 “Talqin va tadqiqotlar” Respublika ilmiy-uslubiy jurnali №7 Fizika fani mazmuni fizikaning rivojlanish bosqichlari bo’yicha tanlansa, maqsadga muvofiq bo’ladi[8]. Chunki fizika fanining rivojlanishi jamiyatning iqtisodiy va siyosiy rivojlanishi bilan birgalikda qaralishi lozim. Ma’lumki, har qanday fanning rivojlanishi jamiyat ehtiyojlarini qondirish bilan uzviy bog’liqdir. Shunday qilib fizika fani rivojlanish tarixini o’rganish o’quv jarayoni va tarbiyaning ko’pgina muammolarini hal etish bilan birgalikda, fizika fanini o’qitish uslubining ham rivojlanishida muhim omil hisoblanadi. Tarixiy hujjatlardan dars jarayonida quyidagi shakllarda foydalanish tavsiya etiladi: - tarixiy voqyea va hujjatlar asosida kirish so’zi bilan muammoli vaziyatni yuzaga keltirish orqali darsning samaradorligini oshirish; - o’tilgan mavzuni yakunlashda o’quvchilar bilimini bir tizimga keltirish maqsadida tarixiy hujjatlardan foydalanish; - o’quvchilar bilimining ishonarli bo’lishiga erishish uchun buyuk kashfiyotlar, fundamental tajribalar tarixini bayon etish; - o’quvchilar shaxsini takomillashtirish, milliy mafkura va tarbiyani amalga oshirish maqsadida buyuk olimlar hayoti va ilmiy faoliyatidan ayrim lavhalarni keltirish; - tarixiy mazmundagi masalalardan dars jaryonida foydalanish. Tarixiy hujjatlarni ushbu shaklda bayon etish shartli bo’lib, ba’zan, bir necha usullar birgalikda qo’llanilishi mumkin[9]. O’tilayotgan mavzu ishonarli bo’lishi uchun tarixiy tajribalar, kashfiyotlar, chizmalar, grafiklar, rasmlar, modellar orqali tushuntiriladi. Xuddi shuningdek, moddiy va abstrakt modellar, kinofilmlardan foydalanish ham mumkin. O’rta maktab fizika kursida 100 dan ortiq fizik olimlar nomi qayd etiladi. Ushbu olimlarning hayoti va ilmiy kashfiyotlari qanchalik darajada va qanday tartibda berilishi kerak. Albatta, o’quv rejasida keltirilgan vaqt davomida barcha olimlarning hayoti va ilmiy faoliyatini to’la yoritishning imkoniyati yo’q. Shu sababli ba’zi olimlar hayotidan ayrim lavhalarni, jumladan, dars mavzusiga mos kelganlarini tanlab olish maqsadga muvofiqdir. Ba’zan olimlar tomonidan aytilgan hikmatli so’zlar, masalan,T.Yung aytganidek, «Odam qilgan ishni boshqa odam ham bajara oladi» yoki olimlar tomonidan aytilgan hazil-mutoyibalar darsning qiziqarli o’tishiga sabab bo’ladi. Buyuk olimlar hayotidan lavhalar keltirish orqali yoshlarda mehnatsevarlik, halollik, vatanparvarlik kabi tuyg’ularni shakllantirishga erishiladi. Masalan: alXorazmiy, Ibn Sino, Beruniy, Forobiy, Ulug’bek, Qori Niyoziy, R.Bekjonov, A.K. Otaxo’jayev kabi olimlarimizning hayoti va ilmiy faoliyatini yoritish orqali milliy g’urur, iftixor, mustaqillik, ozodlik g’oyalarini o’quvchilar ongiga singdirish imkoniyati yaratiladi. Olimlar hayoti bilan tanishish jarayonida ularning faqat ijobiy 176 “Talqin va tadqiqotlar” Respublika ilmiy-uslubiy jurnali №7 tomonlari olinmasdan, balki, turmushda va ilmiy izlanishda yo’l qo’ygan xato va kamchiliklari to’g’risida ham to’xtash maqsadga muvofiqdir. Masalan V. Tomson va Klauziuslarning «Olamning issiqlik halokati» to’g’risidagi fikri yoki "Efir", "teplorod" nazariyasi tarafdorlarining noilmiy fikrlari darsning qizikarli bo’lishiga xizmat qiladi. Xulosa qilib shuni aytish mumkinki, dars jarayonida buyuk fizik olimlarning dunyoqarashi va ilmiy kashfiyotlari haqida ma’lumotlar berish orqali darsni qiziqarli o’tish va uning samaradorligini oshirish kabi bir qator didaktik vazifalarni amalga oshirish mumkin. Foydalanilgan adabiyotlar: 1. Kudryavsev P.S. Kurs istorii fiziki. M. 1989; 2. Ziyaxanov R.F., Mirzayev A.T., Astronomiya va fizika tarixi, I qism, Toshkent, 2008; 3. Pannekuk A, Istoriya astronomii, M., Nauka, 1966; 4. Mirzayev A.T. Astronomiya tarixi. (ma’ruzalar matni). T. О‘zMU. 2000; 5. Mamadazimov M.M. Astronomiya va fizika tarixi. (ma’ruzalar matni), T. TDPU, 2001; 6. Mamadazimov M.M. Ulug‘bek va uning rasadxonasi, T. О‘zbekiston, 1994; 7. Gorbatskiy V.G., Leksii po istorii astronomii. SPgu, 2002; 8. Astronomicheskiye dati i otkritiya, http://astro.websib.ru/istor/istor.htm; 9. http://naturalhistory.narod.ru/Page_1.htm. 177 

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Bangabandhu Sheikh Mujib Medical University Journal 2023;16(4):198-204 bsmmuj.org ORIGINAL ARTICLE Quality of life among adolescents with substance use disorders Rubaiya Khan1, Mohammad Shamsul Ahsan2, Md. Tanvir Rahman Shah2, Bijoy Kumar Dutta3, Mukul Chandra Nath4 1 Department of Psychiatry, Khwaja Yunus Ali Medical College and Hospital, Sirajganj, Bangladesh of Psychiatry, Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh 3Department of Psychiatry, Chottogram Medical College Hospital, Chottogram, Bangladesh 4Department of Psychiatry, Marine City Medical College and Hospital, Chottogram, Bangladesh 2Department Correspondence to: Dr. Rubaiya Khan, Email: rubaiyakhan1201@yahoo.com ABSTRACT Background: Adolescents are vulnerable to substance use, which profoundly affects their func on, feelings and quality of life (QoL). This study aimed to assess the physical health, psychological, social rela onships, and environmental domains of QoL among adolescents with substance use disorders (SUDs). Methods: A cross-sec onal study was done from October 2020 to September 2021 among 44 adolescents selected purposively from Central Drug Addic on Treatment Center (CDC), Dhaka and Ashok Punorbashon Nibash (APON), Manikganj. Clinical diagnosis was assigned using The Diagnos c and Sta s cal Manual of Mental Disorders, Fi h Edi on (DSM-5) by psychiatrists. Then, they were interviewed with a ques onnaire and a Bengali Version of the World Health Organiza on quality of life scale brief version (WHOQOL-BREF). Results: Among the 44 adolescents with SUD, 18 (40.9%) rated their QoL as poor, 14 (31.8%) as very poor, 8 (18.2%) as neither poor nor good, and only 4 (9.1%) as good. Nearly half of them (47.7%) reported that they were dissa sfied, one third (29.5%) were very dissa sfied with their health. Among the four domains, the psychological domain (9.8±2.3) had the lowest mean score, followed by physical health (10.5±2.7), social rela onships (10.6±2.6), and environmental domain (11.5±2.2) of the WHOQOL-BREF. Mul ple drug users had poorer scores of their QoL than single drug user. Conclusion: Our study demonstrated that all domains of QoL (physical, psychological, social, and environmental) are impaired in adolescents with SUDs. Keywords: quality of life, substance use disorders, adolescents National Institute of Mental Health, Dhaka during the INTRODUCTION Substance use disorders (SUDs) are one of the alarming mental health issues in adolescents throughout the world, which lead to impairment and distress. There periods of 2017 to 2018 showed a 2.9% prevalence of substance use among seven years and above children and adolescents.3 are substantial geographical variations in the prevalence In several studies, it was found that quality of life (QoL) of SUD among adolescents. A study done in the United is poorer among substance-dependent individuals and States using data from the National Household Survey substance use disorders treatment seekers than among on Drug Use and Health (NSDUH) shows that 7-9% of cohorts without substance use disorders.4, 5 SUDs are adolescents in the US between the ages of 12-17 years associated with a wide range of serious health, social, disorders.1 and economic complications. The health status of In a community-based cross-sectional study carried out alcohol and drug abusers is generally affected by their among 3564 children of Bangladesh aged 5 to 17 years abuse. Consequently, their life expectancy is often much in 2009, substance-related disorders was found in 0.8% lower of respondents. In the case of adolescents aged 12 years relational, and judicial problems are also well- and above, substance abuse was 2.1%.2 A nationwide documented among substance abusers. Drug and met the criteria for an alcohol or illicit drug household-based cross-sectional study conducted by the than the general population.6-9 Housing, alcohol abuse further causes high costs due to frequent Received: 18 June 2022; Revised version received: 16 Oct 2023; Accepted: 4 Nov 2023; Published online: 23 Nov 2023 DOI: https://doi.org/10.3329/bsmmuj.v16i4.60341; ISSN 2074-2908, eISSN 2224-7750; © CC-BY-NC-4.0 This work encompasses the MD thesis of Dr Rubaiya Khan Quality of life among adolescents with substance use disorders 199 Research instruments HIGHLIGHTS 1. Quality of life is lower among adolescents with substance use disorders. 2. Among the four domains of the WHOQOL-BREF scale, the psychological domain is affected most by adolescent substance usage. 3. Multiple substance use affect QoL more adversely than single substance use. a. Questionnaire for socio-demographic and related variables for the study of QoL A semi-structured questionnaire in Bangla was designed by the researcher to collect information regarding sociodemographic variables such as age, sex, residence, religion, level of education, family type, family history of substance use, age of onset of taking the drug, the and multiple hospitalizations and treatment episodes.10 reason for initiation of substance use, name of substance currently using, route of administration, A study revealed that adolescents who met the criteria monthly expenditure, history of receiving treatment and for SUDs had reduced scores across all domains of QoL, admission in hospital for substance use, etc. with the most observable effects on academic achievement and school adjustment.11 Stevanovic et al. b. DSM-5 criteria for SUD found that adolescents who reported using substances The DSM-5 criteria for substance use disorders were had significantly lower scores in total, physical, used to diagnose SUDs in adolescents. The DSM-5 emotional, social, and school functioning domains of recognizes SUDs resulting from the use of ten separate the Pediatric Quality of Life Inventory (PedsQL) than classes those who did not report using any substance.12 On the hallucinogens, inhalants, opioids, sedatives-hypnotic-or other hand, a moderate reduction in the frequency of anxiolytics, stimulants, tobacco, and other unknown substance use among adolescents was associated with substances.14 improvements in of drugs: alcohol, caffeine, cannabis, QoL.13 c. World Health Organization quality of life scale, brief The primary objective of our study was to assess the version (WHOQOL-BREF) quality of life in relation to sociodemographic factors among adolescents with SUDs. Our secondary objective This study used WHOQOL-BREF to assess the quality was to compare the quality of life among adolescents of life of adolescents with SUD. The scale had 26 items, using single and multiple substances. among which the first two items are used to measure an METHODS Study design and participants This cross-sectional study was done from October 2020 to September 2021 in the Central Drug Addiction Treatment Centre (CDC), Tejgaon, Dhaka, and Ashokti Punorbashon Nibash (APON), Singair, Manikganj. A total of 44 adolescents within the range of 11 to 19 years of either sex with SUD who attended the inpatient and outpatient department of the CDC and APON were purposively included in the study. However, adolescents in an intoxication or withdrawal state who were mute and those with non-communicable were excluded from the study. We obtained ethical approval from the Institutional Review Board of Bangabandhu Sheikh Mujib Medical University. individual’s overall perception of QoL and overall perception of their health. The remaining 24 items measured the following four domains: physical health, psychological, social relationships, and environment.15 The WHOQOL-BREF has been translated into Bangla and validated for Bangladeshi adolescents.16 Data collection Prior permission was taken from the authority of two institutions: CDC and APON. Participants and their parents were informed about the study's purpose, method, and outcome. Informed written assent/consent was taken from the patients and one of their parents before data collection, as appropriate, by face-to-face interviews. Qualified psychiatrists made clinical diagnoses by applying DSM-5 criteria of SUD. Then, socio-demographic information was documented using the semi-structured questionnaire. The Bangla version Khan R et al. Bangabandhu Sheikh Mujib Medical University Journal 2023; https://doi.org/10.3329/bsmmuj.v16i4.60341 Quality of life among adolescents with substance use disorders of the WHOQOL-BREF was applied to evaluate the QoL. One of the investigators (RK) read out the questions and recorded the answers given by the participants. Data analysis TABLE 1 Socio-demographic characteristics and substance use among the study participants with substance use disorder (n=44) Variables Age, years Age of onset of substance use, years Monthly expenditure due to substance use (BDT) Descriptive statistics such as frequency (percent) or mean (standard deviation) were used as appropriate. Student’s t test was used to compare the mean score of four domains (physical health, psychological health, social relationship, and environment) of the WHOQOLBREF categories. The analysis of variance was used to compare more than two categories (e.g., education). P<0.05 was considered statistically significant. RESULTS Forty-four adolescents with SUDs were included in this study, among which 24 were from APON and 20 from CDC. The mean age of the study participants was 16.5 (2.3), 88.6% being male. The majority (79.6%) of the respondents were from urban areas. About half (43.2) of the parents were divorced, widows, widowers, or separated. About 80% were from nuclear families (TABLE 1). About 47.7% of the respondents had a family history of substance use. The mean age of onset of substance use was 12.2 years. The mean monthly expenditure due to substance use was BDT 11,579. A little less than half (40.9%) of them rated their QoL as poor, and 31.8% as very poor. About 48.0% of the respondents were dissatisfied with their health, but 9.1% were satisfied (FIGURE 1). The most commonly used substance was tobacco (75.0%), followed by cannabis (65.9%), stimulant (45.5%), alcohol (38.6%), inhalant (18.2%), sedative (15.9%), and opioid (9.1%) (FIGURE 2). The mean standard deviation scores of physical health, 200 Sex Male Female Residence Urban Rural Education Primary or below Secondary school Higher secondary or above Type of family Nuclear Joint Marital status of parents Married Separated/ divorced/ widow/ widower Family history of substance use Yes No Reason for initiation of substance use* Peer pressure Curiosity Broken family Family negligence Failure in study Failure in a love affair Number of substances currently used Single Multiple Route of administration* Smoking Swallowing Inhalation Injection History of treatment for substance use Yes No History of hospital admission for substance use Yes No Findings Mean 16.5 12.2 SD 2.3 3.5 11,579 Number 11,117 Percent 39 5 88.6 11.4 35 9 79.5 20.5 16 13 15 36.4 29.5 34.1 35 9 79.5 20.5 25 19 56.8 43.2 21 23 47.7 52.3 18 19 9 11 2 6 40.9 43.2 20.5 25.0 4.5 13.6 10 34 22.7 77.3 40 25 17 1 90.9 56.8 38.6 2.3 24 20 54.5 45.5 17 27 38.6 61.4 SD indicates standard deviation *Multiple responses DISCUSSION psychological, social relationships, and environmental domains were 10.5 (2.7), 9.8 (2.3), 10.6 (2.6), and 11.5 SUDs (2.2), respectively. According to the WHOQOL-BREF uncontrolled use of a particular substance despite are complex condition characterized by scale, a higher score indicates a better quality of life. harmful consequences. Adolescents are at greater risk of Therefore, the psychological domain was most impaired substance use. During adolescence, development occurs among the four domains, followed by the physical in multiple domains like-emotional, social, cognitive, health, social relationships, and environmental domain and biological, and conditions like SUDs may affect (TABLE 2). adolescents’ behavior, lifestyle, and future life. Nowadays, SUD has increased significantly among adolescents and Khan R et al. Bangabandhu Sheikh Mujib Medical University Journal 2023; https://doi.org/10.3329/bsmmuj.v16i4.60341 Quality of life among adolescents with substance use disorders 201 young adults.17 SUD impairs individuals’ physical and psychological health and also jeopardizes general safety and social performance and, thereby, overall quality of TABLE 2 Relationship of WHOQOL-BREF scores and sociodemographic variables of the respondents (n=44) Variables Mean (standard deviation) scores of Quality of Life Physical Health Psychological Social Relationships Environmental Overall 10.5 (2.7) Gender Male 10.5 (2.7) Female 10.6 (2.9) P* 0.97 Residence Urban 10.7 (2.5) Rural 10.6 (2.9) P* 0.74 Education Primary or below 9.6 (2.5) Secondary school 11.1 (2.4) Higher secondary or 11.1 (2.6) above P* 0.18 Type of family Nuclear 10.5 (2.5) Joint 10.6 (3.5) P* 0.90 Marital Status of Parents Married 10.9 (3.0) Separated/divorced/ 9.3 (2.7) widow/widower P* 0.27 History of receiving treatment Yes 9.4 (2.3) No 12.0 (2.5) P* 0.001 History of admission to the hospital Yes 9.4 (2.3) No 11.3 (2.7) P* 0.02 Number of substances Single 13.0 (1.9) Multiple 9.8 (2.5) P* 0.001 9.8 (2.3) 10.6 (2.6) 11.5 (2.2) 9.8 (2.3) 9.6 (2.3) 0.85 10.6 (2.6) 10.2 (2.3) 0.74 11.4 (2.2) 11.4 (2.0) 0.91 9.8 (2.3) 10.1 (2.3) 0.57 11.0 (2.4) 11.0 (3.1) 0.61 11.8 (2.3) 11.6 (2.0) 0.32 9.2 (2.3) 10.0 (2.1) 10.3 (2.5) 11.0 (2.6) 11.5 (1.8) 11.1 (2.8) 12.3 (2.8) 11.5 (2.4) 12.5 (2.5) 0.41 0.88 0.64 9.9 (2.4) 9.2 (1.8) 0.35 10.9 (2.6) 9.1 (1.1) 0.03 11.6 (2.2) 10.5 (1.5) 0.09 10.2 (2.7) 9.2 (1.7) 10.2 (2.2) 9.8 (2.1) 12.0 (2.3) 11.4 (2.8) 0.03 0.37 0.98 8.8 (2.3) 10.1 (2.2) 0.002 10.5 (2.1) 10.7 (3.1) 0.90 11.2 (2.1) 11.5 (2.3) 0.90 9.1 (2.2) 10.3 (2.4) 0.10 10.7 (2.1) 10.6 (2.9) 0.81 11.4 (2.3) 11.5 (2.1) 0.81 11.8 (2.2) 9.2 (2.1) 0.005 12.1 (2.3) 10.1 (2.5) 0.03 12.5 (2.2) 11.1 (2.1) 0.11 life. Thus, the present study aimed to evaluate the quality of life among adolescents with SUD. FIGURE 1 Perception of quality of life and satisfaction about health among the participants of substance use disorder (n=44). Our study's mean scores for four domains were from 9.8 to 11.5 According to the WHOQOL-BREF scale, the higher the mean scores, the better the quality of life. We found that the mean scores of physical health, psychological, social relationships, and environmental domains of WHOQOL-BREF were higher in the healthy male adolescent population of both slum and residential areas of Dhaka city than the mean scores of these four domains of our respondents who were substance users.18 This indicates that all four domains of the QoL of adolescents with SUD were low compared to the healthy adolescent male population. *Student t-test or ANOVA relationships, and environmental domains were low.19 An international study involving 2393 adolescents from A study done in Etawah, India, on 145 street children 13 seven countries using the Pediatric Quality of Life -18 years of age abusing substances revealed that mean Inventory scores substances have a significantly lower score in PedsQL of physical health, psychological, social (PedQL) showed adolescents using physical, emotional, social, and school functioning domains.12 Most adolescents rated their QoL as poor or very poor. Most of the substance-dependent adolescents were dissatisfied with their health, and only a few were satisfied. Though the QoL among adolescents with SUD is a major public health concern, QOL assessment using the WHOQOL-BREF among adolescents was minimal. Therefore, comparing this study’s results concerning the perception of quality of life and satisfaction with health FIGURE 2 Proportion of type of substance use among the respondents with substance use disorders (n=44). with other studies was impossible. Khan R et al. Bangabandhu Sheikh Mujib Medical University Journal 2023; https://doi.org/10.3329/bsmmuj.v16i4.60341 Quality of life among adolescents with substance use disorders 202 Among the four domains, the psychological domain was educational background is almost identical to the mostly affected in adolescents with SUD. Psychological national data. Moreover, it is similar to other studies.25 distress is a common experience for patients with SUD, Broken family and history of substance use in the family partly due to substance use and its consequences and increase the risk of adolescent substance use.26 partly due to psychiatric disorders that often coexist with SUDs, resulting in a double burden of symptoms and problems.20 Approximately 70-80 percent of youths presenting to treatment for SUD are duly diagnosed.21 A study in Bangladesh revealed that among the patients In the present study, the average age of onset of substance use was 12 years, and the reasons for initiation were peer pressure, curiosity, broken family, family negligence, academic failure, and relationship with first-episode psychosis, lifetime SUD was double failure. Sawhney and Kaur also reported similar age of that of control.22 The temporal relationship between onset, and the reasons were enjoyment, showing substance use and psychological symptoms, such as manhood and fun, frustration, curiosity, peer pressure, depression, anxiety, or psychosis, should be considered. and imitating their father and siblings.25 Substance use may be a perpetuating factor for The finding of this research suggests that adolescents deteriorating mental health. On the other hand, with SUD have reduced QoL across all domains. impaired mental health may also have preexisted before the substance use, precipitated the substance use, and may have deteriorated further.23 Therefore, assessment of the psychological domain and providing appropriate interventions toward treating both SUD and psychiatric disorders simultaneously is mandatory for a better QoL. Therefore, a biopsychosocial approach through a multidisciplinary team should be implemented while managing such patients. Management should include treatment of co-morbid physical and psychiatric illness, psychological intervention, and social rehabilitation to improve and maintain QoL. Proper psychological The impairment in physical health may be due to assessment by a psychiatrist and a more integrated increased risk for injury and violence associated with approach are needed for a better QoL. Awareness substance use, respiratory problems due to smoking, or programs for society about the disease, treatment, and inhalation of substances. Co-occurring psychiatric outcome of SUD should be implemented. Psychiatrists, disorders may have contributed to lower scores in the governmental organizations, and non-governmental psychological social working on drug use should deal with this condition relationships, adolescents with multiple substance use holistically. The QoL assessment, therefore, should be may be more neglected by the family or society due to considered in health care. QoL measures are needed to social stigma. Those who go to hospital might have be routinely included in the evaluation of treatments. domain. In an aspect of severe problems of SUD as indicated by our data. Clear male dominance was (88.6%) This study has several limitations too. First, SUDs were observed. diagnosed by psychiatrists using DSM-5 criteria, where Throughout the world, the prevalence of substance use a structured interview could give a more accurate is higher in males than in females.24 Due to social diagnosis. Second, small number of participants from restrictions and the society's attitude, male dominance selected drug addiction treatment centres, lack the is also observed in our country. The majority (79.6%) of representativeness, and therefore, findings cannot be the patients hailed from urban areas. As the sample was generalized. Third, a cross-sectional study design collected from two centres near Dhaka city, most cannot provide a causal relationship. attendants belonged to the metropolitan area. It may also refer that adolescents from urban areas have better The quality of life is impaired in adolescents with SUD. access to services. Education might have some link to adolescents' addiction behaviour. Conclusion However, our participant's The psychological domain is the most impaired among the four domains of the WHOQOL-BREF scale. In addition, physical health, psychological, and social Khan R et al. Bangabandhu Sheikh Mujib Medical University Journal 2023; https://doi.org/10.3329/bsmmuj.v16i4.60341 Quality of life among adolescents with substance use disorders 203 relationships domains are significantly impaired in 4. Donovan D, Mattson ME, Cisler RA, Longabaugh R, Zweben A. Quality of life as an outcome measure in alcoholism treatment research. Journal of Studies on Alcohol, Supplement. 2005 Jul(15):119-39. DOI: https:// doi.org/10.15288/jsas.2005.s15.119. 5. Smith KW, Larson MJ. Quality of life assessments by adult substance abusers receiving publicly funded treatment in Massachusetts. Am J Drug Alcohol Abuse. 2003 May;29 (2):323-35. DOI: https://doi.org/10.1081/ada-120020517. 6. De Alba I, Samet JH, Saitz R. Burden of medical illness in drug- and alcohol-dependent persons without primary care. Am J Addict. 2004 Jan-Feb;13(1):33-45. DOI: https:// doi.org/10.1080/10550490490265307. 7. Price RK, Risk NK, Murray KS, Virgo KS, Spitznagel EL. Twenty-five year mortality of US servicemen deployed in Vietnam: predictive utility of early drug use. Drug Alcohol Depend. 2001 Nov 1;64(3):309-318. DOI: https:// doi.org/10.1016/S0376-8716(01)00134-X. 8. Sørensen HJ, Jepsen PW, Haastrup S, Juel K. Drug-use pattern, comorbid psychosis and mortality in people with a history of opioid addiction. Acta Psychiatr Scand. 2005 Mar;111(3):244-249. DOI: https://doi.org/10.1111/j.16000447.2004.00445.x. 9. Wahren CA, Brandt L, Allebeck P. Has mortality in drug addicts increased? A comparison between two hospitalized cohorts in Stockholm. Int J Epidemiol. 1997 Dec;26(6):12191226. DOI: https://doi.org/10.1093/ije/26.6.1219. 10. Xie X, Rehm J, Single E, Robson L, Paul J. The economic costs of alcohol abuse in Ontario. Pharmacol Res. 1998 Mar;37(3):241-249. DOI: https://doi.org/10.1006/ phrs.1998.0293. 11. Clark DB, Kirisci L. Posttraumatic stress disorder, depression, alcohol use disorders and quality of life in adolescents. Anxiety. 1996;2(5):226-233. DOI: https://doi.org/10.1002/ (SICI)1522-7154(1996)2:5%3C226::AID-ANXI4%3E3.0.CO;2 -K. 12. Stevanovic D, Atilola O, Balhara YP, Avicenna M, Kandemir H, Vostanis P, Knez R, Petrov P. The relationships between alcohol/drug use and quality of life among adolescents: An international, cross-sectional study. Journal of child & adolescent substance abuse. 2015 Jul 4;24(4):177-85. DOI: https://doi.org/10.1080/1067828X.2013.773864. 13. Becker SJ, Curry JF, Yang C. Longitudinal association between frequency of substance use and quality of life among adolescents receiving a brief outpatient intervention. Psychol Addict Behav. 2009 Sep;23(3):482-490. DOI: https:// psycnet.apa.org/doi/10.1037/a0016579. 14. American Psychiatric Association. Substance-related and Addictive Disorders. Desk Reference to the Diagnostic Criteria from DSM-5. American Psychiatric Association; 2013:227–284. 15. Whoqol Group. Development of the World Health Development of the World Health Organization WHOQOLBREF quality of life assessment. The WHOQOL Group. Psychol Med. 1998 May;28(3):551-558. DOI: https:// doi.org/10.1017/S0033291798006667. 16. Izutsu T, Tsutsumi A, Islam A, Matsuo Y, Yamada HS, Kurita H, Wakai S. Validity and reliability of the Bangla version of WHOQOL-BREF on an adolescent population in Bangladesh. Qual Life Res. 2005 Sep;14(7):1783-1789. DOI: https:// doi.org/10.1007/s11136-005-1744-z. multiple substance users. The physical health domain is significantly lower among those who seek services. A comprehensive management plan is necessary to improve the health of adolescents with SUDs. Longitudinal studies using a more representative sample are warranted. Acknowledgments The authors are grateful to all the adolescents who participated in this study. Author Contributions Conception and design: RK, MSA. Acquisition, analysis and interpretation of data: RK, MSA, MTRS, BKD, MCN. Manuscript drafting and revising it critically: RK, MSA. Approval of the final version of the manuscript: RK, MSA, MTRS,BKD, MCN. Guarantor accuracy and integrity of the work: RK, MSA. Funding The study received financial support from Bangabandhu Sheikh Mujib Medical University. Conflict of Interest Authors declare no conflict of interest. Ethical Approval The study was approved by the Institutional Review Board of Bangabandhu Sheikh Mujib Medical University (Memo No: BSMMU/2021/126 issued on 13 February 2021). ORCID iD Rubaiya Khan https://orcid.org/0009-0007-4193-6090 REFERENCES 1. 2. 3. Mericle AA, Arria AM, Meyers K, Cacciola J, Winters KC, Kirby K. National Trends in Adolescent Substance Use Disorders and Treatment Availability: 2003-2010. J Child Adolesc Subst Abuse. 2015;24(5):255-263. DOI: https:// doi.org/10.1080/1067828X.2013.829008. Rabbani, M.G., Alam M.F., Ahmed HU, Sarker M, Chowdhury WA, Zaman MM, Chowdhury S, Chowdhury MWA, Das SK, Hamid MA, Islam MT, Mohir MA, Jahan NA, Rahman AHM, Choudhury S, Chowdhury KP, Wahab MA, Rahman F, Mandal MC, Hossain MD, Bhowmik AD, Bashar MK, Khan NM, Uddin MJ, Khan MJR. Prevalence of mental disorders, mental retardation, epilepsy, and substance abuse in children. 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Adolescent substance abuse: Psychiatric comorbidity and high-risk behaviors. Taylor & Francis; 2008. https://psycnet.apa.org/record/2008-05667000 (Access date: 18 Nov 23). 204 22. Ahsan MS, Mullick MS, Begum K, Arafat SM, Shah MA, Podder BR, Roy J. Substance Use among the Patients with First Episode Psychosis. Mymensingh Med J. 2018 Apr;27 (2):313-320. URL: https:// pubmed.ncbi.nlm.nih.gov/29769496/. 23. Vederhus JK, Pripp AH, Clausen T. Quality of Life in Patients with Substance Use Disorders Admitted to Detoxification Compared with Those Admitted to Hospitals for Medical Disorders: Follow-Up Results. Subst Abuse. 2016 May 18;10:31-37. DOI: https://doi.org/10.4137/SART.S39192. 24. Gureje O, Degenhardt L, Olley B, Uwakwe R, Udofia O, Wakil A, Adeyemi O, Bohnert KM, Anthony JC. A descriptive epidemiology of substance use and substance use disorders in Nigeria during the early 21st century. Drug Alcohol Depend. 2007 Nov 2;91(1):1-9. 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Adaptive divergence despite small population sizes and strong genetic drift in English yew (Taxus baccata L.) M. Mayol, M. Riba, D. Grivet, Giovanni G Vendramin, L. Vincenot, Santiago C. González-Martínez To cite this version: M. Mayol, M. Riba, D. Grivet, Giovanni G Vendramin, L. Vincenot, et al.. Adaptive divergence despite small population sizes and strong genetic drift in English yew (Taxus baccata L.). 14. MEDECOS & 13. AEET meeting. Human driven scenarios for evolutionary and ecological changes, Jan 2017, Séville, Spain. 268 p., 2017, XIV MEDECOS & XIII AEET meeting. Human driven scenarios for evolutionary and ecological changes. �hal-02735247� HAL Id: hal-02735247 https://hal.inrae.fr/hal-02735247 Submitted on 2 Jun 2020 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 XIV MEDECOS & XIII AEET meeting Human driven scenarios for evolutionary and ecological changes Abstract book 31st January - 4th February 2017 Seville, Spain Abstract book of the XIV MEDECOS & XIII AEET meeting, Seville, Spain, 31st January - 4th February 2017 Issued in Open Access, under the terms of Commons attribution Non Commercial License Doi.: 10.7818/MEDECOSandAEETCongress.2017 Editorial deadline: 20th December, 2016. Publisher: Asociación Española de Ecología Terrestre (AEET) C/ Tulipán s/n, 28399, Móstoles, Madrid, Spain Layout: Imprenta Sand, S.L. Seville. Spain. Symposium 09 XIV MEDECOS & XIII AEET meeting. Human driven scenarios for evolutionary and ecological changes Seville, Spain, 31st January - 4th February 2017 Sp.09: Local adaptation in the Mediterranean Sp.09-1-Main talk Why do we need to incorporate phenotypic variation into species distribution models? Benito Garzon, M.1 (1) INRA, France Correspondence e-mail: [marta.benito@gmail.com] Here I will focus on trees and explore i) how considering phenotypic variation can change species distributions ii) the difficulties arising for accounting for phenotypic variation due to the experimental designs and iii) how species and populations models based on the phenotypic variation can be used in management decisions. Extensive experiments with translocations of several tree species across Europe do exist and can be used to estimate the amount of the variation due to plasticity and to local adaptation with statistical approaches that account for the origins of phenotypic variation. However, in some cases, these experiments do not cover the entire range of the species because they have not been designed to understand the ecophysiological limits of the species. . Including phenotypic variation into species distribution models gives a more realistic output than classical species distribution models and allows us to visualize the phenotypic variation at species and population levels, a useful tool to design assisted migration programs for the future. Sp.09-2-Oral Ecological mechanisms underlying hybridization and its constraint in genus Erysimum Abdelaziz, M.1, Muñoz-Pajares, A.J.2, Perfectti, F.3, Gómez Reyes, J.M.4 (1) University of Granada. (2) CIBIO-InBIO, Universidade do Porto. (3) University of Granada. (4) CSIC Correspondence e-mail: [mabdelazizm@ugr.es] Hybrid zones have the potential to shed light on evolutionary processes driving local adaptation and speciation. Secondary contact hybrid zones are particularly powerful natural systems where study the interaction between divergent genomes to understand the mode and rate at which reproductive isolation accumulates during speciation. Using 10 microsatellites markers we studied five populations from two Erysimum (Brassicaceae) species presenting a secondary contact zone in Sierra Nevada Mountains (SE Spain). We have estimated the genetic differentiation, the genetic structure, and the amount of recent gene flow between populations. We found a narrow unimodal hybrid zone where the hybrid genotypes were more frequent than the parental genotypes. Afterwards, we carried out two experiments to explore the pre-zygotic and post-zygotic reproductive barriers underlying the origin and maintenance of this narrow hybrid zone. Post-zygotic barriers were evaluated performing over 1000 hand crosses on 44 plants from both species till the second generation. While to evaluate the pre-zygotic barriers, a plot of parental and hybrid flowering plants were presented to the pollinators in the natural populations to evaluate their preferences. We did not identify any significant reproductive barrier at the generation of F1 hybrids nor the pre-zygotic barriers. However, we identify a strong and significant postzygotic reproductive barrier in the F2 hybrid production. Our results suggest that the pollinators are the main promoters of the hybrid zone, but a constant migration of plants from the parental population would be necessary for the long-term maintenance of that narrow hybrid zone. 173 Symposium 09 XIV MEDECOS & XIII AEET meeting. Human driven scenarios for evolutionary and ecological changes Seville, Spain, 31st January - 4th February 2017 Sp.09-3-Poster Spatio-temporal flowering patterns in Mediterranean Poaceae. A community study in SW Spain Cebrino Cruz, J.1, García-Castaño, J.L.2, Domínguez-Vilches, E.3, Galán, C.4 (1) University of Córdoba. (2) University of Seville. (3) University of Córdoba. (4) University of Córdoba Correspondence e-mail: [jcebrinocruz@gmail.com] Grasses are one of the largest plant families and they are worldwide distributed. Numerous studies have focused on the phenology of different plants. However, lower attention has been given to grass species and to the effects of different land covers. The goals of this work were (i) to study the grass community composition in different land covers; and (ii) to assess the importance of the species and the land cover in the grass flowering phenology start and duration in two study years. Twenty-nine sampling points were randomly selected in three different land covers. We found differences among plants covers in grass species composition, showing the scrub the highest number of species. In addition, we detected differences on the flowering start date for some species –i.e. Hyparrhenia hirta (L.) Stapf was the earliest species whereas Melica magnolii Gren. & Godr. was the latest one-. Moreover, we detected differences for the land cover type and the study year. Species in the scrub flowered the latest whereas they flowered the earliest in the pasture in 2014 and in the riverbank in 2015. In general, the flowering start occurred later in 2014 than in 2015. In relation to the phenological length, differences for some study species were detected, being the shortest season for Aegilops triuncialis L. and the longest one for Hyparrhenia hirta. This study provides information for species and, especially, a spatially dependent answer, which might be important in the scenario of the predicted climate change. Sp.09-4-Oral Clinal variation in phenological behavior, size at flowering and biomass allocation reveals adaptation to climatic divergence in annual Thrincia hispida across its range Pedro, M. de1, Mayol, M.2, González-Martínez, S.C.3, Regalado, I.4, Riba, M.5 (1) CREAF (Centre for Ecological Research and Forestry). (2) CREAF (Centre for Ecological Research and Forestry). (3) INRA. (4) CREAF (Centre for Ecological Research and Forestry). (5) CREAF (Centre for Ecological Research and Forestry) Correspondence e-mail: [manueldepedrorodriguez@gmail.com] Understanding and predicting the consequences of global change on species persistence and distribution requires considering the extent and potential for local adaptations. It is generally accepted that local adaptation in plants is a widespread phenomenon. However, recent theoretical and empirical work suggests that adaptation could be constrained in short-lived plants during range expansions due to demographic processes and phenotypic and genetic correlations among traits. Our aim is to produce new experimental evidence of evolutionary mechanisms during plant range expansions. Here we present preliminary data on population differentiation in several life history traits: time to germination and flowering, size at flowering and biomass allocation in 42 populations covering the whole range of the short-lived colonizing plant Thrincia hispida. We found significant phenotypic variation among population in all measured traits in a common garden experiment. The timing of germination and flowering followed environmental clines with temperature and inter-annual variation of precipitation, indicating potential drivers of selection. Size at flowering was strongly correlated with phenology. Compared with plants from northern populations, plants from southern populations germinated later and flowered earlier, were smaller and allocated more biomass to reproduction. We conclude that southern populations have evolved to face short growing periods by evolving traits favoring short life cycles. This study provides clear evidence for local adaptation in phenological traits in response to environmental varying selection, and highlights the role of selection on phenological traits in explaining the variability in other correlated traits (e.g. size) closely linked to fitness. 174 Symposium 09 XIV MEDECOS & XIII AEET meeting. Human driven scenarios for evolutionary and ecological changes Seville, Spain, 31st January - 4th February 2017 Sp.09-5-Oral Reproductive isolation of colour morphs in Lysimachia arvensis through a combination of incomplete pre- and postzygotic barriers Jiménez-López, F.J.1, Pannell, J.R.2, Ortiz, P.L.3, Arista, M.4 (1) Universidad de Sevilla. (2) Université de Lausanne. (3) Universidad de Sevila. (4) Universidad de Sevilla Correspondence e-mail: [fjimenez16@us.es] The boundaries that define plant species have been much debated. Although absolute reproductive isolation is accepted as an unequivocal sign of speciation, partial isolation between divergent populations can also lead to speciation. Evolution of reproductive isolation depends on a considerable number of barriers. Tto understand the speciation process, it is necessary to identify all reproductive barriers that limit hybridization. In this study, we estimated the strength of prezygotic and postzygotic barriers between the two flower colour phenotypes, red and blue, of the herb Lysimachia arvensis. Our study points to a temporal separation between morphs at two stages: the blue morph begins to flower 10-20 days earlier than the red morph; and blue flowers open about 1 hour earlier in the morning and close about 1 hour later in the evening that red flowers. Pollen flow between morphs carried out by pollinators was always less frequent than expected, based on morph frequencies. Pollen of each morph showed the same fertilization capacity on the ovules of the other morph. F1 and F2 hybrids had lower seed production than parental plants. Our results indicate that none of these barriers to hybridization are complete, but it seems that a sum of incomplete barriers may cause effective isolation of each morph, reducing the chance of F1 hybridisation and contributing to the speciation process. Sp.09-6-Poster Adaptive divergence despite small population sizes and strong genetic drift in English yew (Taxus baccata L.) Mayol, M.1, Riba, M.2, Grivet, D.3, Vendramin, G.G.4, Vincenot, L.5, González-Martínez, S.C.6 (1) Centre de Recerca Ecològica i Aplicacions Forestals (CREAF-UAB). (2) CREAF-UAB. (3) INIA-CIFOR. (4) National Research Council of Italy. (5) Université de Rouen. (6) INRA Correspondence e-mail: [maria.mayol@uab.es] English yew (Taxus baccata L.) is a dioecious long-lived gymnosperm occurring in a great diversity of habitats in most of the European continent. However, populations are often small and isolated, show irregular size/age structures and biased sex ratios, and neutral genetic diversity is highly structured, so that the ability of this species to cope with local changing conditions in the face of impending climate change may be severely reduced. Here we provide an overview of our research on adaptive patterns of variation in this conifer, both at the phenotypic and molecular levels. Results from a clonal common garden experiment revealed the existence of two basic adaptive phenological patterns according to the regional origin (“mild-type” and “continental-type”) associated to temperature and precipitation clines. At the molecular level, new-generation outlier detection and environmental association analysis of 25,726 SNPs (1,210 candidate genes) in 120 individuals distributed along an environmental gradient in Europe identified several outliers associated with temperature and precipitation. In particular, 4 SNPs located on an early-responsive to dehydration stress protein (ERD4) were identified as a target of selection by all the different approaches used. Our results suggest that, despite small population sizes and strong genetic drift, divergent selection have contributed to population divergence in this conifer. However, we found that both current and past climatic data could explain the environmental associations detected, so adaptive patterns might be likely reflecting processes occurred in the distant past. 175 

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life Communication Ferlins and TgDOC2 in Toxoplasma Microneme, Rhoptry and Dense Granule Secretion Daniel N. A. Tagoe 1,† , Allison A. Drozda 1,† , Julia A. Falco 2 , Tyler J. Bechtel 2,‡ , Eranthie Weerapana 2 and Marc-Jan Gubbels 1, * 1 2 * † ‡



 Citation: Tagoe, D.N.A.; Drozda, A.A.; Falco, J.A.; Bechtel, T.J.; Weerapana, E.; Gubbels, M.-J. Ferlins and TgDOC2 in Toxoplasma Microneme, Rhoptry and Dense Granule Secretion. Life 2021, 11, 217. Department of Biology, Boston College, Chestnut Hill, MA 02467, USA; dnatagoe@gmail.com (D.N.A.T.); drozda@bc.edu (A.A.D.) Department of Chemistry, Boston College, Chestnut Hill, MA 02467, USA; falcoju@bc.edu (J.A.F.); tylerbechtel@gmail.com (T.J.B.); eranthie@bc.edu (E.W.) Correspondence: gubbelsj@bc.edu; Tel.: +1-617-552-8722 These authors contributed equally. Present Address: Merck & Co., Inc., Cambridge, MA 02141, USA. Abstract: The host cell invasion process of apicomplexan parasites like Toxoplasma gondii is facilitated by sequential exocytosis of the microneme, rhoptry and dense granule organelles. Exocytosis is facilitated by a double C2 domain (DOC2) protein family. This class of C2 domains is derived from an ancestral calcium (Ca2+ ) binding archetype, although this feature is optional in extant C2 domains. DOC2 domains provide combinatorial power to the C2 domain, which is further enhanced in ferlins that harbor 5–7 C2 domains. Ca2+ conditionally engages the C2 domain with lipids, membranes, and/or proteins to facilitating vesicular trafficking and membrane fusion. The widely conserved T. gondii ferlins 1 (FER1) and 2 (FER2) are responsible for microneme and rhoptry exocytosis, respectively, whereas an unconventional TgDOC2 is essential for microneme exocytosis. The general role of ferlins in endolysosmal pathways is consistent with the repurposed apicomplexan endosomal pathways in lineage specific secretory organelles. Ferlins can facilitate membrane fusion without SNAREs, again pertinent to the Apicomplexa. How temporal raises in Ca2+ combined with spatiotemporally available membrane lipids and post-translational modifications mesh to facilitate sequential exocytosis events is discussed. In addition, new data on cross-talk between secretion events together with the identification of a new microneme protein, MIC21, is presented. https://doi.org/10.3390/life11030217 Keywords: Apicomplexa; Toxoplasma; microneme; rhoptry; dense granule; exocytosis; DOC2 Academic Editor: Stanislas Tomavo Received: 14 February 2021 Accepted: 5 March 2021 1. Introduction Published: 9 March 2021 The opportunistic apicomplexan parasite Toxoplasma gondii causes significant disease in people with immature or compromised immune systems [1]. Central to the pathogenesis of all apicomplexan parasites, including malaria causing Plasmodium falciparum, is the rapid serial completion of lytic replication rounds causing tissue damage and inflammation. Completion of the lytic cycle requires host cell invasion and egress, which are mediated by sequential exocytosis of, respectively, the apically located micronemes and rhoptries and the more scattered dense granules organelles [2,3] (Figure 1A,B). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Life 2021, 11, 217. https://doi.org/10.3390/life11030217 https://www.mdpi.com/journal/life Life 2021, 11, 217 2 of 22 Figure 1. Schematic representation of the function and timing of secretion events in the T. gondii lytic cycle. (A). Schematic representation of T. gondii and its three secretory organelles, serving as legend for the other panels. (B). Schematic representation of secretion events throughout the lytic cycle. (C). Schematic representation of the levels of secretion of each organelle and the levels of intracellular calcium [Ca2+ ]c corresponding with the secretion events throughout the lytic cycle. Timeline on the X-axis corresponds with the events in panel B. Note that the invasion process completes in less than a minute and is shown in a stretched-out time window. The two spikes in [Ca2+ ]c at the time of egress correspond with the release of intracellular stores followed by the influx of extracellular Ca2+ , while the [Ca2+ ]c oscillates during gliding in concert with bursts of motility [4]. The activity of DOC2 and FER1 and FER2 is also indicated. Microneme protein function ranges from pore-forming protein (PLP1) facilitating egress, to a spectrum of adhesion molecules enabling the gliding motility and invasion machinery that mature through the action of microneme secreted-serine proteases (subtilisins) [5,6]. Microneme secretion is dosed as long as parasites are between host cells, a necessity to support sustained gliding motility, which also powers host cell invasion. Upon contact with an appropriate host cell the parasite injects the contents of its rhoptry organelles directly into the host cell cytosol. Rhoptry neck proteins (RONs) residing closest to the site of ejection, are released first to establish the moving junction, which is critical to efficient invasion. The rhoptry bulb proteins (ROPs) are released in the same secretion event (Figure 1B,C), and subvert a variety of host cell pathways to secure parasite replication and survival [7–9]. In addition, the rhoptries contain cholesterol and phosphatidylcholine presumably required to establish the parasitophorous vacuole membrane [10,11]. Upon abortion of an invasion event, rhoptries are still secreted as ‘evacuoles’ into noninvaded host cells and enable manipulation of these host cells similar to invaded host cells [12,13]. Dense granule proteins (GRA) are lastly secreted after PVM formation into the PV lumen from where they traffic to the PVM, intravacuolar network (IVN), host cell cytoplasm and even to the nucleus [14]. Dense granules with non-overlapping contents have been described, although it is not known whether these have different functions and/or different secretion kinetics [15]. GRA proteins have been implicated in several functions from Life 2021, 11, 217 3 of 22 interference with host cell signaling [16], transfer of small molecules between host cells and PV [17], manipulation of host cell immune response [18] and even parasite egress [19]. C2 domains are composed of 80–160 amino-acids that fold into β-sheets with specific binding characteristics that are found in a large and diverse set of eukaryotic proteins that can be grouped into several families [20]. Most C2 domains have lipid binding characteristics that are family specific [21], but they can also interact with other proteins [22–24]. The most diverse C2 domain protein family is derived from a protein kinase C (PKC) archetype that is unique among C2 domains in that it can stabilize Ca2+ ions (up to 3 per C2 domain) in a pocket composed of three loops emanating from the β-sheets. This family almost exclusively functions in signal transduction, membrane trafficking and fusion through conditionally engaging lipids and/or membranes upon changes in [Ca2+ ]c . However, not all members have retained the Ca2+ -binding capacity and their membrane association could be conditional upon the presence of lipids in certain membranes. In principle, these C2 domain have two different lipid or membrane binding sites, which are not variably conserved: one in the Ca2+ -binding loop region, and one in a lateral concavity in the β sheets [20]. The versatility of the PKC-C2 domain family is further enhanced by domains of tandemly positioned C2 domains creating a double C2 or DOC2 domain. This provides more flexibility in the interaction repertoire by creating cooperatively functioning C2 domains. Importantly this effect can modulate the response to varying [Ca2+ ]c . DOC2 proteins control numerous Ca2+ -dependent exocytic processes, but have been studied in most detail for the Ca2+ -dependent release of neurotransmitter from neurons [22,25–27] (Figure 2). For Ca2+ mediated fusion of vesicles to the plasma membrane to occur in general, a DOC2 Ca2+ -sensor with a transmembrane (TM) domain anchored in the secretory vesicle is required, represented by the synaptotagmin (Syt) family [22,28–30]. These typically respond to fast, high Ca2+ fluxes and are required for fast-synchronous neurotransmitter release [23,31–33]. Soluble sensors like mammalian DOC2 proteins (Figure 2B: note that their domain structure is very different from TgDOC2) are sensitive to lower [Ca2+ ]c concentration and regulate constitutive and spontaneous asynchronous neurotransmitter release [23,34–37]. Upon the correct Ca2+ concentration, DOC2 proteins act on congregating and clamping on SNARE proteins residing in the secretory vesicle (v-SNARE) and target membrane (t-SNARE) and result in fusion of the two membranes, thereby releasing the vesicle contents. The ferlin family comprises a distinct family of DOC2 proteins absent from neurons that facilitate secretory processes in other cell types. Ferlins are defined by a C-terminal TM domain and 5–7 C2 domains, typically organized in three DOC2 domains (Figure 2). Vertebrate ferlins function in the secretory, endocytic and lysosomal pathways with structural and functional importance in health and disease [38]. Secretion of synaptic vesicles from the inner ear cells upon mechanical stimulation by sound, only a single Ca2+ -sensor, otoferlin, is required for exocytosis, at the site of secretion, thereby deviating significantly from the standard model of Ca2+ -dependent exocytosis established in neurons [39–41]. Humans encode six ferlins that cluster closely together compared to the three different apicomplexan ferlins (Figure 2C). This suggests that the apicomplexan diversity represents unique clades that originated independently of the mammalian ferlins, which appear to have radiated out of a single ancestral mammalian ferlin. Here, we discuss the apicomplexan ferlins in light of newly emerging insights regarding the triggers and machinery facilitating microneme and rhoptry exocytosis. In addition. T. gondii ferlin and TgDOC2 mutants were used to explore their respective contributions to the various secretion events, which fortuitously led to the identification of a novel microneme protein family, MIC21, conserved in the Coccidia sub-class of the Apicomplexa. Life 2021, 11, 217 4 of 22 Figure 2. Comparative topologies of Ca2+ -mediated exocytosis machinery in Toxoplasma and humans and ferlin phylogeny. (A). Toxoplasma encodes four DOC2 domain proteins: TgDOC2 and three ferlin proteins. Ferlins are defined by 5 to 7 C2 domains. TgFER3 contains an N-terminal signal peptide (SP), which, in combination with the C-terminal TM domain, could signal GPI-anchor addition at the C-terminus. Yellow shades in TgFER3 represent coiled-coil domains. Light blue C2 domains are degenerate (defined as having a p-value below the cut-off in PFam database searches); light blue shaded Ca2+ binding to C2 is based on modeling. The C2 domains labeled A–F by established conventions [28,30,42]. TM: C-terminal transmembrane domain; FerI: conserved ferlin domain of unknown function. Modified from [43]. (B). The spectrum of types of C2 domain containing proteins in humans with roles in Ca2+ -dependent secretion. Calcium sensors are present in the Syt-x, DOC-x, and ferlin (otoferlin shown as representative) families, though not all family members are Ca2+ sensors. MID = MUN Interaction Domain. MUN = Mammalian Uncoordinated domain. ZF = Zn Finger; PxxP = Pro-rich. (C). Phylogenetic analysis of apicomplexan, chromerid and human ferlins. The following abbreviations are used: human ferlins “L1-L5” FR1L1-5 (FR1L1 (dysferlin; O75923.1), FR1L2 (otoferlin; Q9HC10.3), FR1L3 (myoferlin; Q9NZM1.1), FR1L4 (A9Z1Z3.1), FR1L5 (A0AVI2.2), FR1L6 (Q2WGJ9.2)), Ot: green algae Ostreococcus tauri (Q01FJ7); Chromerids: “Vb” Vitrella brassicaformis (VbFER1 (Vbre_12074 + Vbra_12075), VbFER2 (Vbra_9198)) and “Cv” Chromera velia (CvFER1 (Cvel_17519.2) and CvFER2 (Cvel_9223)); Apicomplexa: “Tg” Toxoplasma gondii (TgFER1 (TGME49_309420), TgFER2 (TGME49_260470), TgFER3 (TGME49_295472 + TGME49_295468)) “Nc”, Neospora caninum (NcFER1 (NCLIV_053770), NcFER2 (NCLIV_026570), NcFER3 (NCLIV_002280)), “Em” Eimeria maxima (EmFER1 (EMWEY_00002120), EmFER2 (EMWEY_00009280), EmFER3 (EMWEY_00017650)), “Pf” Plasmodium falciparum (PfFER1 (PF3D7_0806300), PfFER2 (PF3D7_1455600)), “Pb” Plasmodium berghei (PbFER1 (PBANKA_122440), PbFER2 (PBANKA_131930)), “Cp” Cryptosporidium parvum (CpFER1 (cgd8_2910), CpFER2 (cgd2_2320)), “Gn” Gregarina niphandrodes (GnFER1 (GNI_063830), and GnFER2 (GNI_073830)). Alignment and unrooted Jukes-Cantor phylogenetic tree were generated in Geneious (v.6.1.6) [44]) from a MUSCLE alignment using neighbor-joining. Note that the FER1 and FER2 nodes for Tg and Nc are barely discernable at this scale. From [43]. 2. Materials and Methods 2.1. Parasites and Host Cells Transgenic derivatives of the RH strain were maintained in human foreskin fibroblasts (HFF) or hTERT immortalized HFF cells as previously described [45]. The following previously reported parasite lines were used: line ts-DOC2 is the recapitulated temperature sensitive (ts) mutation [46] in a RH∆HX background with an addition of a C-terminal 5xTY tag on DOC2 [47], parasite line DN-FER1 is conditional overexpression of an exogenous FER1 gene controlled by the destabilization DD domain and the ligand Shield-1 with an Life 2021, 11, 217 5 of 22 additional Myc-tag [46], and in parasite line FER2-cKD the FER2 gene is placed under a tetracycline regulatable promoter with an additional N-terminal Myc-tag [47]. 2.2. Cloning Plasmid pmic21-MIC21-YFP(MCS)/sagCAT was cloned by PCR amplification of genomic DNA containing the ORF and the 1400 bp promoter upstream of the newly annotated start codon using primers #5085 gPro234380YFP-F GCCGGTGTTAGGAGATGACGAGACGTTTAAACCGCTGCCAGTGGATATATCTACGG and #5086 gPro234380YFP-R CTCCTCGCCCTTGCTCACCATCCTAGGAAGGTATTTCTTCAAAAGGTGTCAAGGG (restriction sites underlined) which was cloned into PmeI/AvrII digested ptub-YFP-YFP(MCS)-30 dhfr/ sagCATsag [48] to replace the α-tubulin promoter and the first YFP ORF by Gibson assembly (NEB). 2.3. SILAC and ESA Collection We adapted published methods to differentially label T. gondii cultures with isotopes [49]. Parasites grown at the permissive condition (35 ◦ C) were metabolically labeled with heavy (C13, N15) Lys and Arg (L-Lysine-2-HCl (Thermo/Life Cat # 89987) and LArginine-HCl (Thermo/Life Cat # 88210;) for two lysis rounds using established T. gondii SILAC protocols [49]. Medium was prepared in phenol free DMEM Media for SILAC (Thermo/Life Cat # 89985). Specifically, parasites were grown in 5 mL medium (containing 1% FBS) in a T25 flask that was pre-equilibrated with isotope containing medium 24 hours (hrs) prior to parasite inoculation. Following lysis 48 hrs later, parasites were passed in one T25 flask under similar conditions. 5 mL freshly lysed parasites were passed into two T175 flasks per condition, pre-equilibrated for 24 hrs in isotope containing medium. Following growth for 48 hrs at the restrictive condition (40 ◦ C, 1 µg/mL anhydrous tetracycline (ATc), or 1 µM Shield-1) in isotope labeled medium, or 48 hrs at the permissive condition (35 ◦ C or no ligands) in non-isotope labeled medium, monolayers were scraped with a rubber policeman and parasites released by passage through a 21 gauge needle. Parasites were collected by centrifugation at 1000× g for 20 min at RT, resuspended in 1 mL of serum free SILAC medium containing 10 mM Hepes pH 7.3 and centrifuged again. Following one additional wash step, parasites were resuspended to 1.2 × 108 parasites/mL in serum free SILAC medium. 200 µL aliquots were placed in round bottom 96 well plates ((CELLTREAT Scientific Products)). Secretion was triggered by 2% ethanol incubation at 37 ◦ C for 15 min as described [50,51]. The plate was centrifuged at 1000× g at 4 ◦ C for 10 min to pellet cells and 100 µL of the supernatant was transferred to an Eppendorf tube followed by addition of 10 µL protease inhibitor (100× stock; Sigma # P88490). Parasites and supernatant remaining in the plate were resuspended and transferred into a separate set of tubes, centrifuged at 1000× g at 4 ◦ C for 10 min, supernatant discarded and pellet resuspended in 180 µL PBS before addition of 20 µL 10% SDS as control for parasite number. Protein concentrations were determined using the BCA plate assay (Pierce #23227). 2.4. Silver Staining Silver staining of SDS-PAGE gels was performed using a Silver Stain Kit (Pierce, Thermo Scientific) following the manufacturer’s protocol. Briefly, 25 µg each of Heavy Labelled (HL) and Light Labeled (LL) ESA proteins were run on 8–12% SDS-PAGE MOPS gel. The gel was washed twice 5 min in water and fixed in 30% ethanol/10% acetic acid for 2 × 15 min. The gel was washed twice in 10% ethanol for 10 min followed by 2 washes for 1 min in water. The gel was sensitized for 1 min in Sensitizer working solution followed by 2 × 1 min washes with water. Proteins were stained for 30 min in Stain working solution followed by 2 × 20 sec washes with water. Gels were developed in Developer Working Solution for 3 min and the reaction stopped with 5% acetic acid for 10 min. Life 2021, 11, 217 6 of 22 2.5. LC-MS/MS Analysis LC–MS/MS analysis was performed on an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher) coupled to an EASY-nLC 1000 nanoLC (Thermo Fisher). Samples were pressure loaded onto a 250 µm fused silica desalting column packed with 4 cm of Aqua C18 reverse-phase resin (Phenomenex). The peptides were then pushed onto a column (100 µm fused silica with a 5 µm tip, packed with 10 cm C18) and eluted with a gradient of 0–55% Buffer B in Buffer A (Buffer A: 95% water, 5% acetonitrile, 0.1% formic acid; Buffer B: 20% water, 80% acetonitrile, 0.1% formic acid). The flow rate through the column was set to 400 nl/min and the spray voltage was set to 3.5 kV. One full MS1 scan (FTMS; 400–1800 MW) was followed by seven data dependent scans (ITMS) of the nth most intense ions with dynamic exclusion enabled. The generated tandem MS data were searched using the SEQUEST algorithm [52] using a concatenated target/decoy variant of the T. gondii GT1 ToxoDB-V28 database combined with a target/decoy non-redundant variant of the human UniProt database. Data sets were searched independently with the following parameter files; for the light search, all amino acids were left at default masses; for the heavy search, static modifications on lysine (+6.02013) and arginine (+10.00826) were specified. A static modification of +57.02146 on cysteine was specified in all searches to account for iodoacetamide alkylation. SEQUEST output files were filtered using DTASelect 2.0 [53]. Reported peptides were required to be unique to the assigned protein (cannot be attributed to both human and T. gondii derived protein), with a minimum of two unique peptides per protein. Discriminant analyses were performed using the CIMAGE quantification package as previously described [54]. The H/L ratios generated for unique peptides were grouped by protein with the median H/L ratio chosen as the representative ratio for that protein. Human proteins were removed from the dataset. Ratios from each sample were normalized to the median ratio of T. gondii derived proteins within that sample in order to correct for variations in isotope labeling. 2.6. Fluorescence Microscopy RH∆HXGPRT parasites were co-transfected with 15 µg each of plasmid pmic21MIC21-YFP(MCS)/sagCAT and PG53 encoding MIC8-mCherry ([55] a kind gift from Markus Meissner) or pMIC2-mCherry-Myc (Luc in plasmid pMIC2-Luc-Myc, kindly provided by David Sibley [56], was replaced with mCherry). 18 hrs post transfection, parasites were imaged with a Zeiss Axiovert microscope and a ORCA-FLASH 4.0 camera was used in capturing images using 100x objective with oil and 0.55 Numerical Aperture (NA). 2.7. Western Blots SDS-PAGE (8–12%, MOPS) separated ESA samples and corresponding total parasite lysates were blotted onto PVDF membrane and probed with mouse α-MIC2 (1:8000) [57], mouse α-TY (MAb BB2; 1:1000; a kind gift from Sebastian Lourido), mouse α-tubulin (12G10) (1:2000; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), rabbit α-GRA7 (1:2000; a gift from John Boothroyd) [58]. Procedures and signal intensity quantification as previously described [43,47]. A two-tailed, two sample equal variance Student’s t-test was performed on protein levels normalized to α-tubulin. 2.8. MIC21 Phylogeny The reannotated TGGT1_234380 with signal peptide was used for a BLASTP search of select representative Apicomplexa against EuPathDB [59]. Manually curated orthoand para-logs were aligned using ClustalO 3.0 (CLC Main Genomic Workbench 20, Qiagen). The alignment was used to generate an unrooted phylogenetic tree using the Neighbor joining Algorithm, Jukes-Cantor protein distance and 100x bootstrap analysis (CLC Main Genomic Workbench 20, Qiagen). The following sequences were included in the analysis: MIC21: TGGT1_234380, HHA_234380, NCLIV_049520, CSUI_003785; MIC21L1: TGGT1_222188, HHA_222188, NCLIV_005580, SN3_01200570, CSUI_000781 (2a), Life 2021, 11, 217 7 of 22 CSUI_009221 (2b); MIC21-L2: EBH_0032420, ETH_00028875, EAH_00066010, LOC34621018; MIC21-L3: ETH_00004565, LOC34624209. 3. Results 3.1. Apicomplexan Protein Trafficking to the Secretory Organelles The secretory pathway that subsequently passes through the endoplasmic reticulum (ER) and Golgi apparatus mediates protein trafficking to the three secretory organelles. Targeting proteins to the dense granules does not require any additional signals and comprises the default pathway secretory organelle. Specifics on the protein trafficking pathways to the micronemes [60] and rhoptries [61] and their biogenesis were recently reviewed. Although some rhoptry specific targeting signals have been identified [62,63], specific sorting signals for microneme proteins have remained elusive. Following the Golgi compartment, microneme and rhoptry proteins traffic through an endosome like compartment (ELC) (reviewed in [64,65]), where they undergo maturation by proteolytic removal of a pro-peptide [66]. Specific Rab GTPases have been associated with the first leg of the trafficking through the endosomal pathway, but are absent from the mature organelles or their sites of secretion [67]. Peculiarly, a subset of microneme proteins (MIC3, MIC5, MIC8 and MIC11) traffic, in Rab5A/C-dependent fashion while the others are Rab5A/C-independent [67]. Sustained microneme secretion in gliding extracellular parasites is balanced with active endocytosis [68]. During cell division, micronemes of the mother are re-directed into the newly forming daughters, which hints at a mechanism that supports trafficking of micronemes in more than one direction [69]. 3.2. Organization of Organelle Secretion: Small Molecules and Machinery Microneme release is turned off during intracellular replication and only occurs during egress, gliding and host cell invasion (Figure 1C). Pharmacological studies allowed for dissection of the controls of microneme exocytosis and established a role for three signaling molecules: cytoplasmic Ca2+ , phosphatidic acid (PA) and cGMP [70]. The levels of cytoplasmic calcium ([Ca2+ ]c ) correlate with the level of microneme secretion [71] (Figure 1C), and the micronemes are considered a bona fide Ca2+ -dependent secretory organelle [72]. Ca2+ mediators are involved at several levels. Firstly, the exocytosis event has been associated with Centrin2 [73], which has four EF-hand domains putatively binding Ca2+ , and two double C2 domain (DOC2) proteins, TgDOC2 [46] and FER1 [47]. An indirect role is also found in the requirement of a Ca2+ -dependent protein kinase, CDPK1, for microneme exocytosis, but the key phosphorylation events are still unknown [74,75]. A parallel set of controls is mediated by phosphatidic acid (PA) produced upon activation of egress which is deposited in the plasma membrane. PA is recognized by an essential acylated pleckstrin homology (PH) domain-containing protein (APH) on the surface of the micronemes [76]. Micronemes are exocytosed at the very apical tip of the parasite, where the rhoptries are discharged as well. The triggers for rhoptry exocytosis are not well understood besides that this requires microneme secretion first, and that it only occurs upon contact with a host cell. The connection to the micronemes resides in critical roles for microneme proteins MIC8 [55], AMA1 [77] and CLAMP [78], but the detailed mechanisms have not been resolved. In addition, rhoptry secretion is mediated by PA and phosphatidylinositol 4,5 bisphosphate (PIP2) [79], which accumulate at the plasma membrane following induction of the microneme secretion [77]. C2 and PH domains in Rhoptry Associated Surface Proteins (RASPs) engage with PA and PIP2, bringing the rhoptry in close contact with the plasma membrane and/or apical vesicle residing at the very apical between the rhoptry and the plasma membrane. [70]. In addition, a machinery shared with the release of trichocysts by ciliates comprises several Nd proteins, names for their ‘not discharged’ genetic phenotype in the ciliate Paramecium [80]. The Nd proteins are critical for the apical rosette, a structure in the plasma membrane through which the rhoptries and trichocysts are secreted. The apical vesicle is positioned on the cytoplasmic side of the rosette opposed to the rhoptry tip. Life 2021, 11, 217 8 of 22 It is of note that microneme secretion is not mediated by the rosette as they are positioned at the plasma membrane next to the apical vesicle and, moreover, microneme secretion is also not affected by disruption of the rosette structure in TgNdP1 and TgNdP2 mutants [80]. Some Nd proteins reside in the cytoplasm and others at the apical vesicle, but the Nd proteins are either directly critical for the rosette structure, or for the exocytosis event [80]. Furthermore, the Nd proteins interact with another DOC2 domain protein, FER2, which is essential for rhoptry secretion [43]. Two models have been proposed for the role of Nd proteins in controlling the fusion step: 1. the correct assembly of Nd6, Nd9, NdP1 and NdP2 as a complex together with a GTPase (TGME49_277840) is mediated by the Nd6 guanine exchange factor (GEF) domain and Ca2+ -dependent action of FER2, or; 2. cytosolic Nd9 and/or NdP1 interact transiently with the apical vesicle associated Nd6 to trigger the assembly of the rosette [80]. Although no pharmacological compounds are known to trigger rhoptry secretion, an inhibitor, 4-bromophenacyl bromide (4-BPB), is available [81]. This compound typically inhibits A2 phospholipases, but its target in T. gondii is unknown [81]. Overall, the structural architecture of the site of rhoptry secretion comprises a critical role for several C2 domain proteins engaging with various lipids, while providing a putative role for Ca2+ through FER2 [79]. Dense granule secretion is the least understood. Not even the site of secretion has been conclusively established. Although there is evidence that the dense granules are secreted somewhere at the apical end of the parasite [82,83], their secretion has also been inferred from a specialized invagination at the posterior end of the parasite where the basal complex resides [84]. However, an alternative interpretation is that dense granule proteins are secreted at an apical site from the parasite and subsequently transition to the posterior end [85]. In either scenario dense granule proteins facilitate the assembly of multilamellar vesicles in the vacuole at the posterior end of the parasite, which subsequently transition into tubulated structures making up an intravacuolar tubulovesicular network [84]. Since dense granules with different contents are present, it is also possible these might have distinct secretion sites and/or kinetics. 3.3. Ferlins in Mammals The mammalian ferlins come in two basic flavors differentiated by their sub-cellular localization at either the plasma membrane or on intracellular compartments. Localization relates to their function in either late endosomal transit or trans-Golgi recycling [86]. Dysferlin and otoferlin have been the most studied. Dysferlin is critical for muscle development and functions in vesicular trafficking and lysosome exocytosis during muscle plasma membrane damage repair [38,87]. Dysferlin does not localize to lysosomes in intact myotubes, but dysferlin-containing vesicles fuse with lysosomes upon sarcolemma damage, possibly in response to a raise in [Ca2+ ]c [88]. This two-step process might mimic the role of the apical vesicle wedged between the rhoptry tip and the plasma membrane, which also required a two-step fusion event [80]. A further parallel with the Apicomplexa is the indication that rhoptry targeting tyrosine motifs are shared with mammalian lysosomal trafficking signals [63,89]. This suggests a kinship at several levels between mammalian lysosomes and rhoptries, with a possibly comparable role for dysferlin and FER2, respectively. Otoferlin functions as both a scaffolding protein in the secretory pathway as well as in the actual membrane fusion during exocytosis [39,41,90,91]. Otoferlin is expressed in many tissues but in Cochlear Hair Cells (CHCs) in the inner ear auditory systems where is localizes to synaptic vesicles and the plasma membrane and is essential in neurotransmitter release upon mechanical triggers: a variety of mutations across otoferlin lead to hearing loss [90,92]. Under low [Ca2+ ]c interactions among C2C, C2D, C2E, and C2F is promoted, whereas high [Ca2+ ]c leads to interaction with phospholipids, the prelude to membrane fusion [90]. Life 2021, 11, 217 9 of 22 3.4. DOC2 Protein Repertoire in T. gondii The Apicomplexa encode two widely conserved ferlin proteins, FER1 and FER2 ([43] and one quite unconventional alveolate-wide conserved DOC2 protein without a TM domain [46] (Figure 2A,C). Some parasites, including T. gondii, encode a degenerate third ferlin, TgFER3 [43]. We were able to knock out TgFER3 without any effect on the lytic cycle and concluded that it is not involved in organelle secretion, at least not in the tachyzoite stage (Tagoe and Gubbels, unpublished). TgFER1 and TgFER2 mediate exocytosis of the micronemes and rhoptries, respectively [43,47]. Besides T. gondii, ferlins have only been studied in one other apicomplexan parasite, Plasmodium berghei. The ferlin-like protein, PbFLP, the TgFER1 ortholog, is essential for male gametocyte egress, and its knock out phenotype was consistent with a role in exocytosis [93]. The conserved DOC2 protein without TM domain, TgDOC2, is unconventional in its relatively long length without other recognizable domains (Figure 2). TgDOC2 is essential for microneme protein secretion, a function that is conserved in Plasmodium falciparum [46]. PfDOC2 has been shown to bind Ca2+ and is present in the membrane fraction of replicating parasites [94]. In conclusion, all conserved DOC2 domain proteins function in the exocytosis of the specialized apicomplexan secretory organelles defining the obligate intracellular lifestyle of these parasites. Comparing the function and behavior of mammalian ferlins to the T. gondii repertoire points at otoferlin as relevant to understand FER1 and dysferlin as a role model for FER2. Regarding the function of TgDOC2, comparison with the mammalian repertoire points to the DOC2 family: cytoplasmic Ca2+ sensors like mammalian DOC2 proteins (Figure 2B: note that their domain structure is very different from TgDOC2) which are sensitive to lower [Ca2+ ]c concentration and regulate constitutive and spontaneous, asynchronous neurotransmitter release [34–37]. They act in concert with a membrane bound, TM domain containing Ca2+ sensor like the synaptotagmins or otoferlin that typically respond to fast, high Ca2+ fluxes required for fast-synchronous neurotransmitter release [23,31–33]. Tentatively, this dual player DOC2 system seems to fit the micronemes with their dynamic secretory nature (Figure 1C), whereas as single DOC2 protein seems sufficient for the single burst in rhoptry release. However, based on the unconventional structure of TgDOC2 we cannot exclude other potential roles fulfilled by different mammalian multiple C2 domain proteins such as MUNC13 or RIMs outlined in Figure 2B. Munc13 (mammalian ortholog of C. elegans uncoordinated protein) stabilizes the SNARE and Ca2+ -sensor complex and is absolutely essential for any type of Ca2+ -mediated exocytosis [95,96]. MUNC13 is not a Ca2+ sensor but a CAPS (Calcium-dependent Activator Protein for Secretion), which prime vesicles for Ca2+ -triggered exocytosis. MUNC13s harbor 2 or 3 C2 domains and a diacylglycerol-binding C1 domain [97] (Figure 2B). The MUN domain stabilizes the SNARE complex [97,98]. However, the T. gondii genome does not encode a protein with a domain reminiscent of a MUN domain, which indicates a mechanism distinct from the well-studied systems. RIMs are involved in vesicle priming, docking and organization at the site of secretion. RIM stands for Rab3 Interacting Molecules. They harbor multiple C2 domains (Figure 2B) and bind multiple presynaptic proteins, e.g., SNAREs, Rab3, Munc13’s, mediated by the C2A, C2B and Pro-rich domains [99]. RIMs receive the secretory vesicle from the Rab-mediated trafficking machinery and relay it to the fusion proteins. Knock-out of RIMs reduces neurotransmitter release to 60% [100]. The relevance to T. gondii goes off the rails by the absence of Rabs and SNAREs at the sites of microneme and rhoptry secretion [67]. However, the unconventional nature might bypass the need for these players, as ferlins equally have the capacity to function without. Moreover, otoferlin is even considered to be the one and only Ca2+ -sensor for secretory vesicle exocytosis in CHCs, since stimulus–secretion coupling is eliminated in otoferlin knock-out mice [39–41]. In conclusion, the role of apicomplexan ferlins in microneme and rhoptry exocytosis is consistent with mammalian ferlins acting on the endosomal pathway, wherein the micronemes and rhoptries reside. However, given the specialized nature of the apicomplexan secretory organelles combined with the ancient divergence between FER1, FER2 and the Life 2021, 11, 217 10 of 22 mammalian ferlin families (Figure 2C), this comparison is only at a high level. The ferlins act in the context of lineage specific proteins like TgDOC2 [46] and APH [101] for the micronemes, and RASP [79] and Nd [80] proteins in rhoptry exocytosis. 3.5. FER1 and FER2 in Microneme and Rhoptry Fusion with the Plasma Membrane The fine details on how FER1, FER2 and TgDOC2 orchestrate the exocytosis of micronemes and rhoptries is associated with several questions. In particular, T. gondii Rabs are absent from the microneme and rhoptry secretion site in the apical conoid [67]. Although Rab11A has been shown to be critical in constitutive secretion of dense granules, this is not a triggered event [102]. In addition, no Toxoplasma’s SNARE proteins have been detected at the site of microneme and rhoptry secretion site in the very apical plasma membrane [103]. The most forward localizing SNARE in the secretory pathway identified is a syntaxin, TgStx12, which is critical in trafficking of proteins from ELC to the micronemes and rhoptries, but not for their secretion. Thus, this is different from neurotransmitter release where SNAREs play a key role in the membrane fusion event facilitating exocytosis. In general, the absolute requirement for SNAREs in exocytosis, certainly regarding ferlin-mediated exocytosis, has come under debate. It has been postulated that fast, Ca2+ dependent exocytosis is inconsistent with the role of SNAREs [104] and some exocytosis in absence of SNAREs is possible [21,105]. Furthermore, some neurotransmitter is still released when all relevant SNAREs are depleted [105], and alternative models for SNARE independent neurotransmitter release have been postulated [104]. Most germane to the situation in parasites is the absence of SNAREs from the site of otoferlin-mediated neurotransmitter release in the auditory hair cells [106]. Furthermore, at the sites of membrane lesions in cultured myotubes SNAP23, syntaxin4 and VAMP4 do not accumulate, but myoferlin does and is required for membrane repair [107,108]. Thus, these ferlins are essential for specialized membrane fusion events, but are not dependent on SNARE proteins. This leaves the door open for SNARE-independent fusion, or alternatively, an as yet uncharacterized class of SNAREs [90,106]. Regarding the modular nature of the DOC2 domains in ferlins, the multiple C2 domains have been proposed to be able to support membrane binding integrating the Ca2+ sensing and membrane fusion events [31,42]. Indeed, pairs of C2 domain can interact cooperatively to take on a closed or open conformation [109] or coordinate a driver-facilitator relationship [110,111]. In this manner C2-C2 interactions facilitate protein scaffolding, determine Ca2+ sensitivity and direct lipid binding affinity. In this division of labor, a single C2 domain may bind a recruited protein, while others within the ferlin are responsible for binding Ca2+ or lipid. In this way all domains of the protein coordinate to allow fusion of membranes [112,113]. The combination of several C2 domains in a single protein allows for dynamic conformational changes that reveal or hide different C2 domains and thus change its function and as such ferlins might facilitate Toxoplasma exocytosis in absence of SNAREs. Our working model is therefore that TgDOC2 and FER1 and FER2 can fulfill the missing function of SNAREs and Rabs at the plasma membrane. 3.6. Ferlins in Organellar Trafficking FER1 does not only function in microneme exocytosis but also in microneme organelle trafficking [47]. Two different phenotypes in different mutants were observed: conditional overexpression of dominant negative FER1 allele without its TM domain results in a reversible, retrograde microneme transport away from the apical cortex to a pile up of micronemes in an apical cytoplasmic location, whereas conditional overexpression of the full-length FER1 resulted in an anterograde transport of the micronemes into the very apical tip of the parasite and even premature exocytosis [47]. The retrograde transport was interpreted as being germane to the process of microneme recycling from the mother into the emerging daughters during the second half of cell division [69]. The actin-dependent trafficking also provides a tentative connection between FER1 and a myosin. The anterograde trafficking supports a role in replenishment of secretory vesicles at the site of Life 2021, 11, 217 11 of 22 secretion. It is assumed that micronemes travel along the sub-pellicular microtubules as they follow this organization pattern in their cortical localization pattern [67]. However, there is no direct experimental data supporting this assessment. The microtubule based motor, dynein TgDLC8a, has been associated with replenishment, but since it is only observed in the very apical tip, is unlikely to transport the organelles along the sub-pellicular microtubules, but more likely through the conoid [73]. Either way, dual roles in membrane fusion and vesicle trafficking are also seen in the vertebrate ferlins [86], such as otoferlin in replenishment of synaptic vesicles [40,41] through myosin VI [114,115]. The role of dysferlin in vesicular trafficking in quite diverse [38], but is also dependent on actin myosin for plasma membrane repair [116]. Overall, these observations and similarities with vertebrate ferlins indicate that the role of FER1 changes under different conditions organellar trafficking during intracellular replication, and microneme exocytosis upon activation of egress, during gliding and host cell invasion. Currently, we have no indication that FER2 has such a dual role. The physical feature of the rhoptries are in alignment with this model: they are constitutively docked at the apical end and do not traffic [80] whereas their relatively large size would also not be very amendable to an organelle recycling pathway [61]. 3.7. T. gondii DOC2 Proteins Act Differentially on the Various Secretory Organelles We performed knock-out or dominant negative allele studies on FER1 [47], FER2 [6], and TgDOC2 [46] to reveal the organelles these factors are acting on. However, to obtain more subtle, quantitative data on the exact nature of secretion events and to refine the mechanistic models we performed Stable Isotope Labeling with Amino acids in Cell culture (SILAC) proteomics on the proteins released in our set of secretion mutants. We induced all conditional mutant phenotypes overnight followed by mechanical release of the parasites from their host cells and then stimulated exocytosis with 2% ethanol for 15 min. We collected the excreted and secreted antigens (ESA) under both induced and non-induced control conditions, which were differentially labelled with light- and heavyisotope containing amino acids, respectively, before mixing in equal ratios and analysis by mass spectrometry (Figure S1). Changes in the heavy:light ratio reveal which proteins are differentially secreted in each mutant. For the temperature sensitive (ts)-TgDOC2 lines, the results confirmed the dominant role of TgDOC2 on micronemes (Figure 3A). We also confirmed that the ts allele results in protein instability and loss of TgDOC2 from the parasites at restrictive, high temperature (Figure S2). However, we also identified significantly less secretion of both the dense granules, and, to a lesser but still significant extent, the rhoptries. Induction of DN-FER1, which redirects the vast majority of micronemes away from the cortex thereby inhibiting their secretion, results in a significant reduction in secretion of many microneme proteins, whereas the ones below the significance cut-off trend strongly to reduced secretion (Figure 3B). There is no effect on the rhoptries. However, we observed a secretion trend for the dense granules, mimicking the micronemes, albeit largely below the statical relevance limit. Life 2021, 11, 217 12 of 22 Figure 3. SILAC data for T. gondii DOC2 and Ferlin mutants. (A). ts-DOC2 is a recreated temperature sensitive allele of DOC2 conditionally treated at 35 ◦ C and 40 ◦ C [43,47]; (B). DN-FER1 is a DD-Myc conditional overexpression allele induced with 1 µM Shield-1 [47]; (C). cKD-FER2 is tetracycline regulated promoter replacement induced with 1 µM ATc [59]; (D). the wild type control consisted of RH∆Ku80 parasites treated at 35 ◦ C and 40 ◦ C. All inductions were performed for 48 hrs on intracellularly replicating parasites. For ESA collection, parasites were mechanically lysed from the host cell, and stimulated with 2% ethanol for 15 min. Parasites grown under permissive conditions were grown in light-isotope (L) labeled amino acids; parasites at restrictive conditions in heavy-isotope (H) labeled amino acids. Equal amounts of ESA collected under permissive and restrictive conditions were mixed and subjected to mass spectrometry. Averages of two biological repeats are shown, except for cKD-FER2 which was a single experiment. Marked in pink, the cKD-FER2 experiment is the only one in which some ER proteins (ER-2 pool defined in [117]) were detected (TGGT1_229480: putative calcium binding protein precursor; TGGT1_221210: cyclophilin; TGGT1_211680; protein disulfide isomerase). Dotted lines mark the −0.5 and +0.5 Log2 arbitrary cut-offs for decreased or increased significant changes, respectively, in excretion/secretion above background. 3.8. Do DOC2 Proteins Act on Multiple Secretory Organelles? The suggestive role of TgDOC2 in microneme and rhoptry discharge and that of FER1 in dense granule exocytosis can be interpreted in two different ways: 1. a direct role for TgDOC2 in rhoptry and dense granule exocytosis and for FER1 in dense granule exocytosis; 2. induced dense granule and rhoptry release requires secretion of the micronemes first. Discharge of rhoptries indeed requires microneme secretion first [54], which would explain the strong effect seen in the ts-DOC2 line. However, the dependence of dense granules on either TgDOC2 or FER1 is less clear. In fact, an opposite correlation between induced microneme and dense granule exocytosis has been reported [118]. However, dense granule exocytosis was not completely inhibited, and the differences in protein secretion we detect are of course relative to the non-induced condition. The secretion drop we see therefore drops even below the reported reduced level [118]. To validate the reduction in dense granule exocytosis we performed Western blots and quantified the differential secretion of the microneme and dense granules protein reporters MIC2 and GRA7, respectively (Figure 4). This revealed that like MIC2, GRA7 secretion is completely inhibited. The other notable new insight is that the expression Life 2021, 11, 217 13 of 22 levels of both MIC2 and GRA7 are trending upward upon TgDOC2 depletion. Although this upregulation does not reach a statistically significant difference, it trends contrary to the complete disruption of secretion, thereby indicating the potential presence of a feedback loop to increase protein expression as a compensatory mechanism for the block in exocytosis. Figure 4. Dense granule secretion is TgDOC2 dependent. (A). Representative Western blots of ts-DOC2 secretion assays as performed for the ESA-SILAC experiment. “total” represents the pellet of the ESA assay. Antiserum against α-tubulin was used as loading control. Relative to the first lane for each condition, only half the protein amount was loaded in the second lane. (B). Quantification of secreted and total MIC2 normalized against α-tubulin shows that MIC2 expression is 3-fold upregulated in ts-DOC2 at the restrictive condition (40 ◦ C), yet none is secreted. (C). Quantification of secreted and total GRA7 normalized against α-tubulin shows that GRA7 is not upregulated, but its stimulated secretion is dependent on DOC2. All blots used for quantification are provided in Supplementary Figure S2 and the quantification data in Supplementary Table S2. Statistics was performed with a two-tailed, two sample equal variance Student’s t-test; n.s.: non significant. 3.9. Identification of a Novel Microneme Protein: MIC21 Besides the annotated microneme, rhoptry and dense granule proteins, we also identified differentially secreted proteins outside these categories. These were mostly typical contaminants such as SAG surface antigen, ribosomal proteins, as well as mitochondrial or cytoplasmic proteins (although we cannot exclude the possibility that their secretion is truly dependent on the DOC2 domain proteins; Figure 5 and Table S1). Either way, one protein in the ts-DOC2 dataset stood out for its extremely strong differential secretion putting it right between the other microneme proteins: TGGT1_234380 (named MIC21; Figure 3A). This protein lacked a signal peptide prediction in ToxoDB [119], but was identified in the microneme fraction of the recent subcellular compartment proteome hyperLOPIT study of T. gondii [117]. Furthermore, we detected orthologs and a paralog across the Coccidia, which in contrast to TGGT1_234380 all contained a signal peptide. We therefore scrutinized the TGGT1_234380 annotation and noted a continuous open reading frame omitting the single intron that results in inclusion of a signal peptide comparable to the para- and orthologs (Figure 5A). We further identified two conserved domains (CD1 and CD2), without significant homology to known domains, which therefore does not direct further functional roles. Life 2021, 11, 217 14 of 22 Figure 5. Characterization of putative microneme protein TGGT1_234380. TGGT1_234380 was the top hit in the ts-DOC2 SILAC experiment and annotated as a hypothetical protein on ToxoDB. Recently, the protein was detected in the microneme fraction by HyperLOPIT [117]. Querying select, representative apicomplexan and chromerid annotated genomes in EuPathDB identified only orthologs in the Coccidia. All except the T. gondii annotation of the predicted gene contained an N-terminal signal peptide (SP), which upon closer examination, was also present in the T. gondii gene if the gene was not spliced. This re-annotated version of the protein, which we named microneme protein 21 as this is the next available number in the microneme protein family (MIC21), was used to assemble a protein alignment that is the basis of data presented in panels A-C. (A). Schematic representation of MIC21. Two highly conserved domains (CD1 and CD2) were identified whose conservation across orthologs and paralogs is shown in the magnified panel as % conservation and amino acid logo plots. A PFam search identified domain defined in the peroxisomal acyl-CoA oxidase-II domains 3 and 4 (SCOP domain d1is2a3) with a non-significant e-value of 1, and furthermore this domain is not conserved across ortho- and para-logs. (B). Unrooted (neighbor-joining, Jukes-Cantor, CLC Workbench) phylogenetic tree of representative MIC21 related sequences identified in the Coccidia resolves into 4 branches of ortho- and paralogs (MIC21 cluster next to MIC21-like proteins MIC21-L1, MIC21-L2, and MIC21-L3 clusters). Bootstrap analysis (100x) supported all branches with over 70%, except for the two branch points as indicated. Hh: Hammondia hammondi; Nc: Neospora caninum; Cs: Cystoisospora suis; Sn: Sarcocystis neurona; Ea, Eb, Et: Eimeria acervulina, brunetti and tenella, respectively; Cc: Cyclospora cayetanensis. (C). Heatmap representing the percentage of sequence similarity across the DSP1 protein family. Percentage similarity based on ClustalOmega alignment. (D). RNAseq data available on ToxoDB reveals MIC21-L1 is not expressed in tachyzoite but only in the enteric stages. MIC21 is robustly expressed tachyzoites, bradyzoites and the enteric stages [120]. Neither MIC21 nor MIC21-L1 is expressed in sporulating oocysts [121]. Error bars represent standard error [120]. (E,F). Exogenous, transient expression of genomic DNA spanning the TgMIC21 promoter (1.4 kb) and coding sequence N-terminally fused to the YFP reporter co-transfected with a MIC8-mCherry (E) or MIC2-mCherry (F) reporter indicates MIC21 resided in the micronemes though does not perfectly co-localize with either MIC8 or MIC2. Life 2021, 11, 217 15 of 22 Phylogenic analysis revealed that the ortho- and paralogs formed four subgroups, two of which span the cyst-forming coccidia (Hammondia hammondi, Neospora caninum, Sarcocystis neurona, Cystoisospora suis) whereas the other two are present in the non-cyst forming coccidia (Eimeria spp. and Cyclospora cayetanensis) (Figure 5B,C). Analysis of T. gondii stage specific gene expression data revealed that TGGT1_234380 is expressed in tachyzoites, bradyzoites and merozoites but not sporozoites, whereas the paralog TGGT1_222188 (MIC21-L1) is restricted to the merozoites (Figure 5D). Finally, we experimentally validated localization of MIC21 to the micronemes by exogenous expression of a C-terminal YFP fusion construct under control of the endogenous MIC21 promoter. MIC21 localized to the apical region reminiscent of the micronemes. Co-transfections with either MIC2-mCherry or MIC8-mCherry reporters showed partial co-localization, whereas MIC21 always was present in larger apical puncta (Figure 5E,F). These puncta likely reflect some mis-trafficking induced by the fusion protein, which fits with the inability to obtain parasites stably expressing the MIC21-YFP protein. Altogether, we conclude that TGGT1_234380 is a bona fide microneme protein and we named it MIC21; the as yet unvalidated paralogs were named MIC21-like proteins MIC21-L1, MIC21-L2, and MIC21-L3. 4. Discussion Pharmacological triggering of microneme release facilitated studies on the proteins discharged by the parasite from this organelle [50,51]. Our quantitative proteomics using the DOC2 and ferlin secretion mutants confirmed the previously assigned roles for TgDOC2 and FER1 in microneme exocytosis, but in addition revealed cross-organelle impacts on the discharge dynamics of the various organelles. Notably, TgDOC2 depletion also resulted in decreased rhoptry and dense granule protein levels in the ESA (Figures 3A and 4). The former might not be surprising given that microneme exocytosis is a requirement for rhoptry expulsion, but the connection to reduced dense granules release was not immediately obvious. This hints at a potential role for TgDOC2 as a general factor in secretory organelle fusion with the plasma membrane. We envision a dual function for TgDOC2, with a Ca2+ -dependent role on microneme membrane fusion with the plasma membrane, whereas it might be a general factor, less dependent on Ca2+ , in the membrane fusion event of rhoptries and dense granules, e.g., as a MUNC13-like clamp to stabilize the complex of molecules required for membrane fusion. The dynamic of the overexpression of a dominant negative FER1 allele indicated this was acting equally on micronemes and dense granules, but not on the rhoptries at all. The microneme phenotype in the DN-FER1 line originates in mistrafficking of micronemes, while the presence of a wild type FER1 allele still facilitates a low level of microneme protein secretion [47]. We did not assess dense granule trafficking in the DNFER1 line and therefore we can currently not differentiate a role for FER1 in dense granule trafficking effect from a function in actual dense granule membrane fusion with the plasma membrane. A putative model should consider the two modes of dense granule exocytosis: a steady, constitutive level of dense granule release, and a specifically regulated burst upon completion of host cell invasion (Figure 1). It is possible that FER1 plays a differential role in these different events. TgDOC2 and the ferlins are derived from the ancestral PKC family of C2 domains that binds Ca2+ . However, not all extant C2 domains retained the Ca2+ -binding capacity, and this is an unsettled question for the apicomplexan family members. We analyzed the canonical C2 domains and determined that at least one C2 domain has a minimal set of negatively charged aspartic acid or glutamic acid residues in the key position in the C2 domain loops (FER1-C2D, FER2-C2F and TgDOC2-C2B: Supplementary Figure S3). Overexpression of FER1 allele with a C2D domain wherein the Ca2+ -binding residues were mutated resulted in distinct microneme trafficking phenotype, suggesting physiological relevance of this in sillico analysis [47]. However, these assignments need experimental validation. It is of note that dysferlin and otoferlin each only have three predicted Ca2+ binding sites, but experimentally, all seven dysferlin C2 domains and five of the six otoferlin Life 2021, 11, 217 16 of 22 C2 domains were able to bind Ca2+ , albeit, to variable degrees [122,123]. Hence, the presented prediction could underestimate the Ca2+ -biding capacity in the T. gondii proteins. Details on lipid and membrane binding capacities are another open question key to understand the role of the ferlins and TgDOC2 in membrane fusion. In particular the possibility to ‘sculpt’ the membrane structure of phosphatidylserine membranes as a prelude to membrane fusion [124] is of interest for FER1 since this is a membrane lipid preferentially flipped by the guanyl-cyclase-flippase required for microneme discharge [125]. Furthermore, PIP2 and PIP4 are common lipids bound by ferlins [123,126], which again are lipids with critical roles in apicomplexan exocytosis [127]. Furthermore, (conditional) lipid binding of the key RASP and Nd proteins associated with rhoptry release suggest that lipid binding by FER2 is quite likely an essential event in this process [79,80]. Furthermore, rhoptry secretion requires recruitment of all Nd proteins to the apical vesicle or rosette, suggestion protein-protein interactions are another critical aspect wherein FER2 could participate. Thus, the spatiotemporal conditions that could change ferlin conformation range from the local availability of lipids in the plasma membrane (e.g., phosphatidylserine [125] and phosphatidic acid [101] for the micronemes) to the increase in [Ca2+ ]c as well the potential contribution of Ca2+ -dependent phosphorylation by, e.g., CDPK1 [74], PKA [128] or PKG [129,130]. For example, phosphorylation of otoferlin regulates the Ca2+ -sensitivity of C2C and C2F domains [131]. Currently there is no data supporting phosphorylation of FER1 or FER2, but three phosphorylated residues of TgDOC2 are contained in the tachyzoite phosphoproteome [132]. Furthermore, a vacuolar H+ -ATPase (VP1) in a compartment at the very apical tip of the parasite has been related to a critical role in microneme secretion [133,134]. The identity of this compartment is not known, but it is unlikely the apical vesicle that is not essential for microneme secretion. In summary, the DOC2 proteins of T. gondii are woven deeply throughout the fabric of the exocytic organelles and events. The endosomal ancestry of these organelles fits well with endolysosomal context wherein the vertebrate ferlins facilitate vesicular trafficking and Ca2+ -dependent exocytosis. Open questions regarding the mechanism of how the DOC2 proteins exert their function pertain to their Ca2+ , lipid and protein binding capacities together with conditional conformational changes that might integrate environmental conditions and post-translational modifications. Supplementary Materials: The following are available online at https://www.mdpi.com/2075-1 729/11/3/217/s1, Figure S1: Silver stained SDS-PAGE gels of ESAs, Figure S2: Western blots of ESAs, Figure S3: In sillico analysis of Ca2+ -binding potential of C2 domains, Table S1: ESA mass spectrometry SILAC data, Table S2: Western blot quantification data. Author Contributions: Conceptualization, M.-J.G. and E.W.; methodology, D.N.A.T., A.A.D., T.J.B., J.A.F., M.-J.G. and E.W.; validation, D.N.A.T. and M.-J.G.; data curation, T.J.B., J.A.F. and. E.W.; writing, A.A.D. and M.-J.G.; visualization, D.N.A.T., A.A.D. and M.-J.G.; supervision, E.W. and M.-J.G.; project administration, M.-J.G.; funding acquisition, M.-J.G. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by National Institutes of Health grants AI122042, AI099658, and AI122923, and American Cancer Society grant RSG-12-175-01-MPC. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [135] partner repository with the dataset identifier PXD024210. 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Article citation info: 45 Ostapchuk O, Kuznetsov V, Kruczek W, Kuznetsov V, Tsyplenkov D. Analysis of the neutral grounding modes influence on the reliability characteristics of local systems with renewable energy sources. Diagnostyka 2021; 22(1): 45-56. https://doi.org/10.29354/diag/132834 DIAGNOSTYKA, 2021, Vol. 22, No. 1 ISSN 1641-6414 e-ISSN 2449-5220 DOI: 10.29354/diag/132834 ANALYSIS OF THE NEUTRAL GROUNDING MODES INFLUENCE ON THE RELIABILITY CHARACTERISTICS OF LOCAL SYSTEMS WITH RENEWABLE ENERGY SOURCES Oleksandr OSTAPCHUK 1, Valeriy KUZNETSOV 2, Włodzimierz KRUCZEK 3, Vitaliy KUZNETSOV 4, Dmytro TSYPLENKOV 5 1 Department of Renewable Energy, Igor Sikorsky Kyiv Polytechnic Institute, Prosp. Peremohy, 37, Kyiv, Ukraine, 03056, e-mail: O.Ostapchuk@kpi.ua 2 Electric Power Department, Railway Research Institute, Chlopickiego str., 50, Warsaw, Poland, e-mail: vkuznetsov@ikolej.pl 3 Electric Power Department, Railway Research Institute, Chlopickiego str., 50, Warsaw, Poland, e-mail: wkruczek@ikolej.pl 4 Department of the Electrical Engineering and Electromechanic, National Metallurgical Academy of Ukraine, Gagarina ave., Dnipro, Ukraine, 49600, e-mail: wit1975@i.ua 5 Department of Electrical Engineering, Dnipro University of Technology, av. Dmytra Yavornytskoho, 19, Dnipro, Ukraine, 49005, e-mail: tsyplenkov.dv@nmu.one Abstract When comparing the performance indicators of electrical networks with different types of neutral grounding, along with the fulfilment of the requirement to ensure reliability of power supply to consumers, serious attention is drawn to the main network parameters influencing the performance of power supply systems. Analysis of research and its results, reported above, on the influence of the neutral ground of power networks on the reliability and electrical safety conditions of the power supply systems as a whole, on the damage of distribution networks and electrical equipment as well as on the working capacity of relay protection devices, provides an opportunity to estimate each specific operation mode of the neutral and to develop recommendations aimed at strengthening the positive indicators of the corresponding modes. Other things being equal, reliability of power supply to electrical receivers or reliability of distribution networks is mainly determined by the damage to network elements and the performance of relay protection devices. The degree of influence of these factors on the reliability of distribution networks depends on the neutral mode, which in turn determines the level of internal overvoltages and the nature of transient processes at earth fault. Keywords: electric networks, protection, reliability, ground failure current, electrical safety, system with isolated neutral, system with resonant grounding, system with low-impedance neutral grounding. 1. RELEVANCE The rapid development of renewable energy systems in Europe and the world creates new directions for their application. One of these areas in recent years has been the use of a distributed generation system to provide power to non-traction consumers of railway transport [1-4]. Such systems are called local systems and in their specificity of work, structure and execution: they significantly differ from general-purpose systems [5-9]. A feature of the work of these systems is the provision of power to consumers of the first and second categories, namely: circuits of automatic and semiautomatic blocking, signalling, communications, outdoor track lighting, automatic heating control systems for buildings etc. [10-12]. Accordingly, for such networks it is necessary to determine the indicators on which the reliability of providing electric energy depends [13-15]. The reliability and safety of maintenance of these systems is largely determined by the neutral grounding method, which is established according to the instructions of normative and technical documents and by technical and economic comparison according to established criteria. In many countries of the world, these conditions have developed historically, under the influence of various factors that changed during the development of networks [16-19]. Today, prerequisites have arisen to analyse these factors of influence on the reliability, efficiency and safety of servicing local networks. In accordance with [16] medium voltage (MV) networks can have system with isolated neutral or system with resonant grounding or system with low-Impedance neutral grounding (resistance) [20]. When comparing the performance indicators of electrical networks with different types of neutral 46 DIAGNOSTYKA, Vol. 22, No. 1 (2021) Ostapchuk O, Kuznetsov V, Kruczek W, Kuznetsov V, Tsyplenkov D.: Analysis of the neutral modes ... grounding, along with the fulfilment of the requirement to ensure reliability of power supply to consumers, serious attention is drawn to the main network parameters influencing the performance of power supply systems, these include: - insulation levels and overvoltage resistibility (overvoltage protection) [21, 22]; - provision of single-phase-to-ground failure protection and its performance; - selectivity of protection actions against ground failures; - safety protection from the effect of touch and step voltages at earth failure. Other things being equal, reliability of power supply to electrical receivers or reliability of distribution networks is mainly determined by the damage to network elements and the performance of relay protection devices. At the same time, the main indicators of failure will be following: - the probability of failure Q (t); - MTTF T 0 ; - the average recovery time T R. The degree of influence of these factors on the reliability of distribution networks depends on the neutral mode, which in turn determines the level of internal overvoltages and the nature of transient processes at earth failure. The level of overvoltage has a decisive influence on the first and second indicators, and the nature of transient processes - on the third [23]. Distribution networks MV with system with isolated neutral are most common, but such operation mode of the network neutral is not always optimal in terms of the specified earlier criteria. Power supply systems with a fully insulated neutral, as compared to networks with other neutral modes, do not require additional capital expenditures. However, the operational costs of such networks due to their greater damage rate as well as due to the power outages, are significantly greater than those in the networks operating with other neutral modes [20, 24]. The efficiency of the capacitive current compensation and the performance system with resonant grounding largely depends on the tuning mode of the compensating device. In this context, most researchers give preference to the resonant inductance tuning (it is extremely difficult to provide it in dynamic networks) of the compensating device with a network capacity relative to the earth. From an economic point of view, distribution networks with the compensation of the capacitive ground failure current require additional capital expenditures on the arc suppression reactors and devices for their connection. With regard to operating costs, they are significantly less than those in the networks with an isolated neutral due to a lower level of damage to the elements of the system. With resonance tuning of the compensating device and minor disorders of compensation in electric networks, the value of dielectric strength in relation to the operating overvoltages increases up to 30% [25, 26]. Electrical networks with a neutral resistor have, in comparison with system isolated neutral and resonant grounding, higher reliability because of the suppression of transient processes accompanying single-phase ground failures, the reduction of damage to the elements of the power supply system (the latter is caused by a significant decrease of internal overvoltages) and the elimination of ferro-resonance processes. Considerable additional capital costs for the implementation of networks with a neutral resistor, as compared to networks with a fully isolated neutral, at the earth failure currents will be greater than 5 ... 10 A. In this case, it is required to include a high-voltage resistor and devices for its connection into the neutral network, which in addition to switching devices may also include special transformers needed for the connection of resistors. In networks with ground-failure currents up to 5 A, additional capital costs decrease practically to zero, since in this case it is possible to manage available in the network measuring transformers and low-voltage resistors [21]. 2. EFFECTS OF THE NEUTRAL MODE ON THE PHASE CHARACTERISTICS OF EMERGENCY CURRENTS In local systems, the earth failures are the most common damages (up to 70-85% of the total number of accidents) [27, 23]. In such networks, possible overvoltages can reach 3.95 the phase voltage value. Hence greater attention and higher requirements to the operational performance of protection devices, otherwise the unreasonable interruptions in power supply to responsible consumers (under the mistaken a protective actions) or further development of the failure is probable (in case of protection failure, transition of a singlephase earth failure into a double one is possible). To study phase characteristics of voltage and zero sequence currents, we use the equivalent circuit (Fig. 1), which is presented in the form of two attachments connected to one power transformer [28]. Given that, we believe that conductance of appropriate phase-to-ground insulation of the controlled connection ( Y A1 = Y B1 = Y C1 = Y 1 ) and the rest of the distribution network ( Y 'A = Y 'B = Y C' = Y ' formula Y 1' + Y ' ) are related by = Y ... 1 1 Then Y = + jC; Y 1 = + jC1; R1 R 1 1 1 1 YK = −j ; Y = 1 ; y = ; yi = . RK  L N RN ri r the DIAGNOSTYKA, Vol. 22, No. 1 (2021) 47 Ostapchuk O, Kuznetsov V, Kruczek W, Kuznetsov V, Tsyplenkov D.: Analysis of the neutral modes ... The Figure 1 shows the zero sequence current filter TA installed on the damaged connection and intended to isolate the zero sequence current I 0 in the monitored line; currents flowing through the conductance of phase-to-ground insulation of the monitored line ( I A1 , I B1 , I C1 ), and currents flowing through the appropriate phase conductance of the external network ( I 'A , I 'B , I C' ) ; I g 1 , I g - single- phase ground failure currents in the monitored line and in the external network, respectively; I N current flowing through the conductance of the network neutral point with respect to ground. We proceed from the fact that a single-phase earth failure can occur in the supervised line ( y1  0 ) or in the external network ( y  0 ). L1 L2 L3 I0 Q ТA IN Q1 I A1 I B1 I C1 YH I A I B IC Q2 I g1 I g y1 YA1 YB1 YC1 y Y A Y B YC Fig. 1. The equivalent circuit diagram of the distribution network for study of phase characteristics of voltage and zero-sequence currents 2.1 System with isolated neutral ( YN = 0 ) By using the basic laws of electrical engineering for the presented equivalent circuit diagram, we have done necessary transformations and obtained a mathematical equation for the zero-sequence current in the case of an error in the monitored line: I 0C = −3U 0  Y ' = −3U 0  (Y − Y 1 ) or  ( R − R)+ j(C −C1 ) R R1  (1) I 0 = −3U 0  1 , R R1   Where R and C are, respectively, the insulation resistance and the capacity of the whole electrically connected network with respect to ground; R1 and C1 are, respectively, the active insulation resistance and the capacity of the supervised connection with respect to ground. Equations for the intrinsic current of the monitored line (zero-sequence current in the monitored line with an external earth failure) are as follows: I 0C = 3  U 0  Y 1 or 1 + j  C1  R1 R1 The analysis of the equations (1) and (2) shows that in the network with the fully isolated neutral, the zero-sequence current in the failure line is determined by the zero-sequence voltage and insulation parameters of the external network with respect to ground, that is, by the isolation parameters of the entire network with respect to ground, excluding insulation of the faulted connection, whereas the intrinsic current of the monitored connection (zero-sequence current in the supervised line with external faults) is determined by the zero-sequence voltage and insulation parameters with respect to ground, only in relation to the supervised connection. From the obtained expressions, it can be concluded that on the basis of the appearance of voltage and current of the zero sequence it is possible to establish the fact of asymmetric insulation damage, and by their magnitude to assess the degree of this failure. The presence of these indicators can be used to build diagnostic models. Using these equations, with account of their imaginary and real parts, we obtain formulas that determine the phase dependence of the zerosequence current vector: I 0C = 3  U 0 01 = 1800 + arctg (C −C1 ) R1 R R1 − R (3) , and the phase dependence of the intrinsic current vector of the monitored line (the current of the zero sequence in the monitored line with the external single-phase-to-ground fault) 02 = arctg (  C1  R1 ), (4) in relation to the zero sequence voltage vector on the insulation parameters with respect to ground of the whole network and of the controlled connection. The analysis of the obtained equations (3) and (4) shows that taking into account the real values of the insulation parameters, relative to the earth, of the whole network and of a separate connection, and also taking into account the actual interrelation of the capacitive and active insulation resistance, the following conclusions can be drawn: - the angle between the zero-sequence current vector and the zero-sequence voltage vector does not depend on the completeness of the closure (the transient resistance at the closing point) and it is almost 270 el. degrees, or minus 90 el. degrees; - the angle between the intrinsic vector of the monitored line (zero-sequence current in the monitored line with the external single-phase-toground fault) and the zero-sequence voltage vector is determined by the isolation parameters relative to the ground only of the controlled connection and it is almost 90 el. degrees. 48 DIAGNOSTYKA, Vol. 22, No. 1 (2021) Ostapchuk O, Kuznetsov V, Kruczek W, Kuznetsov V, Tsyplenkov D.: Analysis of the neutral modes ... 2.2. Compensated neutral network upon 1 1 condition ( Y N = Y K = ) −j RK L For this network, we similarly obtain the equations [6, 18] for the zero-sequence current in case of damage in the controlled line:  L  (3R1  RK − 3R  RK + R  R1 ) − I 0 = −U 0  [ R  R1  RK   L −j R  R1  RK  ( − 3   2  C1L) ] R  R1  RK   L , (5) for the intrinsic current of the monitored line (zero sequence current in the monitored line with the external earth fault): 1 + j  C1  R1 I 0C = −3  U 0 , (6) R1 and also the formulas determining the phase dependence of the zero sequence current vector: 01=180 − − arctg ( R  Rk  R1  − 3 2  C1  L ) , (7)   L ( 3Rk  R1 − 3Rk  R + R  R1 ) and the phase of the intrinsic current vector of the monitored line with respect to the zero-sequence voltage vector on the insulation parameters 02 = arctg (  C1  R1 ) (8) with respect to the zero sequence voltage vector from the insulation parameters relative to the earth of the whole network, the controlled connection and the degree of the compensating device detuning = 1 − 3  2  C  L from the resonance mode with the network capacity. The analysis of the equations (5) - (8) shows that at the single-phase earth fault in the network with a compensated neutral, taking into account the actual values of insulation parameters relative to the ground of the whole network and of the separate connection as well as taking into account the actual values of the arc suppression reactor parameters, we can state: - the zero sequence current in the faulty line is determined by the zero sequence voltage and the insulation parameters of the external network relative to the ground, i.e. the insulation parameters of the entire network relative to the ground (including the compensating device parameters), minus the insulation parameters of the damaged connection; - the angle between the zero-sequence current vector and the zero-sequence voltage vector does not depend on the completeness of the closure (transient resistance at a closing point) and is determined to a large extent by the value of detuning of the compensating device from the resonant mode and for resonance tuning it is approximately 180 el. degrees; when the compensating device is detuned from the resonance mode both in the direction of overcompensation and under compensation, the current vector deviates by an angle of respectively up to plus 90 el. degrees and minus 90 el. degrees, so the range of angle variation between the zero-sequence current vector and the zero-sequence voltage vector is theoretically 180 el. degrees; - the intrinsic current of the controlled connection is determined by the zero-sequence voltage and the insulation parameters with respect to ground (5) only of the controlled connection; - the angle between the intrinsic current vector of the monitored line and the zero-sequence voltage vector is determined by the insulation parameters with respect to ground only of the controlled connection and it is almost 90 el. degrees. 2.3 Network with a neutral resistor ( Y N = Y R = 1 / R N ). For such a network, in accordance with the equivalent circuit diagram (Fig. 1), the formulas [3, 4, 7] for the studied currents and their phase characteristics are as follows: I 0 = −U 0  [  L  (3RN  R1 − 3R  RN + R  R1 ) R  R1  RN R  R1  RN  (C − C1 ) ]; R  R1  RN 1 + j  C1  R1 I 0C = −3 U 0 ; R1 − (9) −j 01=180 + arctg (10) R  RN  R1   ( C − C1 ) ; (11) 3RN  R1 − 3R  RN + R  R1 02 = arctg (  C1  R1 ), (12) and their analysis shows that in case of a singlephase earth fault in the network with a neutral resistor: − the zero-sequence current in the faulty line is determined by the zero sequence voltage and the insulation parameters of the external network with respect to ground as well as by the resistance value of the neutral resistor, and the phase of this current in relation to the zero sequence voltage vector does not depend on the completeness of the closure and in contrast to the networks with a fully insulated neutral it is not fixed and depends on the resistance value of the resistor, besides it can theoretically take values in the range from 180 to 270 el. degrees in relation to the zero-sequence voltage vector; for the real network insulation parameters with respect to ground and the recommended resistance value of the resistor, this angle is approximately 225…240 el. degrees, i.e. in contrast to the network with a fully isolated neutral, a shift of the emergency current vector is possible by an angle from 30 to 45 el. degrees; DIAGNOSTYKA, Vol. 22, No. 1 (2021) 49 Ostapchuk O, Kuznetsov V, Kruczek W, Kuznetsov V, Tsyplenkov D.: Analysis of the neutral modes ... − the intrinsic current of the controlled connection is determined by the zero-sequence voltage and insulation parameters with respect to ground only of the controlled connection, and the angle between the vector of this current and the zerosequence voltage vector is determined in the same way as in the networks with a fully isolated and compensated neutral − by the insulation parameters relative to the ground only of the controlled connection and it is almost 90 el. degrees. When constructing diagnostic models, it’s must be taken into account the fact that the position of the zero-sequence current vector with respect to the zero-sequence voltage vector is generally determined by the nature and mode of the network neutral ground, and the phase of the intrinsic current of the controlled connection (zero-sequence current in the monitored line with the external single phase-to-ground fault does not depend on the neutral operation mode − it is determined only by the parameters of the directly controlled connection and practically it is rigidly linked to the zerosequence voltage. 3. EFFECTS OF THE NEUTRAL MODE ON THE PERFORMANCE OF GROUNDFAILURE PROTECTION At present, in the distribution networks, the ground failure protection devices, responsive to the steady-state parameters of the emergency mode, which can conditionally be divided into three types: current (maximum) protection - devices that react to the zero sequence current; voltage protection devices that respond to the zero sequence voltage; directional protection - devices that respond to the zero sequence power [17, 24, 29]. Studies have shown that the use of simple current protection in networks with a fully isolated neutral is justified by the sensitivity conditions under the capacitance of the protected line 5…7.5 times less than the capacity of the entire electrically connected network. If the specified condition is not observed, selectivity of its operation is disrupted. The necessity of fulfilling this condition significantly limits the application area of current protection devices, particularly if to take into consideration the fact that, while in operation, the capacity of network, as well as of individual lines, varies greatly. The ratio of the zero sequence currents in case of external earth fault and in the protection zone, as well as the conditions ensuring selectivity performance of the current protection devices, make it difficult to use them in networks with a compensated neutral. In networks with the capacitance current compensation in the steadystate ground-fault mode, when adjusting the compensating device in resonance with the network capacity relative to the earth, as well as with insignificant disorders from the resonance mode, the zero sequence current of the protected connection (intrinsic current) is nearly always greater than the closing current and the zero sequence current in the case of an error in the protected connection. This circumstance practically excludes the possibility of applying current protection devices in networks with a compensated neutral. The most favourable conditions for the application of simple residual-current protective devices are created in networks with a neutral resistor due to the suppression of transient processes, however it is difficult to achieve the operational performance of current protection devices in case of faults through the transition resistances. Protective devices responding to the zerosequence voltage are characterized by the simplicity of their circuits and elements; in local networks they are mainly used either in the form of backup units for networks with high security requirements The principal drawback of such protective devices is an inability to ensure the performance selectivity. In system with resonant grounding, application of such protectors is also practically impossible because of the high value of the neutral bias voltage at resonance (or close to resonance) tuning of the arc suppression reactor. Devices of directional protection against singlephase ground failure, responding to the parameters of the steady-state closing mode, ensure the operation selectivity due to the phase comparison of the zero sequence current and voltage. Such devices are largely used only for networks with a fully isolated neutral, since in such networks the angles between zero sequence currents and voltages practically do not depend on the parameters of the network insulation and the transition resistance at the fault point in case of one- phase ground failure and they are practically fixed. The presence of transient processes accompanying the occurrence of a phase-to-ground failure and primarily the shutdown of the damaged connection should be considered as the determining reasons for the unsatisfactory operation of the directional protection devices in networks with a fully isolated neutral. The reason for this lies in the transient process which accompanies the process of recovery of phase-to-ground voltage and which is oscillating, damping and rather lengthy, with a frequency close to the industrial one. The reasons, causing false operation of existing directional protection devices, should also include the imperfection of circuitries. From this group of reasons special attention should go to the following ones [30, 31]: - insufficient detuning of devices through the zero sequence current and voltage channels from higher harmonic components, the level of which can be significantly high, especially at closures through the alternating arc; − a wide angular operating area that is approximately 180−210°, which leads to a time coincidence of the compared signals due to their 50 DIAGNOSTYKA, Vol. 22, No. 1 (2021) Ostapchuk O, Kuznetsov V, Kruczek W, Kuznetsov V, Tsyplenkov D.: Analysis of the neutral modes ... phase distortions caused by angular errors of current and voltage transformers as well as phase shifts of signals directly in the device circuit, etc. In networks with a compensated neutral, the specified principle of protection implementation is impossible, as in these networks the angles between zero sequence currents and voltages are determined mainly by the tuning mode of the compensating device and the value of the transition resistance at a point of damage and it may vary within 180 ° (from minus 90° to plus 90°) which practically excludes the selectivity principle of the protection operation [29]. In networks with a neutral resistor, perspectives of using the directional protection devices from single-phase earth faults, operating on the basis of phase comparison of the zero sequence current and voltage can be considered good for the following reasons: - in such networks, the false operation of devices from transient processes is practically excluded, because the presence of a neutral resistor greatly suppresses the duration and amplitude of the transient processes; - the angular error for the values, recommended for quarry networks and introduced by superimposing an active component of the earth fault current on the network, is in the range of 30 ° ... 40 ° and it refers exclusively to the zero sequence current in the faulty line. The current ground-failure protection devices, which received their application in networks with a compensated neutral, are devices responding to the higher harmonic components of the zero sequence current. Reliable functioning of the devices under consideration is possible only at a sufficiently high level and stability of the natural harmonics used for the protection operation. The absence of a compensating device (network with a fully insulated neutral) and the inclusion of the resistor in the neutral network contribute to a decrease in the level and spectrum of natural harmonics. A general lack of the devices responsive to higher harmonics in the zero sequence current is a complex dependence of the structure and level of natural harmonics on: the number of lines, the tuning mode of the compensating reactor, the resistance at the point of fault, as well as the presence of interferences. Investigations have shown that for the singlephase ground failure through the transition resistance of several tens of ohms, the level of the higher harmonics components sharply decreases. So, with a change in the coefficient of the closure completeness from 1 to 0.4, the zero sequence current for the 5th harmonic decreases 11.5 times, for the 7th harmonic − 16 times, for the 11th harmonic − 25 times [27,32]. Protection devices responding to the transient process parameters are used in system with resonant grounding, since for the protection implementation of the ground fault capacitive current compensation doesn't allow, as a rule, the usage of simpler and more reliable principles based on the parameter checkout of the zero sequence currents and voltages. To the shortcomings of the earth-fault protection devices, responding to the amplitude and wave characteristics of the transient process, which largely limit their spread in the quarry distribution networks, it is necessary to include the following ones: - the inrushes of the initial capacitive current as well as the currents and voltages of waves of the transient discharge process are determined, to a significant extent, by the values of voltage and its phase at the moment the earth fault occurs. At the same time, the aerial quarry electrical networks are characterized by a high probability of a mechanical insulation damage, which can occur at the moments when the voltage of the damaged phase hasn't reached its highest value yet and when the transient process hasn't actually arisen; - the presence of a transition resistance at the point of the phase-to-ground fault leads to a decrease in the amplitude and time characteristics of the transient process. Operational experience with distribution networks shows that when the value of the transient resistance is of the order of hundreds of ohms, the transient process practically does not arise; - the absence of repeated actions of the protective devices when the signal is acknowledged in the conditions of a steady earth fault. The ground-failure protection devices that react to the network-imposed currents of non-industrial frequency can be used in networks with any neutral mode. The disadvantage of such devices is the absence of operation selectivity (the transformer is usually switched off). For the devices reacting to the superimposed alternating current of non-industrial frequency, the value of the superimposed current is composed of the components determined, in addition to the voltage level of the source Us, by the value of the transition resistance r at the fault location and the sum of the phase network capacities with respect to ground: I v.s = I v.s + I v.s = U v .s U v.s + , z + r z + 3 xc (13) where z - is the value of the total resistance of transformer windings, line resistance and ground resistance for the current of the superimposed frequency; xc - is the capacitive resistance of one phase of the entire network with respect to ground (for the superimposed frequency). The second component of the superimposed current is apart from the transition resistance value and rises with an increase of the network capacity and of the control voltage frequency. This DIAGNOSTYKA, Vol. 22, No. 1 (2021) 51 Ostapchuk O, Kuznetsov V, Kruczek W, Kuznetsov V, Tsyplenkov D.: Analysis of the neutral modes ... component is distributed across all transmission lines of the network in proportion to their capacities. A general lack of the protection reacting to the superimposed current of both low and high frequency is the impossibility of creating highly sensitive devices, since it is necessary to avoid the leakage of operating current through the capacitance of the protected line. In addition, one must note the possibility of false operation of protection devices during the transient processes, since the spectrum of currents in the transient process can contain components of operating frequency [33]. The principle of superimposing the operational voltage of non-industrial frequency on the protected network is most preferable for system with resonant grounding both by the simplicity implementation of the protection device itself and operational source. 4. INFLUENCE OF THE NETWORK NEUTRAL MODE ON ELECTRICAL SAFETY CONDITIONS. In accordance with the electrical safety conditions, when a person directly touches the current-carrying parts of the distribution networks, none of the proposed neutral operation modes can be considered as an advantageous one. Regardless of the neutral mode with account of the real insulation parameters of the distribution networks, as well as the action time of protection devices and used switching equipment, the current value passing through the human body will significantly exceed the safe levels. None of the protective devices can guarantee safe outcomes when a person touches current-carrying parts in networks with any neutral mode at a voltage of 6 kV and higher. Even in the networks with the compensation of the capacitive fault current, the presence of a transient process arising at the moment of touching one of the phases, the benefits of such networks, in respect to electrical safety, are practically reduced to zero. At the moment of touching, the so-called free current of the compensating device will pass through the human body, the frequency of which is determined by the physical parameters of the electrical network and the arc-suppression device, and its value is approximately 25% of the capacitive fault current [28]. Therefore, with regard to the distribution networks MV, one should speak about the indirect electrical safety of people from the effect of touch and step voltage, when operating electrical networks. The degree of danger of an electrical network for a person who touches the energized electrical equipment and installations housings because of the damage of one of the phases depends, to a large extent, on the neutral mode. Especially this study is of interest for the widespread mode of unstable closure (through an alternating arc). In this mode, which in networks with an isolated neutral at certain network parameters poses danger to humans, in networks with a neutral resistor the danger of touching the frames of damaged equipment significantly decreases due to a sharp reduction in the duration of the transient process as well as to a decrease in the amplitude of the earth fault current inrushes. When superimposing an additional active current equal to at least 50% of the capacitive current at the earth fault location, it is interesting to analyse the touch and step voltages arising on the earthed enclosures of electrical equipment with the alternating arc faults in the network. Since the touch and step voltages are determined by the earth fault current (flowing from the grounding electrode), it makes sense to consider the effect of the active neutral resistance on the value of this current in the transient mode. For the air-cable distribution network, which can be represented in the form of L, C, r of the contour (without taking into account the longitudinal active resistance of the network), connected to the instantaneous phase voltage U = U m cos(t ) ; at the beginning of the closure as well as throughout the entire process of burning of the alternating (or intermittent) arc free and steadystate components of the emergency current arise. The maximum inrush of a free capacitive current with sufficient accuracy for practical calculations is determined by the formula [21]: U i fr = m  e− t  sin (   t ) ,  L or i fr = 2 I C  K  e− t   2fr  sin (   t ) , (14)   where Um – is the maximum value of the voltage on the damaged phase; K – is the voltage multiplicity of the damaged phase at the moment of breakdown; δ = r/(2L) – is the damping coefficient of the free r2 1 – is − 4 L2 3L  C the angular frequency of natural oscillations; 1 ; L – is the zero-sequence equivalent  fr = 3L  C inductance of one phase; r – is the earth fault resistance, keeping in mind that the minimum possible resistance for the arc combustion is taken equal to the resistance of the quarry grounding network − 4 Ohm. Calculations performed by the expression (14) show that initial values of the free circuit current can be tens of times greater than the amplitude of the steady-state capacitive current IC in the event of a ground failure. At several repeated one after another extinctions and ignitions of the arc, free current surges are also repeated. The effective value of the current for one period of the industrial circuit current;  =  2 −  fr = 52 DIAGNOSTYKA, Vol. 22, No. 1 (2021) Ostapchuk O, Kuznetsov V, Kruczek W, Kuznetsov V, Tsyplenkov D.: Analysis of the neutral modes ... frequency (assuming that the arc goes out and ignites once per period) with the accuracy sufficient for practical purposes can be determined by the formula [21]: 0.013 i fr = K  IC , (15) rC So, for example, in the network voltage of 6 kV with IC = 10 А, r = 4 Ω and K=1 the effective value of free current will be Ifr = 38 А. When grounding the network neutral through the resistor that provides creation of an active component of the failure current at the level of 50 – 100 % of the capacitive one, in case of an arc closure to grounded electrical equipment, the effective value of the resulting failure current will decrease 1.4…2.8 times compared to the possible value in the same network with a fully isolated network neutral [22]. This reduction occurs owing to the effective limitation of overvoltages in the network after each break in the arc of the earth failure. |In view of the overvoltage reduction on the undamaged phases (up to 2.2…2.4 U), as a result of the superimposition of the active current, the probability of developing single-phase failures in the closures of two phases at different points of the network also significantly decreases. 5. COMPARATIVE ESTIMATION OF THE NEUTRAL MODE EFFICIENCY IN TERMS OF LOCAL DISTRIBUTION NETWORKS An analysis of the above results of studies of the degree of influence of the neutral grounding method on the operational characteristics of autonomous power supply systems, as well as the performance of relay protection devices, allows us to evaluate each method and develop practical recommendations aimed at their further development. 5.1. System with isolated neutral An insulated neutral system is widespread and has extensive operating experience. However, such a grounding method network neutral mode is not always optimal in terms reviewed of the criteria specified earlier. Taking into account possible changes in the lowering coefficients, for the actual parameters of distribution networks, the maximum voltage between the healthy phases and the ground is at a level of 4.5 of phase voltage. For these same networks, the theoretical maximum of the neutral point displacement voltage is a threefold phase voltage. Besides, the single-phase earth failures in networks with an insulated neutral are accompanied by transient processes arising at the moment of failure occurrence and at the moment of disconnection of the damaged section (the process of voltage recovery in the network), which support a significant part of false responses of the groundfailure protective devices. The disadvantages of networks with an insulated neutral may include: the instability of the neutral voltage, favourable conditions for the occurrence of arc closures, ferro-resonance phenomena, increased touch and step voltages in case of arc ground failures, increased multiplicities of internal overvoltages, etc. 5.2. System with resonant grounding When grounding the neutral through the arc suppression reactor (tuned inductance), the overvoltages of arc closures is smaller and, what is especially important for insulation, they last for a relatively short time interval (usually about half a period). However, this advantage is minimized when the compensation mode is detuned by more than 5% of the resonance value. Also, the disadvantage of the neutral grounding system through the arc suppression reactor is the complexity of providing resonant tuning. Therefore, for small currents, the use of this type of grounding is not economically feasible. If to assess the reliability of power supply to electric consumers by the damage of network elements and by the operating quality of the relay protection, it should be noted that application of compensated networks, where the protective shutdown action is required, is constrained by the second condition. As for the damage rate of the elements of distribution networks, one must note that this indicator directly relates to the tuning mode of the compensating device, since it is the degree of the compensating device detuning from a resonant mode that determines the level of overvoltages in the network under single-phase earth failures. Figure 2 shows that with the resonant tuning of the compensating device, as well as with its detuning within 5%, even theoretically, the overvoltages on the undamaged phases cannot exceed more than 2.75Uf. The decrease in the level of overvoltage is due to the creation of a convenient path for the drainage of static charges in phases due to the inclusion of the inductance of the arc suppression reactor in the neutral of the network [27]. An increase in the degree of compensation detuning from 5 to 30...40% leads to a rapid increase in the level of overvoltages. It should be noted that with a detuning of the compensating device by 20% of the resonant one, the effectiveness of the compensating devices in terms of limiting overvoltages during earth faults is practically not felt in comparison with networks with a completely isolated neutral. The overvoltage in the neutral of the network is approximately 1.5...2 times less than the frequency of overvoltages in the undamaged phases, that also DIAGNOSTYKA, Vol. 22, No. 1 (2021) 53 Ostapchuk O, Kuznetsov V, Kruczek W, Kuznetsov V, Tsyplenkov D.: Analysis of the neutral modes ... helps to reduce the damage to the elements of the power supply system. From an economic point of view, distribution networks with capacitive earth fault current compensation require additional capital costs for arc suppression reactors and devices for their connection. With regard to operating costs, they are significantly less than in networks with a completely isolated neutral due to the less damage to the system elements. With a resonant adjustment of the compensating device and with insignificant compensation mismatches in electrical networks, the margin of dielectric strength of insulation in relation to the impacting overvoltages increases to 30%. Fig. 2. Dependences of the maximum overvoltage multiplicity on the degree of compensation detuning in the mode: 1 - overcompensation; 2 - undercompensation 5.3. System with low-Impedance neutral grounding (active resistance) The system with low-impedance neutral grounding (active resistance), as compared to other neutral grounding systems, have higher reliability due to the improved performance of the protection devices against single-phase earth failures (suppression of post-accidental transient processes), the elimination of ferro-resonance processes and the reduction of the damage to the elements of the power supply system. The latter is conditioned by a significant decrease in internal overvoltages. As the active component of the failure current increases with respect to the capacitive component, the overvoltage multiplicity decreases to 2.4 provided that active and capacitive failure currents are equal. The magnitude of overvoltage in a three-phase network with an active resistance in the neutral is determined by the expression: 0 U ov = 3 U f  sin ( ig + 30 ) + +U f sin   ig − (sin  arc − 0.2 )e− k   a ( ig − arc )   (16)  ( − ) C 1 d , C +CM where  ig – phase of voltage of the damaged phase at the moment of ignition, rad.;  arc – phase of the voltage of the damaged phase at the moment of arc extinction, at which the bias voltage reaches its maximum; ka is the ratio of active and capacitive fault current components. Figure 3 shows the dependence of the maximum multiplicity of internal overvoltages in the network with a resistor in the neutral on the ratio of the active and capacitive components of the single-phase earth fault current, obtained taking into account the most unfavorable conditions according to expression (16). Fig. 3. Dependence of the maximum multiplicity of overvoltage in a network with a resistor in neutral from the ratio of the active and capacitive components of the fault current As the active component of the fault current grows in relation to the capacitive component, the overvoltage ratio decreases to 2.4, with the active and capacitive fault current being equal. Figure 3 shows that a further increase in the active component practically does not lead to a significant decrease in the overvoltages ratio. From the point of view of providing the singlephase ground failure protection, as well as the quality of its performance (which is directly related to the electrical safety and continuity of power supply to electrical receivers), the power networks with a neutral resistor, in other words, with the overlay of the additional active component on the ground failure current, can be considered as the most favourable ones. In such networks, currents and directional devices used in the insulated neutral networks show more reliable and efficient operation by suppressing the transient processes. On condition of providing the single-phase ground failure protection, the electrical networks with a compensated neutral are in the most unfavourable position. 54 DIAGNOSTYKA, Vol. 22, No. 1 (2021) Ostapchuk O, Kuznetsov V, Kruczek W, Kuznetsov V, Tsyplenkov D.: Analysis of the neutral modes ... According to the conditions of electrical safety of power networks, when a person directly touches the current-carrying parts, none of the possible neutral operation modes can be considered as a favourable one. According to the conditions of indirect danger, preference should be given to electrical networks, then the electrical network with the neutral resistor should be considered as the most favourable one. With the development of autonomous networks and an increase in the total capacity of the earth failure, the combined neutral mode of the network is advisable. It takes place when the resistor and the arcing reactor are connected in parallel to the neutral of the network. In this case, in addition to creating the inductive component of the current of a single-phase earth failure, the active component is simultaneously superimposed on the network. The value of the active component imposed on the compensated network should be at the level of 30 50% of the capacitive component, that is, be selected from the condition: I a = (0.3...0.5)  IC . (17) The recommended values of the emergency current active component, ensure the suppression of transient processes in the event of ground failures, the increased efficiency of ground-failure protective devices (signalling), the exclusion of the ferroresonance phenomena, which leads to an increase in the level of electrical safety and reliability, as well as to the improvement of performance characteristics, adequate to the power supply systems only with the neutral resistor, even at the detuning of an arc suppression reactor to 50% of the resonance compensation mode [34-35]. Fig. 4 shows a comparison between the zone of maximum multiplicity of overvoltages on the degree of detuning compensation from the resonant mode in a network with the compensated neutral (zone 1) and in a network with the combined neutral operation mode (zone 2). 4.4 Uov/U 1 4.0 3.6 3.2 2 2.8 2.4  2.0 0 0.2 0.4 0.6 0.8 Fig. 4. Dependence of the multiplicity of internal overvoltages at earth failures on the degree of detuning compensation mode: 1 - in a network with the compensated neutral; 2 - in a network with the combined neutral grounding The upper and the lower boundaries of the zones correspond to the values of the coefficient overvoltage reduction, equal respectively to 1 and 0.8, which takes into account network physical characteristics, relative damage location, etc. 6. CONCLUSIONS 1.The carried out studies allowed to establish the fact that the reliability of distribution networks in local systems is determined by the failure rate of network elements and the effectiveness of relay protection devices. The degree of influence of these factors on the reliability of distribution networks depends on the neutral mode, which in turn determines the level of internal overvoltages and the nature of transient processes at single-phase-to-ground failures. 2. As regards the provision of earth failure protection as well as of its performance (that is directly related to the electrical safety and the power supply continuity to electrical consumers), electrical networks with the resistor in a neutral can be considered as most advantageous ones, where the current and directional protection devices applied in the networks with the insulated neutral, due to the suppression of transient processes, show more reliable and effective operation. In terms of provision of the single-phase-to-ground failure protection, electric grids with the compensated neutral are in the most unfavourable position. 3. The lowest level of operational reliability corresponds to the networks with the fully isolated neutral, as well as to the ones with the compensated neutral in case of compensation detuning to 20% and higher from the resonant value. This is because of the high level of damage to the elements of power supply systems by the effect of internal overvoltages and of ferroresonance phenomena. 4. In the case of compensated networks in which it is difficult to provide a resonant tuning mode, it is recommended that the combined neutral mode of operation, with the creation of an active component, be at the level of 30 … 50% of the capacitive one, that provides performance indicators adequate to power supply systems with only a resistor in the neutral, even during malfunctions arc suppression reactor up to 50% of the resonant compensation mode. 5. None of the considered methods of neutral grounding allows to achieve the required level of reliability in autonomous systems and requires the use of additional devices that continuously monitor the above indicators. 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Institute of Electrical and Electronics Engineers Inc. https://doi.org/10.1109/UKRCON.2019.8879841. Tifour B, Moussa B, Ahmed H, Camel T. Monitoring and energy management approach for a fuel cell hybrid electric vehicle. Diagnostyka. 2020:21(3):1529. https://doi.org/10.29354/diag/123996 Budahs M, Rozenkrons J, Zviedritis M. Latvia's New Energostandard "Medium Voltage Network Neutral Regimes". 2014 9th International Electric Power Quality and Supply Reliability Conference. 2014: 363-366. Colella P, Napoli R, Pons E, Tommasini R, Barresi A, Cafaro G, De Simone A, Di Silvestre ML, Martirano L, Montegiglio P, Morozova E, Parise G, Parise L, Sanseverino ER, Torelli F, Tummolillo F, Valtorta G, Zizzo G. Current and Voltage Behavior During a Failure in a HV / MV System: Methods and Measurements. 2015 Ieee 15th International Conference on Environment and Electrical Engineering . 2020: 404-409. Mitolo M, Musca R, Tartaglia M, Zizzo G. Electrical safety analysis in the presence of resonant grounding neutral. IEEE Transactions on Industry Applications. 2019; 55(5): 4483-4489. https://doi.org/10.1109/tia.2019.2926236 Wronkowicz A. Concept of diagnostics of energy networks by means of vision system. Diagnostyka. 2014;15(2):13-18. Lowczowski K, Lorenc J, Andruszkiewicz J, Nadolny Z, Zawodniak J. Novel earth failure protection algorithm based on MV cable screen zero sequence current filter. Energies. 2019;12(16): 3190; https://doi.org/10.3390/en12163190 Benguesmia HM, Ziou N, Boubakeur A. Simulation of the potential and electric field distribution on high voltage insulator using the finite element method. Diagnostyka. 2018; 19(2): 41-52, http://dx.doi.org/10.29354/diag/86414 Rouini A, Derbel N, Hafaifa A, Kouzou A. New approach for prediction the DC breakdown voltage using fuzzy logic controller. Diagnostyka 2018; 19 (3): 55-62. https://doi.org/10.29354/diag/92294 Kacejko P, Machowski, J. Short circuits in power networks. Basics of calculations. WNT, Warsaw, 1993. Linčiks J, Baranovskis D. Single phase earth failure location in the medium voltage distribution networks. Electrical, Control and Communication Engineering, 2009;25:13-18. EN 50522: 2011 Groundingof power installations exceeding 1 kV AC. Andersen E. Impact of zero point grounding method on surges in medium voltage networks. Prace Instytutu Energetyki 1986. 56 DIAGNOSTYKA, Vol. 22, No. 1 (2021) Ostapchuk O, Kuznetsov V, Kruczek W, Kuznetsov V, Tsyplenkov D.: Analysis of the neutral modes ... 27. Shkrabets FP,Kovalyov AI. Neutral modes of electrical networks. Naukovy Visnyk of NMU. 2010;1: 78-83. 28. Stojanovic, ZN, & Djuric, MB An algorithm for directional earth-failure relay with no voltage inputs. Electric Power Systems Research,. 2013;96: 144-149. https://doi.org/10.1016/j.epsr.2012.11.004 29. Guo MF, Yang GJ, Huang SY. Adaptive faulty line selection based on transient characteristics for resonant groundingsystem. Electric Power Automation Equipment. 2012; 32(12):25-31. 30. Ayuurzana E, Mambetzhanov AD, Takyrbashev BK, Dzhamagidze D, Petrov MI, Kuzmin AA. Evaluation of the components of the current of a single-phase earth failure during field experiments in mediumvoltage networks for various purposes. Bulletin of the Chuvash University. 2016;3:21-29. 31. Wang Q, Liu X, Liu Z. Research on Diagnosis and Positioning of Single-Phase Arc Grounding Failure in the Distribution Network. 2018 Chinese Automation Congress (Cac), 2018:2141-2146. 32. Kolesnikova KV, Gusev YP, Smotrov NN, Lkhamdondog, AD. The Impact of the Sequence of Measures for the Transition to a Resistive-Grounded Neutral in 6-35kV Cable Networks on Investment Profitability. In 2019 IEEE Conference of Russian Young Researchers in Electrical and Electronic Engineering (EIConRus). 2019; 992-995 33. Ravlić S, Marušić A, Havelka J. Neutral grounding in middle voltage power systems: Simulation model. In 2015 38th International Convention on Information and Communication Technology, Electronics and Microelectronics (MIPRO). 2015 1124-1130. 34. Guldbrand A. System earthing. Report, Sweden. 2006. 35. Vinokurova TY, Dobryagina OA, Shagurina ES, Shuin VA. Selective complex single-phase earth failure protection for distribution medium-voltage networks. Universitätsbibliothek Dortmund. New Method for Calculation of the Harmonics in the Residual Earth Failure Current in Isolated and Compensated Networks. 2015. https://doi.org/10.17877/DE290R-7398 Received 2020-06-30 Accepted 2021-01-25 Available online 2021-01-27 Oleksandr OSTAPCHUK Department of Renewable Energy, Igor Sikorsky Kyiv Polytechnic Institute. Research interests: Power supply systems of industrial enterprises, problems of integration of distributed generation sources into the power system, relay protection, electricity metering systems, intelligent power supply systems, electrical part of stations and substations. E-mail: O.Ostapchuk@kpi.ua Valeriy KUZNETSOV. prof. dr. hab. Electric Power Department, Railway Research Institute, Poland. Obtained dr.hab. degree in electric transport in 2012. His research interests concern: Energy saving methods, energy management, reliability of traction power supply systems. E-mail: vkuznetsov@ikolej.pl Włodzimierz KRUCZEK, M.Sc. Eng: Railway Research Institute, Electric Power Engineering Department. His research interests concern: electric shock protection in traction systems, power equipment of traction substations, traction converters, overhead contact line and traction return system. PhD student at the Warsaw University of Technology Email: wkruczek@ikolej.pl Vitaliy KUZNETSOV, PhD, Department of the electrical engineering and electromechanic, National metallurgical academy of Ukraine. Received PhD in electrical complexes and systems in 2013. His research interests concern: Induction motors, Modelling, Simulation, and Power quality monitoring systems. E-mail: wit1975@i.ua Dmytro TSYPLENKOV Department of Electrical Engineering, Dnipro University of Technology PhD, Associate Professor, Head of the Department of Electrical Engineering. He graduated from the State Mining Academy of Ukraine in 1997. Research interests: Electric machines, wind energy, renewable energy E-mail: tsyplenkov.dv@nmu.one 

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PHYSICAL REVIEW D 107, 115010 (2023) Deconstructing squark contributions to di-Higgs production at the LHC Stefano Moretti,1,2,* Luca Panizzi,2,† Jörgen Sjölin ,3,‡ and Harri Waltari 2,§ 1 School of Physics and Astronomy, University of Southampton, Highfield, Southampton SO17 1BJ, United Kingdom 2 Department of Physics and Astronomy, Uppsala University, Box 516, SE-751 20 Uppsala, Sweden 3 Department of Physics, Stockholm University, 10691, Stockholm, Sweden (Received 14 February 2023; accepted 17 May 2023; published 9 June 2023) We present a novel approach to the study of di-Higgs production via gluon-gluon fusion at the LHC. The relevant Feynman diagrams involving two Standard Model-like Higgs bosons hh are computed within a simplified model approach that enables one to interpret possible signals of new physics in a modelindependent way as well as to map these onto specific theories. This is possible thanks to a decomposition of such a signal process into all its squared amplitudes and their relative interferences, each of which has a well-defined coupling structure. We illustrate the power of this procedure for the case of both a minimal and next-to-minimal representation of supersymmetry, for which the new physics effects are due to top squarks entering the loops of gg → hh. The squarks yield both a change of the integrated cross section and peculiar kinematic features in its differential distributions with respect to the Standard Model. These effects can in turn be traced back to the relevant diagrammatic and coupling structures and allow for a detailed analysis of the process. In order to do so, we perform systematic scans of the parameter spaces of such new physics scenarios and identify benchmark points which exhibit potentially observable features during the current and upcoming runs of the LHC. DOI: 10.1103/PhysRevD.107.115010 I. INTRODUCTION The past few years have seen full data taking at Run 1 & 2 of the Large Hadron Collider (LHC), with Run 3 ongoing, and this has been heralding a new era of precision Higgs physics. The importance of its detailed understanding cannot be overstated since the Higgs properties, originating from the mass generation mechanism, play a key role in searches for new physics (NP) which interacts with the electroweak (EW) sector. The measured Higgs couplings so far still allow for a minimal version of the Higgs mechanism, but one should bear in mind that several of the most important properties directly related to the Higgs potential (i.e., the Higgs self-couplings and Higgs-top couplings) are still very weakly constrained. For this reason, Higgs * s.moretti@soton.ac.uk stefano.moretti@physics.uu.se † luca.panizzi@physics.uu.se ‡ sjolin@fysik.su.se § harri.waltari@physics.uu.se Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. Funded by SCOAP3. 2470-0010=2023=107(11)=115010(17) physics remains one of the highest priorities in the global high energy physics program. Of all Higgs channels where beyond the Standard Model (BSM) effects can be searched for, one which carries particular relevance is di-Higgs production, as it can give direct access to the structure of the scalar potential triggering EW symmetry breaking (EWSB). At the LHC, this process primarily takes place via gg-fusion [1,2], i.e., via the loop subprocess gg → hh involving triangle and box graphs. BSM physics can therefore enter here via loops of new particles at the same perturbative order as those of SM ones, chiefly, the top-quark. In fact, another circumstance which renders these virtual effects from BSM physics potentially accessible specifically in di-Higgs production is that there exists a strong cancellation between the aforementioned triangle and box diagrams involving the top quark [3] at (or near) the SM limit. This is the context of our present study, which therefore dispenses of the case involving “resonant” production of heavy Higgs states decaying into (pairs of) the SM one, as we are concerned here with “nonresonant” di-Higgs production. This has been studied extensively at the LHC to date [4–12] and the standard approach is to parametrize BSM effects via modifications of the Yukawa coupling of the top quark (entering both the triangle and box diagrams) and/or the Higgs self-coupling (entering solely the triangle 115010-1 Published by the American Physical Society MORETTI, PANIZZI, SJÖLIN, and WALTARI PHYS. REV. D 107, 115010 (2023) ones). In turn, these can be interpreted in terms of an effective field theory (EFT), wherein any new particle entering gg → hh production via loops is essentially integrated out. Our approach is different. We allow for a BSM spectrum in which the presence of new states is accounted for exactly, at one-loop level, so that they can have any masses, including those comparable to the dynamical scale of the LHC (of order TeV), for which an EFT approach cannot be adopted. We do so by exploiting a simplified model approach that can easily be translated into any fundamental theory responsible for the EWSB dynamics chosen by Nature. This comes in the form of a numerical toolbox enabling Monte Carlo (MC) studies at a level of sophistication comparable to actual experimental analyses (albeit the version used in the present analysis is one-loop only). Borrowing as reference BSM framework the one of supersymmetry (SUSY), we will prove that the very same particles (i.e., the top quark companions in SUSY, so-called squarks) responsible for enabling extensions to the SM without hierarchy problems can give sizeable effects in diHiggs production, when their typical masses are indeed of order TeV. The plan of the paper is as follows. In the next section we will introduce the realizations of SUSY that we will be adopting for exemplifying our approach, including both a minimal and nonminimal version [13]. We will then describe our toolbox. Numerical results for gg → hh will follow, for both Run 3 and the High-Luminosity LHC (HL-LHC) [14], in turn preceding our conclusions. II. STOPS AND THE HIGGS MASS We shall first consider the minimal supersymmetric standard model (MSSM), which is based on the superpotential W MSSM ¼ yu QHu U c þ yd QH d Dc þ yl LH d Ec þ μHu Hd : ð1Þ The Higgs couples strongly to the top-stop sector, so the (s)tops will have a large impact on both the Higgs mass and its production cross sections. The stop mass matrix at treelevel is 
1 2 2 2 2 2 2 Mt̃L t̃L ¼ mQ̃ þ mt þ mZ cos 2β − sin θW ; ð2Þ 33 2 3 M2t̃L t̃R ¼ mt ðμ cot β − At Þ; M 2t̃R t̃R ¼ m2Ũ 33 2 þ m2t þ m2Z cos 2β sin2 θW : 3 SUSY breaking coupling H0u t̃L t̃R . There are loop corrections to this mass matrix [15], which are potentially large. In the MSSM there is a tree-level bound on the (lightest) SM-like Higgs mass, m2h ≤ m2Z cos2 2β; so large loop corrections are needed to produce the observed Higgs mass of 125 GeV. Since the top Yukawa coupling is the largest coupling in the MSSM, the most economical way to achieve this is to take tan β to be large (so that the tree-level mass is maximal and the SM-like Higgs boson couples maximally to the top/stop), make the stops heavy (so that the top-stop Supersymmetric cancellation is incomplete) and to introduce large stop mixing (which maximizes their loop corrections). Large mixing in the stop sector creates a large mass splitting between the stops. If one of the soft masses m2Q̃ or 33 m2Ũ is somewhat smaller than the other, we might end up 33 with a relatively light stop together with a heavy one. Currently, stop masses down to 600 GeV are allowed if the mass splitting between the stop and the Lightest Supersymmetric Particle (LSP) is small [16,17], since searches based on missing transverse momentum lose their sensitivity in the compressed case. In the MSSM, a large stop mass splitting is a necessity to achieve a 125 GeV Higgs mass, so searches based on missing transverse momentum are sensitive to the heavier stop. Hence, it must be heavy, the lower bound being around 1250 GeV [17,18] Since the stop mixing parameter At needs to be large in the MSSM, the bubble, triangle and box diagrams with trilinear stop couplings (see following section) give a large contribution to Higgs pair production, as known from literature [19–21] and which we will elaborate upon. It is well known that in the MSSM the Higgs trilinear self-coupling is always close to its SM value [22,23]. Numerical scans show that deviations can be at most at the 3% level [24]. Hence, in the MSSM, the modifications of the Higgs self-coupling will not lead to observable effects in Higgs pair production at the LHC as the predicted precision of the di-Higgs cross section measurement is around 40% [25]. In the next-to-MSSM (NMSSM), the superpotential is ð3Þ κ W ¼ W MSSM ðμ ¼ 0Þ þ λSHu Hd þ S3 : 3 ð4Þ Here m2Q̃ and m2Ũ are the soft SUSY breaking squark masses, tan β ¼ hH0u i=hH 0d i and At is the trilinear soft ð5Þ ð6Þ In this case there are additional tree-level contributions to the lightest Higgs boson mass so that the bound reads as [26] 115010-2 DECONSTRUCTING SQUARK CONTRIBUTIONS TO m2h ≤ m2Z
cos2 2β þ  2λ2 2 sin 2β : g2 þ g02 … PHYS. REV. D 107, 115010 (2023) ð7Þ If 1 ≲ tan β ≲ 3 and λ is large, the Higgs mass can be close to 125 GeV without loop corrections. Hence in the NMSSM one can have two light stops [27] but, due to the experimental constraints [17], they need to be nearly degenerate with each other and the LSP. In such a case we need to require stop mixing to be minimal, μ cot β ≃ At [see (3)] and hence the contribution from the trilinear bubble and triangle diagrams will be small. Furthermore, due to the extended Higgs sector, it is possible to have a large deviation from the SM prediction to the trilinear Higgs self-coupling even if other Higgs couplings are SM-like [24]. An enhancement can occur when tan β is close to 1 and λ is large while a suppression requires a second, singlet-dominated scalar to be light and λ to be small. This second option is incompatible with light squarks. We give a qualitative argument for this behavior in Appendix B. In our analysis, we perform a comprehensive scan in the MSSM and then select some benchmark points from the NMSSM to represent cases not present in the MSSM, but relevant for the di-Higgs process. The parameter space of the MSSM is chosen as follows. We choose the mostly right-handed stop to be the light one and to have a small mass splitting, mt̃1 − mχ̃ 01 < 10 GeV; DI-HIGGS ð8Þ with the lightest neutralino, which we take to be Higgsino-like. This mass splitting can always be arranged by choosing the value of the μ-parameter. The mostly left-handed squark doublet will then be heavy (mt̃2 > 1250 GeV). We scan over the values of m2Q̃ , m2Ũ , 33 33 tan β and At , which are the ones that determine the squark contribution to Higgs pair production. The other soft masses are fixed such that the rest of the sparticle spectrum is heavier than the stops and the Higgsinos. We also show a few NMSSM benchmarks representing cases that cannot be realized in the MSSM. One is when we make both stops as light as possible and adjust the Higgs mass to 125 GeV by tuning tan β and λ. For this benchmark the Higgs trilinear self-coupling is about 50% larger than in the SM, but the trilinear Higgs-stop couplings are much smaller than in the MSSM case. A second benchmark point represents a case where the Higgs trilinear self-coupling is about 60% larger than in the SM and the stop masses and mixing are similar to the MSSM points. III. NUMERICAL ANALYSIS The contributions to the di-Higgs final state from the SM are represented by the well-known, destructivelyinterfering topologies shown in Fig. 1, where the only FIG. 1. SM topologies for di-Higgs production at the LHC. coupling parameters entering the amplitudes are proportional to the Higgs trilinear self-coupling λv and the Yukawas yt;b . The SUSY-inspired parameters that are relevant for the di-Higgs process (squark masses and couplings) will be included in a simplified Lagrangian which assumes that every other SUSY particle does not participate significantly in the process: this means all other scalars are too heavy or their couplings are too small and all other squarks are also decoupled. First of all we notice, trivially, that the electric charge of the squarks propagating in the loops does not play any role in the process. What actually matters for a modelindependent treatment of the process is that, besides introducing NP modifications of the SM couplings, there are new colored particles propagating in the loops. Then, the key properties of such particles are their representation under SUð3Þ of QCD, their spin, how many they are and the size of their couplings with the Higgs boson. Only if the new particles are fermions, their electric charge plays a role, due to the fact that heavy quarks with charge 2=3 or −1=3 can mix with the SM quarks. Limiting our treatment to only two scalars in the fundamental representation of SUð3Þ, the simplified squark Lagrangian we consider in this analysis only contains interactions between those two squarks q̃i (with i ¼ 1, 2 labeling the mass eigenstates), h, t, and NP contributions to the couplings h3 and htt̄ (we will neglect modifications to the bottom Yukawa coupling in the following.). The model contains two colored scalars in the loop so it can be adapted to the SUSY scenarios described in the previous section, and also considering that it is realistically very hard (if not plainly inconsistent) to build scenarios in which one of the two scalar top partners is light enough to produce visible effects at the LHC and the other is too heavy and decoupled from low-energy physics. The simplified interaction Lagrangian therefore generically reads as TABLE I. Parameter ranges for the scan in the MSSM. All other soft scalar masses are larger than those mentioned. The μ-parameter is adjusted so that the Higgsino-like LSP is slightly lighter than the lightest stop. Parameter Minimum Maximum tan β At (GeV) m2Ũ (GeV2 ) 7 1500 1.35 × 106 50 3500 2 × 106 2.2 × 106 3.5 × 106 33 m2Q̃ (GeV2 ) 115010-3 33 MORETTI, PANIZZI, SJÖLIN, and WALTARI PHYS. REV. D 107, 115010 (2023) FIG. 2. Representative examples of LO di-Higgs cross sections (shown as a color gradient) as function of pairs of MSSM input parameters (left), stop masses (center), or the parameters of the simplified Lagrangian equation (9) (right), highlighting their correlations. 1 Lq̃NP ¼ −ðλSM þ κ hhh Þvh3 − pffiffiffi ðySM t þ κ htt Þht̄t 2 !
 κ11 κ 12 q̃1 h q̃ q̃ h q̃ q̃ þ vhðq̃1 q̃2 Þ q̃2 · κ 22 hq̃q̃ !
 11 12 κ hhq̃q̃ κ hhq̃q̃ q̃1 þ hhðq̃1 q̃2 Þ ; 22 q̃2 · κ hhq̃q̃ ð9Þ where the trilinear and quartic couplings are kept independent to account in a model-independent way for further NP effects which may alter the relation between the two. In the following, we identify q̃1;2 with t̃1;2 , and all the couplings are assumed to be real. This Lagrangian has been implemented in FeynRules [28] to obtain a UFO [29] output suitable for simulations at nextto-leading order (NLO) in QCD with the MG5_aMC [30] Monte Carlo (MC) generator.1 For di-Higgs production this is obviously necessary as the process is at one-loop at leading order (LO). In all our results we used the NNPDF3.0 LO PDF set [32]. The following analysis is aimed at finding which values of the parameters of NP can alter significantly the signal cross section and at the same time exhibit peculiar kinematic features which could lead to its observation during Run 3 or the HL-LHC. Crucially, our implementation of the gg → hh process starting from the aforementioned simplified Lagrangian is such that we are able to identify each component entering the total cross section, i.e., the contributions of the square of the relevant diagrams as well as the interferences between all of these, thereby 1 The model is publicly available on HEPMDB [31] at this link: https://hepmdb.soton.ac.uk/hepmdb:0223.0337 and it actually contains four new colored scalars in the fundamental of SUð3Þ (recall that we only consider two of these for this analysis). The additional squarks have been included to account for possible extensions. affording us with significant diagnostic power of the emerging signal. While this approach, as emphasized, is model independent, we will be applying it to both the MSSM and NMSSM described in the previous section, in order to show its effectiveness. Studies of squark effects of the kind looked for here have been performed in literature before (e.g., see [21]), but what this approach adds is the ability to directly reverse engineer the dynamics involved and interpret it already at the experimental analysis stage. A. Signal cross section and parametric dependence We first have performed a parametric scan within the MSSM: the scan parameters and their ranges are given in Table I. The spectra were calculated with SPheno v4.0.4 [33,34]. We required the lighter stop to be heavier than 600 GeV and the heavier stop to be heavier than 1250 GeV with the Higgs mass being in the interval 125.2  0.3 GeV (∼2σ around the central value). We set the μ parameter so that the Higgsino-like neutralino LSP is slightly lighter than the lighter stop, hence, we may use 600 GeV as the lower bound for the lighter stop mass. Other parameters are kept fixed such that the rest of the BSM spectrum is decoupled, as previously mentioned. The resulting MSSM couplings and masses have been translated into parameters of the simplified Lagrangian equation (9). With the obtained set of benchmark points we have performed MC simulations and determined correlations between parameters which maximize the cross section, as shown in Fig. 2. We found preferred directions in the parameter space for which the cross section increases, around specific combinations of the stop masses and for specific values of the trilinear parameters, especially the modifiers of SM couplings κ hhh and κ htt . In Fig. 2 we show some of the parametric dependencies. Obviously light squark masses lead to larger deviations from the SM, so the maximal cross sections are seen close 115010-4 DECONSTRUCTING SQUARK CONTRIBUTIONS TO DI-HIGGS to mðt̃1 Þ ¼ 600 GeV, mðt̃2 Þ ¼ 1250 GeV, the lighter stop mass being the more important one in increasing the cross section. Large tan β ≳ 20 is preferred as the top-stop correction to the Higgs mass is maximal and the 125 GeV Higgs mass can then be reached with lighter stops than is the case for low tan β. In the MSSM with fixed squark masses and tan β you either have an interval of possible At values leading to an acceptable Higgs mass (at low tan β) or two intervals of At , where between these intervals the Higgs is too heavy. In the lower of these intervals the squark bubbles and triangles give a smaller contribution and the cross section is SM-like. In the higher interval significant enhancements are possible, if the squarks are light. The region of allowed values for At depends on the average stop mass2: the heavier the stops, the larger the maximum value for At. Therefore, the maximal cross sections are obtained with values of At which are intermediate within the scan range, but which are maximal for the given stop masses and tan β. The point which produces the highest cross section, defined in Table II, will be considered in the following as benchmark point for the kinematic analysis. … PHYS. REV. D 107, 115010 (2023) TABLE II. Benchmark point for the MSSM. Left: tree-level input parameters for SPheno. The loop corrections to the stop mass matrix are large, so the soft masses plus the loop corrections are approximately m2Ũ ¼ 5.3 × 105 GeV2 and m2Q̃ ¼ 1.43 × 33 33 106 GeV2 and the effective value for At ¼ 2750 GeV after loop corrections. Right: mass spectrum and couplings, as defined in the simplified model Lagrangian of Eq. (9). For di-Higgs production the κ12 hht̃ t̃ coupling is not relevant. MSSM benchmark point Input parameter Value tan β At (GeV) m2Ũ (GeV2 ) 45.4 2595 1.547 × 106 33 m2Q̃ (GeV2 ) 2.447 × 106 33 MSSM benchmark point Masses and couplings mt̃1 (GeV) mt̃2 (GeV) κ hhh κ htt  11 12  B. Kinematic distributions  1. Deconstruction of the signal To perform a differential analysis of the signal associated with the propagation of stops in the loops, we deconstruct the signal into basic independent components. The total signal is then obtained as a weighted sum of such components. This procedure allows us to analyze separately their peculiar kinematic features, assess their relevance for the final result and understand semianalytically which range of parameters maximizes the potential observability of the signal. Limiting our treatment to only two stops we can build a limited number of elements. Labeling with a black dot (•) the NP contributions to SM couplings, with a red dot ( ) the purely NP couplings and with red Feynman propagators the new particles (t̃1;2 ), the signal amplitudes can be deconstructed in a small number of elements, listed in Table III. κ ht̃ t̃ κ ht̃ t̃ · κ 22 ht̃ t̃ κ 12 κ 11 hht̃ t̃ hht̃ t̃ · κ 22 hht̃ t̃ Value 600.6 1301.0 3.34 × 10−3 −1.68 × 10−3   
−6.690 7.228 · 8.519  −0.6702 −0.0174 · −0.6374 The signal contributions and their interferences can be parametrized in terms of a sum of different terms, proportional to unique functions of the couplings and to reduced cross sections σ̂ depending exclusively on the stop masses. Labeling as “B” the SM irreducible background, as “M” the contribution of the topologies containing only modified SM couplings (1 to 4 in Table III) and as “S” the contribution of topologies associated with the propagation of squarks (5 to 8) the complete set of contributions is: σ M ¼ κ 2hhh σ̂ 1 þ ðκ hhh κ htt Þ2 σ̂ 3 þ κ 4htt σ̂ 4 ; ð10aÞ X  X jj X jj ii 2 ii 2 2 κ iiht̃ t̃ κht̃ t̃ σ̂ int κ ht̃ t̃ σ̂ int σS ¼ 6i ðmt̃i;j Þ 5i ðmt̃i;j Þ þ κ hhh ðκ ht̃ t̃ Þ σ̂ 6d ðmt̃i Þ þ κ hhh κ ht̃ t̃ i¼1;2 þ j>i ðκ iiht̃ t̃ Þ4 σ̂ 7d ðmt̃i Þ j>i X ij X X ij ii 2 2 int þ ðκht̃ t̃ Þ4 σ̂ 7o ðmt̃i;j Þ þ ðκ iiht̃ t̃ Þ2 ðκjj ðκ ht̃ t̃ Þ2 σ̂ int 7ido ðmt̃i;j Þ ht̃ t̃ Þ σ̂ 7idd ðmt̃i;j Þ þ ðκ ht̃ t̃ Þ j>i  X jj ii 2 ii int þ ðκ hht̃ t̃ Þ σ̂ 8d ðmt̃i Þ þ κ hht̃ t̃ κ hht̃ t̃ σ̂ 8i ðmt̃i;j Þ ; j>i j≠i ð10bÞ j>i This is essentially to avoid a color-breaking vacuum. In general a large ratio of X t =MS , where X t ¼ At − μ cot β and M S is the average tree-level stop mass, can lead to a color-breaking vacuum, though the relation is not so simple if loop corrections are taken into account [35,36]. Our scan ranges have X t =M s < 2.5, which is considered to lead to a stable enough color-conserving vacuum. 2 115010-5 MORETTI, PANIZZI, SJÖLIN, and WALTARI PHYS. REV. D 107, 115010 (2023) σ int MB ¼ κ hhh σ̂ 1B þ κ htt σ̂ 2B ; σ int SB ¼ X κiiht̃ t̃ σ̂ int 5B ðmt̃i Þ ð10cÞ  X ij ii 2 int int þ ðκht̃ t̃ Þ σ̂ 7oB ðmt̃i;j Þ þ κhht̃ t̃ σ̂ 8B ðmt̃i Þ ; 3 3 2 int 2 int int int σ int MM ¼ κ hhh κ htt σ̂ 1;3 þ κ hhh κ htt σ̂ 1;4−2;3 þ κ htt σ̂ 2;4 þ κ hhh κ htt σ̂ 3;4 ; σ int SS ð10dÞ j>i i¼1;2 ð10eÞ X X X jj X ij ii 3 int ii ii int κ hhh κiiht̃ t̃ κ jj ¼ ðκ ht̃ t̃ Þ2 σ̂ int ðκht̃ t̃ Þ2 σ̂ int 5;7i ðmt̃i;j Þ þ κ ht̃ t̃ 5;7o ðmt̃i;j Þ ht̃ t̃ σ̂ 5;6i ðmt̃i;j Þ þ ðκ ht̃ t̃ Þ σ̂ 5;7d ðmt̃i Þ þ κ ht̃ t̃ j>i i¼1;2 j≠i j≠i X jj X jj ii int ii 3 int ii þ κiiht̃ t̃ κ iihht̃ t̃ σ̂ int ðm Þ þ κ κ σ̂ ðm Þ þ κ ðκ Þ σ̂ ðm Þ þ κ κ ðκ ht̃ t̃ Þ2 σ̂ int hhh hhh t̃ t̃ t̃ 6;7d 6;7i ðmt̃i;j Þ 5;8d hht̃ t̃ 5;8i ht̃ t̃ ht̃ t̃ ht̃ t̃ i i;j i j≠i þ κhhh κ iiht̃ t̃ j≠i X ij X jj ii 2 int ii ðκht̃ t̃ Þ2 σ̂ int κ hht̃ t̃ σ̂ int 6;7o ðmt̃i;j Þ þ κ hhh ðκ ht̃ t̃ Þ σ̂ 6;8d ðmt̃i Þ þ κ hhh κ ht̃ t̃ 6;8i ðmt̃i;j Þ j≠i j≠i  X ij X jj ii 2 ii int ii 2 int 2 ii int κ hht̃ t̃ σ̂ 7i;8 ðmt̃i;j Þ þ ðκ ht̃ t̃ Þ κhht̃ t̃ σ̂ 7o;8 ðmt̃i;j Þ ; þ ðκht̃ t̃ Þ κ hht̃ t̃ σ̂ 7d;8 ðmt̃i Þ þ ðκ ht̃ t̃ Þ j≠i σ int MS ð10fÞ j≠i X X ij ii int ii int κ2hhh κ iiht̃ t̃ σ̂ int ¼ ðκht̃ t̃ Þ2 σ̂ int 1;7o ðmt̃i;j Þ þ κ hhh κ hht̃ t̃ σ̂ 1;8 ðmt̃i Þ þ κ htt κ ht̃ t̃ σ̂ 2;5 ðmt̃i Þ 1;6 ðmt̃i Þ þ κ hhh j>i i¼1;2 þ κ hhh κ htt κ iiht̃ t̃ σ̂ int 2;6−3;5 ðmt̃i Þ þ κ 2hhh κ htt κ iiht̃ t̃ σ̂ int 3;6 ðmt̃i Þ þ κ hhh κ htt κ iihht̃ t̃ σ̂ int 3;8 ðmt̃i Þ þ κhtt ðκ iiht̃ t̃ Þ2 σ̂ int 2;7d ðmt̃i Þ þ κ htt X ij ii int ðκ ht̃ t̃ Þ2 σ̂ int 2;7o ðmt̃i;j Þ þ κ htt κ hht̃ t̃ σ̂ 2;8 ðmt̃i Þ j>i þ κhhh κ htt ðκiiht̃ t̃ Þ2 σ̂ int 3;7d ðmt̃i Þ þ κ hhh κhtt X ij ðκht̃ t̃ Þ2 σ̂ int 3;7o ðmt̃i;j Þ j>i þ κ2htt κiiht̃ t̃ σ̂ int 4;5 ðmt̃i Þ þ κhhh κ 2htt κ iiht̃ t̃ σ̂ int 4;6 ðmt̃i Þ  X ij ii 2 2 int 2 int þ κ htt ðκ ht̃ t̃ Þ σ̂ 4;7o ðmt̃i;j Þ þ κ htt κ hht̃ t̃ σ̂ 4;8 ðmt̃i Þ ; þ κ 2htt ðκ iiht̃ t̃ Þ2 σ̂ int 4;7d ðmt̃i Þ ð10gÞ j>i int 2 int σ int MSB ¼ κ hhh κ htt σ̂ 1;2−3B þ κ htt σ̂ 2−4B þ X ii 2 int ii 2 int ½κhhh κ iiht̃ t̃ σ̂ int 1;5−6B ðmt̃i Þ þ κ hhh ðκ ht̃ t̃ Þ σ̂ 1;7d−5;6 ðmt̃i Þ þ ðκ ht̃ t̃ Þ σ̂ 5d−7dB ðmt̃i Þ: ð10hÞ i¼1;2 The reduced cross sections have been labeled according int to the numbering scheme of Table III: σ̂ int x;y and σ̂ xB represent, respectively, the interference terms between the x and y classes of topologies and between x and the int SM background, while σ̂ int x;y−j;k or σ̂ x;y−jB signal combinations proportional to the same function of couplings. Further, σ̂ xd means that the topology class x contains couplings between same quark flavor, σ̂ xo between different ones and finally, σ̂ xi or σ̂ int x;yi mean interference between topologies where two different squarks circulate in the loops. Factors of 2 from interference terms have been included into the σ̂’s. The total cross section induced by NP is given by the sum of all the terms of Eq. (10). This parametrization allows for a model-independent analysis of the process: the kinematics of each individual term of Eq. (10) is determined exclusively by the masses of the particles circulating in the loops, while the couplings only affect the relative proportion between the various terms. Total and differential quantities can be thus determined for multiple benchmarks characterized by the same stop masses and different couplings by performing a unique set of numerical simulations [one for each term of Eq. (10)] for those masses. The simulation syntax is explained in Appendix A.3 In the following sections, the analysis of a specific distribution—the invariant mass of the di-Higgs system—is 3 Notice that, if more colored scalars are present, other terms depending on more than two masses would have to be considP P 2 ered, such as i;k ðκiiht̃ t̃ Þ2 j;k≠i ðκkj ht̃ t̃ Þ σ̂ 7idi ojk ðmt̃i;j;k Þ, and further simulated samples would have to be included in the database. Considering that the topologies of Table III contain at most two different squark propagators, a complete set of σ̂ elements can be obtained considering four colored scalars. The particle content of our UFO model indeed allows one to explore such possibilities. 115010-6 DECONSTRUCTING SQUARK CONTRIBUTIONS TO DI-HIGGS … PHYS. REV. D 107, 115010 (2023) TABLE III. Complete list of topologies describing di-Higgs production with modified SM couplings hhh and htt, and loop propagation of any number of stop squarks t̃i . The topologies are classified according to the different products of new couplings [defined in Eq. (9)] to which the amplitudes are proportional. Topology type Feynman diagrams Amplitude 1 Modified Higgs trilinear coupling Ai ∝ κ hhh 2 One modified Yukawa coupling Ai ∝ κhtt 3 Modified Higgs trilinear coupling and modified Yukawa coupling Ai ∝ κhhh κhtt 4 Two modified Yukawa couplings Ai ∝ κ2htt 5 Bubble and triangle with ht̃t̃ couplings Ai ∝ κiiht̃ t̃ This class of topologies involves only diagonal couplings between the Higgs and the squarks, due to the absence of FCNCs in strong interactions and the presence of one ht̃t̃ coupling. 6 Modified Higgs trilinear coupling þ Ai ∝ κhhh κ iiht̃ t̃ Bubble and triangle with ht̃t̃ coupling 7 8 Only diagonal couplings between the Higgs and the squarks due to the strong interaction. Triangle and box with two ht̃t̃ couplings 2 Ai ∝ jκ ij ht̃ t̃ j Ai ∝ κ iihht̃ t̃ Bubble and triangle with hht̃t̃ coupling Only diagonal couplings between the Higgs and the squarks due to the strong interaction. discussed in detail following the aforementioned procedure, both before and after the decays of the Higgs bosons. 2. Invariant mass of the di-Higgs system before Higgs decay One of the key observables to explore the contributions of NP in di-Higgs production at the LHC is the invariant mass of the di-Higgs system. Its shape is affected by the modification of SM couplings and by the presence of new particles in the loop. In this section we will consider the MSSM benchmark point in Table II to describe the analysis strategy. The invariant mass distribution is built from the individual components of Eq. (10), evaluated for mðt̃1;2 Þ ¼ f600; 1300g GeV, where the masses are approximated to the values on the simulation grid. Considering Eq. (10b) as an example, one can evaluate each differential cross section corresponding to the various σ̂ terms using the corresponding MC samples, as shown in the left panel of Fig. 3. From this, the differential distribution of σ S can be evaluated by weighting each component with the numerical values of the coupling 115010-7 MORETTI, PANIZZI, SJÖLIN, and WALTARI PHYS. REV. D 107, 115010 (2023) FIG. 3. Left panel: dσ̂=dMhh distributions of all components of σ S . Right panel: each component of the left panel is weighted with the associated coupling products corresponding to the MSSM benchmark point of Table II and converted into the number of expected events with at the end of Run 3 with nominal luminosity of 300 fb−1 . The black curve corresponds to the sum of all components and represents the M hh distribution of Eq. (10b). product factor and sum all terms, as shown in the right panel of Fig. 3, where the distributions are also multiplied by the nominal luminosity at the end of Run 3 of the LHC to evaluate the number of expected physical events. From the distributions evaluated before their weighting, it is possible to notice the threshold effects around mhh ¼ 1200 GeV corresponding to the propagation of the 600 GeV stop, and the presence of regions with negative interference. The importance of each term is however reshuffled by the couplings: for example, the negative value of the σ̂ int 5i ðmt̃1;2 Þ contribution is entirely due to the 22 11 negative coupling product (κ 11 ht̃ t̃ κ ht̃ t̃ with κ ht̃ t̃ < 0) in front of it. However, when summed with the other contributions, the overall net effect is a positive cross section in the whole mhh range, with a relevant enhancement of the 1200 GeV threshold peak. The very same procedure is applied to each term in Eq. (10) and the final result for the MSSM benchmark point is shown in Fig. 4. In general there are three types of contributions. Those that involve only modifications to the SM couplings (σ M , σ MM and σ MB ) have a peak at low Mhh and then decay exponentially. Such contributions are small in the MSSM, but can be more pronounced in, e.g., NMSSM, where the trilinear Higgs coupling can deviate from its SM value. Second, we have squark contributions squared (σ S and σ SS ), which are relatively flat over a large range and peak at M hh ¼ 2mt̃ . Finally we have interference contributions between squarks and the SM-type contributions (σ SB , σ MS and σ MSB ), which are positive at low M hh, negative between mt̃ < Mhh < 2mt̃ and turn positive again at high Mhh . This leads to an almost complete cancellation of BSM effects in the range mt̃ < M hh < 2mt̃ . Looking at the MSSM benchmark of Fig. 4 multiple effects are in place: the threshold peak generated by the terms in Eq. (10b) is clearly standing out with respect to the SM irreducible background, even if negative interference contributions from Eq. (10f) slightly reduce its impact, but the increase in the total cross section (SM þ Signal≃ 24.5 fb) is mostly due to the excess of events on the global peak of the distribution, generated by the interference terms of Eq. (10h). Inspecting the elements of Eq. (10h), analogously to what was done in Fig. 3, the largely dominant contribution (in the whole Mhh range) is given by the term 2 int ðκ 11 ht̃ t̃ Þ σ̂ 5d−7dB ðmt̃1 Þ, which sums the pure signal contribution from topology 5 of Table III and the interference FIG. 4. Invariant mass distribution of the di-Higgs system at parton level and before Higgs decay for the MSSM benchmark point of Table II, displaying the number of expected events at the end of Run 3 with nominal luminosity of 300 fb−1 . Curves for the SM intrinsic background (black), pure signal (red) and their sum (blue) are shown as solid lines. For the signal, the individual contributions of its components are also shown as dashed lines, to assess their relative role in the construction of its shape. 115010-8 DECONSTRUCTING SQUARK CONTRIBUTIONS TO FIG. 5. Same as Fig. 4, but showing only the SM and Signal þ SM curves with 1σ uncertainty bands representing the combined scale and PDF systematics. In the bottom panel, the ratio between central values, ðSM þ SignalÞ=SM, is displayed. DI-HIGGS … PHYS. REV. D 107, 115010 (2023) uncertainties are obtained by considering the largest deviations from the central value, while PDF uncertainties are obtained using the standard deviation on the whole PDF set, as described in [38]. The scale and PDF systematics are then linearly summed. In Fig. 5 the distributions for the SM background and the sum of signal and background are shown: it is possible to see that while the excess on the global peak is completely hidden within the uncertainties, the threshold peak can indeed be potentially discriminated. So far, only distributions before Higgs decays have been considered. If no shape discrimination was possible already at this stage, any further investigation would have been pointless. Given the optimistic results, however, the next step of the analysis involves letting the Higgs decay and identify promising final states for experimental detection. 3. Invariant mass of the di-Higgs system after Higgs decay between topology 7 and the SM background, both proportional to the same product of new couplings. Given the small modifications to the SM couplings, the topologies proportional to κ hhh and κ htt have an almost negligible impact on the determination of the final shape of dσ̂ S =dMhh . Despite the excess in the global peak and the presence of a relatively sizeable threshold peak with respect to the background distribution, an estimation of the systematic uncertainties is in order to assess if the different shapes can be potentially discernible using real data. A dedicated MC simulation has been performed for this estimation, validating the results obtained with the deconstruction method as a by-product. The combination of scale and parton distribution function (PDF) systematics is done using the same method of [37]: bin-by-bin, the asymmetric scale FIG. 6. In this part of the analysis we consider three final states, motivated either by large branching ratios or by cleanliness of detection. In all cases we select final states where at least one of the Higgs boson decays to 2 bottom quarks, and the other to either 2 photons, 2τ leptons or other 2 bottom quarks. The reconstruction of final state objects is done using FastJet [39] through MadAnalysis 5 [40–42], using the anti-kT jet clustering algorithm [43] with jet radius parameter R ¼ 0.4. The corresponding distributions of physical events as function of the invariant mass of the di-Higgs decay products are shown in Figs. 6–8: in all figures, the left panels show results at reconstruction level without any cut, while the right panels correspond to the basic selection cuts reported in Table IV. The preselection cuts are inspired by current experimental prospects [44]. For the bbγγ final state Invariant mass distribution of the bbγγ system at reconstruction level (left) and after the cuts of Table IV (right). 115010-9 MORETTI, PANIZZI, SJÖLIN, and WALTARI PHYS. REV. D 107, 115010 (2023) FIG. 7. Same as Fig. 6 but for the bbττ final state. FIG. 8. Same as Fig. 6 but for the 4b final state. a diphoton invariant mass cut around the Higgs boson mass has been applied to reduce the contribution from radiated photons and enhance the contribution of photons from Higgs decay. The depletion in number of events in the bbγγ and bbττ case makes it impossible to discriminate any NP contribution induced by the scenarios we are considering during the Run 3 of the LHC. While the bbbb final state looks more promising, a careful evaluation of all sources of TABLE IV. backgrounds (especially QCD-induced ones) must be performed in order to establish potential discrimination possibilities. IV. DEVIATIONS BEYOND THE MSSM After the first two runs of the LHC, the parameter space of the MSSM has become constrained, especially when it comes to parameters related to Higgs pair production. We Selection and kinematic cuts for the three final states considered in the analysis. bbγγ NðbÞ > 1 NðγÞ > 1 pT ðbÞ > 45ð20Þ GeV jηðbÞj < 2.5 jηðγÞj < 2.5 120 GeV < MðγγÞ < 130 GeV bbττ bbbb NðbÞ > 1 NðτÞ > 1 pT ðbÞ > 45ð20Þ GeV jηðbÞj < 2.5 jηðτÞj < 2.5 NðbÞ > 3 115010-10 pT ðbÞ > 40 GeV jηðbÞj < 2.5 DECONSTRUCTING SQUARK CONTRIBUTIONS TO know that both the top Yukawa coupling and the Higgs trilinear coupling must be close to the SM values, so deviations can mainly arise from a light stop around the threshold of 2mt̃ and the interference effects between squark and SM diagrams. This is not the case in nonminimal Supersymmetric models, as an extended Higgs sector can lead to deviations from the SM in the Higgs trilinear coupling. The top Yukawa is more constrained experimentally as the tt̄h production rate depends on it [45]. The squark contribution arises mainly from the diagrams involving trilinear Higgs-stop-stop couplings. The trilinear coupling at tree-level is proportional to Xt ¼ At − μ cot β. Large stop mixing is needed to get a 125 GeV mass for the SM-like Higgs in the MSSM, so the stop contribution is always relatively large in the MSSM. Large stop mixing also enhances the mass splitting between the stops, so one of the stops needs to be heavy so it will not contribute to the threshold excess. In the NMSSM one can achieve the 125 GeV Higgs mass already at tree-level, so one may try a setup with Xt ≃ 0, which would allow for two light stops, both being insensitive to searches targeting missing transverse momentum. The requirement of mh ¼ 125 GeV requires low tan β and large λ so the term jλj2 jH u Hd j2 leads to an enhancement of the triple Higgs coupling. A larger Higgs trilinear coupling can lead to a first order EW phase transition. In the EFT limit, where all BSM particles are decoupled, a 50% enhancement is needed for a first order phase transition [46]. Additional light degrees of freedom, such as light neutralinos [47], could change the needed enhancement. Nevertheless, an enhancement up to 100% is possible in the NMSSM [24] so it is natural to ask how well could we distinguish such a case. In the left panels of Fig. 9 we show a case with two stops having masses of approximately 600 GeV, tan β ¼ 1.35, λ ¼ 0.64, the trilinear Higgs coupling being 50% larger than in the SM, trilinear Higgs-stop couplings being significantly smaller than those of the MSSM and the top Yukawa being close to the SM value, κ t ≃ 0.985. We see that there is a deficit of events at low M hh : this is due to the enhanced trilinear Higgs coupling. In addition, the excess at the squark threshold has become smaller than the uncertainties4 (while still being around twice the SM rate), which emphasizes the significance of the bubble and triangle diagrams. In the right panel of Fig. 9 we have a “MSSM-like” case in the NMSSM. The stop masses and mixings are similar to those of the MSSM, but we have tan β ¼ 1.38 and λ ¼ 0.69, which again lead to a significant deviation from the SM in the trilinear Higgs coupling, being about 1.6 times the SM value. This results in a deficit of events at low 4 We remark, however, that the uncertainties in the plots are at LO and therefore bound to significantly reduce once higherorders corrections are taken into account. DI-HIGGS … PHYS. REV. D 107, 115010 (2023) Mhh and an excess of events at high M hh , the intermediate range being SM-like due to the cancellation of the various BSM amplitudes. As the bbγγ channel is sensitive to low invariant masses and the 4b channel is sensitive to high invariant masses, we should see a deficit in the former and an excess in the latter. If such a case were interpreted just through the modification of SM couplings, κt and κλ , the results between different channels would show an inconsistency, which implies that the model is not sufficient to describe the physics. Such an effective description can be somewhat misleading, unless a complete EFT basis is used, even in the low Mhh region as the interference between the squark diagrams with the SM ones can lead to a 30% deviation in the event rate as our MSSM benchmark (with essentially κt ¼ κ λ ¼ 1) shows. In the case of a large coupling deviation the κ framework leads to a better estimate, though even there we would have some uncertainty from the nondecoupling squark effects (difference between green (MB) and red (signal) curves in the upper right panel of Fig. 9). In the lowest panels of Fig. 9 the ratios of the event rate compared to the SM are shown. The enhanced trilinear Higgs couplings lead to a deficit of about 60%–70% in the lowest mass bins. Such a precision would be needed to show that a first order EW phase transition is possible without additional light degrees of freedom. V. PROSPECTS AT THE HL-LHC To estimate the prospects for model exclusion/discovery around the 1 TeV region one can, e.g., use information from recent searches for resonant di-Higgs production at the LHC. One such example is an ATLAS search pffiffiin ffi the bb̄bb̄ final state using 139 fb−1 of data collected at s ¼ 13 TeV [48]. In the Mhh mass range 1–1.2 TeV ATLAS expects Oð10Þ signal events over a background of O(100) events resulting in an excluded cross section around 10 pb. The signal acceptance times efficiency is around 10% given a fiducial preselection. Comparing to the particle level preselected yields in Fig. 8 it seems not possible to make any exclusion of the benchmark point using only current LHC Run 2 data. However, with 20 times the data expected from HL-LHC as shown in Fig. 11, compared to the ATLAS search example, the situation for exclusion looks much more promising. This is of course true for the MSSM benchmark point of Table II, which has been selected due to its high cross section. It is important to stress that the cross section strongly depends on the stop masses, as can be seen in the central panel of Fig. 2, and that when the mass of the lightest stop approaches 1 TeV, the MSSM cross section collapses to values similar to the SM case. The threshold effects corresponding to 2mt̃1 in the di-Higgs invariant mass distribution, however, can be seen also when the stop mass approaches 1 TeV: the excess on the stop threshold is in fact be compensated by a smooth and continuous depletion at 115010-11 MORETTI, PANIZZI, SJÖLIN, and WALTARI PHYS. REV. D 107, 115010 (2023) FIG. 9. The invariant mass distributions for the two NMSSM benchmarks described in the text. The integrated luminosity is in this case 3000 fb−1 , corresponding to the nominal reach of the HL-LHC phase. The left panels correspond to a case with two light stops with minimal mixing, while the right panels have a light and a heavy stop. Both have a trilinear Higgs coupling that is larger than that of the SM. FIG. 10. Invariant mass distributions for 3000 fb−1 and for different MSSM benchmark points characterized by different t̃1 masses, from 600 GeV to 1 TeV, and mt̃2 ¼ 1600 GeV. smaller invariant mass values, with higher event counts than the peak, leading to a total cross section which is similar to the SM one. This can be seen in Fig. 10 for different representative MSSM benchmark points characterized by the same value of mt̃2 and different values of mt̃1 , from 600 GeV to 1000 TeV (couplings are not reported as not relevant in this context). For mt̃1 ≳ 800 GeV the number of expected events is however already small (some units) at parton level, and therefore the actual observability of higher masses at HL-LHC is rather challenging, if just not possible. The NMSSM benchmarks also introduce large effects at low invariant Mhh mass. If one focuses only on the MðhhÞ region below the stop mass where an EFT descriptions is valid, the NP contributions are dominated by the modified Higgs triple gauge coupling interfering with the rest of the SM background which is visible in Fig. 9 (labeled MB). This effect is measured in the experiments using coupling modifiers (κ-framework) or more consistently with an EFT, 115010-12 DECONSTRUCTING SQUARK CONTRIBUTIONS TO DI-HIGGS … PHYS. REV. D 107, 115010 (2023) FIG. 11. Invariant mass distributions for the MSSM benchmark point and for 3000 fb−1. The top left panel shows the distribution before Higgs decays, the other panels show the distributions for the 3 final states described in Sec. III B 3 and after application of the cuts of Table IV. and it can, as previously mentioned, have important cosmological implications. The current estimated HLLHC 95% exclusion limits for κ λ using e.g. the ATLAS experiment is [0, 2.5] [44], while the NMSSM benchmarks have κ λ ¼ 1.6. This indicates that the low mass effects might be detectable during HL-LHC with more accurate theory modeling such as an EFT or techniques outlined in this paper, reduced experimental systematic uncertainties and combinations across different experiments. Note that the sensitivity estimates made in this section do not include any di-Higgs signal corrections to account for missing higher order contributions (K-factors). For NP processes these are estimated to be in the range K ≃ 1.6–2.4 [49]. VI. CONCLUSIONS In summary, we have provided a proof-of-concept that nonresonant di-Higgs production at the LHC (involving the SM-like Higgs boson already discovered) can be used as a proxy to viable models of the EW scale, such as SUSY, via loop effects induced by low-mass (i.e., nondecoupled) stops that can appear through both an increase (or decrease) of the inclusive (integrated) rates and significant shape changes in exclusive (differential) distributions of the gg → hh process. Chiefly, among the latter, the invariant mass of the Higgs boson pair may display a large enhancement or depletion just above the 2mh threshold (where the production rates are maximal) and a local maximum when mhh ≈ 2mt̃1 (in correspondence to a loop threshold involving the lightest stop state), both of which can be used for diagnostic purposes of the underlying EWSB scenario, separately or simultaneously depending on the benchmark point being targeted. While near the 2mt threshold the effects are more spread out, the event rate for gg → hh can be enhanced by up to an order of magnitude compared to that of the SM at the 2mt̃ threshold. Quite remarkably, this can happen in the absence of stop cascade signals at the LHC, owing to compressed sparticle mass spectra which can easily be obtained, in compliance with current exclusion limits, in two popular SUSY 115010-13 MORETTI, PANIZZI, SJÖLIN, and WALTARI PHYS. REV. D 107, 115010 (2023) realizations, minimal and nonminimal, like the MSSM and NMSSM, respectively, which we have adopted here. So that, in the end, the effects investigated in this paper could well be the first evidence of SUSY (at least of the squark sector of it) at the LHC during the HL-LHC run. (Unfortunately, Runs 2 and 3 cannot afford one with significant sensitivity in this respect.) In order to prove this, we have resorted to an exact computational framework implementing a simplified model approach that, on the one hand, can easily be translated into any fundamental theory responsible for EWSB dynamics and, on the other hand, dispenses with the necessity of invoking any EFT limit of it, as it can compute (event level) integrated and differential rates for essentially any BSM spectrum of masses and couplings. In fact, the framework relies on a deconstruction method which makes it flexible to study a wide range of scenarios beyond those described here. In this paper, we have shown all this being true while maintaining the hh pair strictly on-shell. However, we have also argued that such stop loop effects might persist after backgrounds (both irreducible and reducible) are accounted for in the most popular channels leading to di-Higgs detection, following a reconstruction-level MC analysis that we have performed. This might be the case even considering both statistic and systematic uncertainties on the signal, which in our analysis are relatively large due to its LO nature. Among these, upon an initial assessment based on existing literature, the 4b channel is likely to be the one affording an actual experimental study with the best sensitivity (above and beyond bb̄γγ and bb̄τþ τ− ), so long that the QCD-induced background can be brought under control. We postpone to a future paper the detailed detector level MC analysis of these final states, including dedicated selections targeting the described kinematic features. ACKNOWLEDGMENTS This work would not have been possible without financing from the Knut and Alice Wallenberg Foundation under the Grant No. KAW 2017.0100, which supported L. P., J. S. and (partially) S. M. within the SHIFT project. S. M. is also supported in part through the NExT Institute and the STFC Consolidated Grant No. ST/ L000296/1. H. W. is supported by Carl Trygger Foundation under Grant No. CTS18:164. L. P. acknowledges the use of the Fysikum HPC Cluster at Stockholm University. All authors thank Arnaud Ferrari, Venugopal Ellajosyula, José Eliel Camargo-Molina and Rikard Enberg for discussions. APPENDIX A: SIMULATION SYNTAX IN MG5_AMC FOR DI-HIGGS WITH THE DECONSTRUCTION METHOD In this appendix we describe how to obtain the various terms of Table III using the UFO model in https://hepmdb .soton.ac.uk/hepmdb:0223.0337. All our simulations have been made using MG5_AMC version 3.4.1. The UFO model contains 4 colored scalars, named sq1, sq2, sq3 and sq4. Their trilinear couplings with the Higgs boson are proportional to the dimensionless parameters KHSQ1SQ1, KHSQ1SQ2..., which multiply a VEV factor, according to Eq. (9). Each coupling is associated to a specific coupling order HSQ1SQ1, HSQ1SQ2... which can be invoked when performing simulations in MG5_aMC to restrict the number of possible topologies. The quadrilinear couplings between the squarks and the Higgs boson are proportional to the dimensionless parameters KHHSQ1SQ1, KHHSQ1SQ2..., according to Eq. (9). Also for the quadrilinears, specific coupling orders have been defined, HHSQ1SQ1, HHSQ1SQ2..., which allow to select or remove these couplings. Finally, the modifications to the SM hhh, htt and hbb couplings are labeled as KHHH, KHTT and KHBB respectively, with coupling orders HHH, HTTMOD and HBBMOD respectively. The convention for these couplings is again the same of Eq. (9). In order to reproduce the results of this paper, the model has to be imported in MG5_AMC with a restriction which removes sq3, sq4 and their corresponding interactions. While not strictly necessary, the restriction greatly speeds up the calculations by ignoring irrelevant interactions and propagators. Alternatively it would be enough to set all coupling orders associated to sq3 and sq4 to zero in the topologygeneration phase, or set all the numerical values of their couplings to zero when generating events. The baseline syntax to generate di-Higgs is completely analogous to what one does in the SM: However, this syntax alone is not sufficient (and MG5_aMC would raise an error message), as the other couplings orders of the model have to be specified. In our analysis we want to isolate specific topologies to apply the deconstruction method: a careful tuning of the coupling orders allow us to do it in an efficient and unambiguous way. 115010-14 DECONSTRUCTING SQUARK CONTRIBUTIONS TO DI-HIGGS … PHYS. REV. D 107, 115010 (2023) The SM irreducible di-Higgs background can be obtained in different ways, either by adding to the baseline syntax all coupling orders associated to NP set to 0: or by removing all squarks from propagation and setting only the SM modified coupling orders to 0: The signal and interferences can then be simulated using different combinations of the coupling orders. One example for each component of Eq. (10) should be sufficient to describe the whole procedure. M: κ2hhh σ̂ 1 22 int S: κ11 ht̃ t̃ κ ht̃ t̃ σ̂ 5i ðmt̃1;2 Þ MB: κ htt σ̂ int 2B int SB: κ 11 ht̃ t̃ σ̂ 5B ðmt̃1 Þ MM: κ2hhh κ htt σ̂ int 1;3 12 2 int SS: κhhh κ 11 ht̃ t̃ ðκ ht̃ t̃ Þ σ̂ 6;7o ðmt̃1;2 Þ int MS: κhhh κ htt κ 11 hht̃ t̃ σ̂ 3;8 ðmt1 Þ 2 int MSB: κ hhh ðκ 11 ht̃ t̃ Þ σ̂ 1;7d−5;6 ðmt̃1 Þ APPENDIX B: TRILINEAR HIGGS COUPLING IN THE MSSM AND THE NMSSM In the SM the Higgs potential is particularly simple. The Higgs self-interactions arise solely from the term λjHj4 . Since λ also determines the Higgs mass, we get a unique prediction λSM ¼ g2 m2h : 8m2W ðB1Þ In the MSSM it is easier to achieve the 125 GeV mass in the large tan β regime. In such a case the SM-like Higgs in the alignment limit is practically the neutral component of the Hu doublet. Thus the only relevant term of the loopcorrected scalar potential that is relevant for the SM-like Higgs mass generation is of the form λeff jHu j4 and hence necessarily λeff ≃ λSM in the MSSM. This is why you never get large deviations in the quartic or trilinear Higgs coupling in the MSSM. 115010-15 MORETTI, PANIZZI, SJÖLIN, and WALTARI PHYS. REV. D 107, 115010 (2023) When the mass matrix is computed, the contribution of the bilinear term comes in a form that leaves no contribution to the SM-like Higgs mass, just like the soft SUSY breaking Bμ Hu Hd term does not contribute to the Higgs mass in the MSSM. Terms of the form of jH0u H0d j2 do not contribute to the diagonal elements of the mass matrix as they get eliminated by the tadpole equations. Hence we get two types of contributions to the mass matrix that affect the SM-like Higgs mass: diagonal elements from jH 0u;d j4 , which come with a factor (from differentiating the potential) of 4 × 3 and off-diagonal elements from jH 0u H 0d j2 , which come with a factor of 2 × 2, in addition there are factors depending on β. As we typically have tan β > 1, the most important contributions are those from jH 0u j4 and jH0u H0d j2 , which have relative weights of sin2 β and cos2 β, respectively. Hence any effect from the jλj2 term gets suppressed as tan β increases. In the trilinear Higgs coupling the relative factors are different, one gets a factor of 4 for the jH0u j4 term and two contributions with factors of 2 from jH 0u H 0d j2 , again the former having a factor of sin2 β and the latter having a factor cos2 β. Therefore the relative contribution of the jH0u H0d j2 term to the trilinear Higgs self interaction is larger than to its mass. Hence if λ is larger than the gauge couplings, it will enhance the Higgs trilinear from its SM value and if it is smaller, it will reduce it. However, this effect is suppressed by cos2 β, so you can only have a significant deviation in the region 1 ≲ tan β ≲ 3. Obviously, the precise computation of the trilinear Higgs coupling will need the loop corrections to the scalar potential (B2), but this tree-level discussion should allow us to understand the results. The enhancement is relatively easy to combine with any stop masses, as the tree-level contribution reduces the need of large loop corrections, so you can always find a suitable level of stop mixing. The parameter space, where the Higgs trilinear is smaller than in the SM is very narrow. The best chances are when the singlet-dominated state is lighter than the SM-like Higgs so that the mixing can push the SM-like Higgs mass up [50]. Even in that case large loop corrections are typically needed, so heavy stops and sbottoms are preferred. In addition, as the singlet is not decoupled, you cannot neglect the trilinear soft interaction term Aλ Hu Hd S, so that scenario is more complex than the case where the trilinear coupling is enhanced. [1] T. Plehn, M. Spira, and P. M. Zerwas, Nucl. Phys. B479, 46 (1996); B531, 655(E) (1998). [2] S. Dawson, S. Dittmaier, and M. Spira, Phys. Rev. D 58, 115012 (1998). [3] E. W. N. Glover and J. J. van der Bij, Nucl. Phys. B309, 282 (1988). [4] A. M. Sirunyan et al. (CMS Collaboration), J. High Energy Phys. 04 (2019) 112. [5] M. Aaboud et al. (ATLAS Collaboration), J. High Energy Phys. 05 (2019) 124. [6] A. M. Sirunyan et al. (CMS Collaboration), J. High Energy Phys. 10 (2019) 125. [7] G. Aad et al. (ATLAS Collaboration), Phys. Lett. B 801, 135145 (2020). [8] A. M. Sirunyan et al. (CMS Collaboration), J. High Energy Phys. 03 (2021) 257. [9] G. Aad et al. (ATLAS Collaboration), Phys. Rev. D 106, 052001 (2022). [10] A. M. Sirunyan et al. (CMS Collaboration), arXiv:2206. 09401. [11] G. Aad et al. (ATLAS Collaboration), arXiv:2209 .10910. [12] ATLAS Collaboration, arXiv:2301.03212. [13] S. Moretti and S. Khalil, Supersymmetry Beyond Minimality: From Theory to Experiment (CRC Press, Boca Raton, Florida, USA, 2019). [14] F. Gianotti et al., Eur. Phys. J. C 39, 293 (2005). [15] A. Donini, Nucl. Phys. B467, 3 (1996). [16] A. M. Sirunyan et al. (CMS Collaboration), Phys. Rev. D 104, 052001 (2021). [17] A. Tumasyan et al. (CMS Collaboration), Eur. Phys. J. C 81, 970 (2021). [18] G. Aad et al. (ATLAS Collaboration), J. High Energy Phys. 04 (2021) 174. [19] J. Cao, Z. Heng, L. Shang, P. Wan, and J. M. Yang, J. High Energy Phys. 04 (2013) 134. In the NMSSM the situation is different. In addition to the standard MSSM D-terms, you get an extra quartic term in the Higgs potential, jλj2 jHu Hd j2 , which is relevant to the Higgs mass generation, if tan β is low or moderate. This leads to the well-known mass bound of Eq. (7). From the previous argument it is obvious that in the large tan β region it is possible to get the correct Higgs mass, but no deviations from the SM value of the Higgs self-coupling. With tan β close to one the situation is different. We now assume that the singlet state is decoupled, so that the soft trilinear interaction Aλ Hu Hd S is irrelevant for the Higgs self-coupling. At tree-level the quartic selfcoupling and the Higgs masses arise from the following terms of the scalar potential VðH0u ; H0d Þ ¼ g2 þ g02 1 jjH0u j2 − jH0d j2 j2 þ jλj2 jH0u H 0d j2 4 8 
2 v v þ Aλ psffiffiffi þ κλ s H0u H0d … ðB2Þ 2 2 115010-16 DECONSTRUCTING SQUARK CONTRIBUTIONS TO [20] B. Batell, M. McCullough, D. Stolarski, and C. B. Verhaaren, J. High Energy Phys. 09 (2015) 216. [21] P. Huang, A. Joglekar, M. Li, and C. E. M. Wagner, Phys. Rev. D 97, 075001 (2018). [22] W. Hollik and S. Penaranda, Eur. Phys. J. C 23, 163 (2002). [23] A. Dobado, M. J. Herrero, W. Hollik, and S. Penaranda, Phys. Rev. D 66, 095016 (2002). [24] L. Wu, J. M. Yang, C.-P. Yuan, and M. Zhang, Phys. Lett. B 747, 378 (2015). [25] M. Cepeda et al., CERN Yellow Rep. Monogr. 7, 221 (2019). [26] M. Drees, Int. J. Mod. Phys. A 04, 3635 (1989). [27] J. Beuria, A. Chatterjee, A. Datta, and S. K. Rai, J. High Energy Phys. 09 (2015) 073. [28] A. Alloul, N. D. Christensen, C. Degrande, C. Duhr, and B. Fuks, Comput. Phys. Commun. 185, 2250 (2014). [29] C. Degrande, C. Duhr, B. Fuks, D. Grellscheid, O. Mattelaer, and T. Reiter, Comput. Phys. Commun. 183, 1201 (2012). [30] J. Alwall, R. Frederix, S. Frixione, V. Hirschi, F. Maltoni, O. Mattelaer, H. S. Shao, T. Stelzer, P. Torrielli, and M. Zaro, J. High Energy Phys. 07 (2014) 079. [31] M. Bondarenko, A. Belyaev, J. Blandford, L. Basso, E. Boos, V. Bunichev et al., arXiv:1203.1488. [32] R. D. Ball et al. (NNPDF Collaboration), J. High Energy Phys. 04 (2015) 040. [33] W. Porod, Comput. Phys. Commun. 153, 275 (2003). [34] W. Porod and F. Staub, Comput. Phys. Commun. 183, 2458 (2012). [35] J. E. Camargo-Molina, B. O’Leary, W. Porod, and F. Staub, J. High Energy Phys. 12 (2013) 103. DI-HIGGS … PHYS. REV. D 107, 115010 (2023) [36] J. E. Camargo-Molina, B. Garbrecht, B. O’Leary, W. Porod, and F. Staub, Phys. Lett. B 737, 156 (2014). [37] A. Deandrea, T. Flacke, B. Fuks, L. Panizzi, and H.-S. Shao, J. High Energy Phys. 08 (2021) 107; 11 (2022) 28. [38] J. Butterworth et al., J. Phys. G 43, 023001 (2016). [39] M. Cacciari, G. P. Salam, and G. Soyez, Eur. Phys. J. C 72, 1896 (2012). [40] E. Conte, B. Fuks, and G. Serret, Comput. Phys. Commun. 184, 222 (2013). [41] E. Conte, B. Dumont, B. Fuks, and C. Wymant, Eur. Phys. J. C 74, 3103 (2014). [42] J. Y. Araz, B. Fuks, and G. Polykratis, Eur. Phys. J. C 81, 329 (2021). [43] M. Cacciari, G. P. Salam, and G. Soyez, J. High Energy Phys. 04 (2008) 063. [44] ATLAS Collaboration, Technical Report No. ATL-PHYSPUB-2022-053, CERN, Geneva, 2022. [45] A. M. Sirunyan et al. (CMS Collaboration), Eur. Phys. J. C 81, 378 (2021). [46] M. Reichert, A. Eichhorn, H. Gies, J. M. Pawlowski, T. Plehn, and M. M. Scherer, Phys. Rev. D 97, 075008 (2018). [47] S. Akula, C. Balázs, L. Dunn, and G. White, J. High Energy Phys. 11 (2017) 051. [48] G. Aad et al. (ATLAS Collaboration), Phys. Rev. D 105, 092002 (2022). [49] G. Buchalla, M. Capozi, A. Celis, G. Heinrich, and L. Scyboz, J. High Energy Phys. 09 (2018) 057. [50] M. Badziak, M. Olechowski, and S. Pokorski, J. High Energy Phys. 06 (2013) 043. 115010-17 

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Lingu@ Nostr@ - Revista Virtual de Estudos de Gramática e Linguística ISSN 2317-2320 PROCESSO DE ALFABETIZAÇÃO: AS RELAÇÕES ENTRE POLÍTICAS E PRÁTICAS DO/NO MUNICÍPIO DE VITÓRIA DA CONQUISTA/BA (2002-2012) LITERACY PROCESS: THE RELATIONSHIP BETWEEN POLICIES AND PRACTICES IN / IN THE MUNICIPALITY OF VITÓRIA DA CONQUISTA / BA (2002-2012) Claudionor Alves da Silva1 Resumo: O início das reflexões sobre o processo de alfabetização se dá a partir das inquietações que surgiram com o constante contato com as professoras alfabetizadoras da rede municipal de ensino de Vitória da Conquista, desde o ano de 2004. Esse contato se deu por meio do desenvolvimento de oficinas de formação continuada e das atividades curriculares desenvolvidas nas disciplinas de Metodologia da Alfabetização e de Prática de ensino do curso de Pedagogia da Universidade Estadual do Sudoeste da Bahia. O constante contato com essas professoras, a escuta atenta de seus relatos sobre as práticas pedagógicas foi motivador para o desenvolvimento de uma investigação intitulada “Processo de alfabetização escolar: concepções de alfabetização e práticas alfabetizadoras de professoras da rede municipal de Vitória da Conquista, Bahia, Brasil”, na qual se insere este trabalho. Entre as perguntas que deram origem a esta investigação, destaca-se: Quais são as concepções de alfabetização das professoras alfabetizadoras e sua relação com os referenciais teóricos-metodológicos que fundamentam suas práticas pedagógicas? Daí, o objetivo deste trabalho é apresentar uma análise das relações entre as políticas e as práticas de alfabetização desenvolvidas na rede municipal de ensino de Vitória da Conquista, Bahia, entre os anos de 2002 a 2012. Para a construção dos dados, realizou-se entrevistas com vinte professoras do Ciclo de alfabetização e análise de documentos. Segundo o que se constatou, as políticas e ou programas implementados entre os anos de 2002 a 2012 pelo sistema municipal de educação de Vitória da Conquista, embora o tenha feito com a justificativa de melhorar os índices educacionais, em especial na alfabetização, as preocupações estavam muito mais voltadas para as questões político-burocráticas do que político-pedagógicas. Como consequência, não houve avanço nas práticas pedagógicas, que permaneceram, até esse período, mecânicas, cujas atividades de leitura e escrita eram destituídas de sentido. Palavras-chave: Alfabetização; Políticas de alfabetização; Práticas de alfabetização. Resumen: El inicio de las reflexiones sobre el proceso de alfabetización se basa en las preocupaciones que surgieron con el contacto constante con los profesores de alfabetización de la red de educación municipal de Vitória da Conquista, desde 2004. Este contacto se produjo a través del desarrollo de talleres de educación continua y las actividades curriculares desarrolladas en las disciplinas de Metodología de Alfabetización y Práctica Docente del curso de Pedagogía de la Universidad Estatal de Sudoeste de la Bahía. El contacto constante con estos profesores, la escucha atenta de sus informes sobre prácticas pedagógicas fue motivador para el desarrollo de una investigación titulada "Proceso de alfabetización escolar: concepciones de alfabetización y prácticas de alfabetización de profesores de la red municipal de Vitória da Conquista, Bahía, Brasil", en la que se inserta este trabajo. Entre las preguntas que dieron origen a esta investigación, destacan las siguientes: ¿Cuáles son las concepciones de alfabetización de los profesores de alfabetización y su relación con las referencias teórico-metodológicas que apoyan sus prácticas pedagógicas? El objetivo de este trabajo es presentar un análisis de las relaciones entre las políticas y prácticas de alfabetización desarrolladas en la red de educación municipal de Vitória da Conquista, Bahía, entre 2002 y 2012. Para la construcción de los datos, se realizaron entrevistas con veinte profesores del Ciclo de alfabetización y análisis de documentos. De acuerdo con lo encontrado, las 1 Mestre em Ciências da Linguagem; Especialista em Psicopedagogia e professor de Alfabetização da Universidade Estadual do Sudoeste da Bahia. Endereço eletrônico: profclaudionor@yahoo.com.br. Língu@ Nostr@, Vitória da Conquista, v. 8, n. 1, p. 405-419, jan-julho. 2020. 405 Lingu@ Nostr@ - Revista Virtual de Estudos de Gramática e Linguística ISSN 2317-2320 políticas y/o programas implementados entre 2002 y 2012 por el sistema educativo municipal de Vitória da Conquista, aunque lo hizo con la justificación de mejorar los índices educativos, especialmente en la alfabetización, las preocupaciones se centraron mucho más en cuestiones político-burocráticas que políticopedagógicas. Como consecuencia, no hubo ningún avance en las prácticas pedagógicas, que permanecieron, hasta este período, mecánicas, cuyas actividades de lectura y escritura no tenían sentido. Palabras-claves: Alfabetización; Políticas de alfabetización; Prácticas de alfabetización. 1. Introdução Presencia-se, no Brasil, desde os anos de 1990, uma expansão quantitativa no sistema educacional brasileiro. Em nome da redemocratização da sociedade, as políticas públicas, entre elas, as educacionais, têm reconhecido a dívida histórica, principalmente com os segmentos sociais menos favorecidos. Em nome do pagamento dessa dívida ou parte dela, ampliou-se o número de vagas e de escolas para o atendimento à população que estava excluída desse cenário. Nesse movimento, conforme percebe Gatti (2008), houve mudanças nos segmentos sociais, que passaram a exigir da formação de professores mudanças radicais. A formação de professores, nessa perspectiva, é vista como uma das variáveis capazes de incidir na melhoria da qualidade do ensino brasileiro. Tal cenário foi propício para a implementação de políticas educacionais, motivadas pela Lei de Diretrizes e Bases da Educação Nacional, no ano de 1996. Daí, surgem novas e ou se ampliam abordagens teórico-metodológicas, sobretudo para o processo de alfabetização. Ampliam-se também, a partir desse momento, os estudos e pesquisas voltados para a alfabetização em função de dois aspectos: primeiro, a ampla divulgação dos estudos acerca da psicogênese da língua escrita de Ferreiro e Tebersoky, a partir da segunda metade dos anos de 1980, que exerceram influências para a implementação dos Parâmetros Curriculares Nacionais de Língua Portuguesa e o segundo refere-se aos sucessivos resultados de exames nacionais e internacionais sobre o desempenho dos estudantes, em leitura e escrita. Esses aspectos são aqui vistos como contraditórios uma vez que não se verifica que os avanços dos estudos, pesquisas e implementação de políticas educacionais específicas contribuíram para a melhoria do desempenho dos estudantes, pois os índices referentes à leitura e escrita permanecem abaixo do desejado. Conforme os resultados das avaliações, a média brasileira ainda está abaixo do esperado; a média do Estado da Bahia está abaixo da média nacional e alguns municípios baianos têm sua média abaixo da média do Estado, inclusive o de Vitória da Conquista, o terceiro maior do Estado. Língu@ Nostr@, Vitória da Conquista, v. 8, n. 1, p. 405-419, jan-julho. 2020. 406 Lingu@ Nostr@ - Revista Virtual de Estudos de Gramática e Linguística ISSN 2317-2320 O início dessas reflexões sobre o processo de alfabetização se dá a partir das inquietações que surgiram com o constante contato com as professoras alfabetizadoras da rede municipal de ensino de Vitória da Conquista, desde o ano de 2004. Esse contato começou com o desenvolvimento de oficinas de formação continuada e das atividades curriculares desenvolvidas nas disciplinas de Metodologia da Alfabetização e de Prática de ensino do curso de Pedagogia da Universidade Estadual do Sudoeste da Bahia. Como exigência da disciplina de Prática de ensino, o professor deveria supervisionar a atividades de regência desenvolvidas pelos discentes do curso de Pedagogia. Esse contato possibilitou, sobretudo, o diálogo com as professoras alfabetizadoras, momento no qual elas tinham a oportunidade de desabafar sobre suas angústias e frustrações em relação ao trabalho pedagógico que desenvolvem. Nessas oportunidades, elas reclamavam de um problema crescente nos últimos anos, relacionado às dificuldades enfrentadas no processo de ensino e aprendizagem da leitura e da escrita. Segundo as professoras, está cada vez mais difícil alfabetizar seus alunos que, segundo elas, enfrentam muitas dificuldades para se alfabetizarem. Isso foi motivador para o desenvolvimento de uma investigação intitulada “Processo de alfabetização escolar: concepções de alfabetização e práticas alfabetizadoras de professoras da rede municipal de Vitória da Conquista, Bahia, Brasil”, na qual se insere este trabalho. Entre as perguntas que deram origem a esta investigação, destaca-se: Quais são as concepções de alfabetização das professoras alfabetizadoras e sua relação com os referenciais teóricosmetodológicos que fundamentam suas práticas pedagógicas? O objetivo deste trabalho é apresentar uma análise das relações entre as políticas e as práticas de alfabetização desenvolvidas na rede municipal de ensino de Vitória da Conquista, Bahia, entre os anos de 2002 a 2012. A construção dos dados aqui analisados se constituiu pela aplicação de entrevistas sobre o processo de alfabetização com professoras do Ciclo de alfabetização e análise de documentos relacionados com a alfabetização proposta pelo sistema municipal de educação. Foi realizada uma reunião em duas escolas, com todas as professoras das classes de alfabetização, com o intuito de apresentar a proposta de investigação para que, por adesão, as professoras participassem, como sujeitos desta investigação. Assim, participaram 20 professoras, sendo 09 de uma escola e 11 de outra. As entrevistas com as professoras foram realizadas em horários e datas previamente agendados, conforme a disponibilidade de cada uma delas. As entrevistas foram gravadas em Língu@ Nostr@, Vitória da Conquista, v. 8, n. 1, p. 405-419, jan-julho. 2020. 407 Lingu@ Nostr@ - Revista Virtual de Estudos de Gramática e Linguística ISSN 2317-2320 áudio, com a permissão de cada uma delas e, em seguida foram transcritas. Para qualificar ainda mais as informações, elaborou-se um roteiro com perguntas sobre a concepção de alfabetização, o método que se utiliza para alfabetizar, os marcos teóricos e sua relação com as práticas de ensino, o trabalho com os gêneros textuais etc. Segundo o que se constatou, as políticas e ou programas implementados entre os anos de 2002 a 2012 pelo sistema municipal de educação de Vitória da Conquista, embora o tenha feito com a justificativa de melhorar os índices educacionais, em especial na alfabetização, as preocupações estavam muito mais voltadas para as questões político-burocráticas do que político-pedagógicas. Evidencia-se daí que não houve preocupação por parte do sistema municipal de ensino em formar os professores, no sentido de muni-los dos referenciais teórico-metodológicos de cada uma das propostas. Como consequência, não houve avanço nas práticas pedagógicas. 2. Antecedentes As políticas públicas educacionais implementadas nas últimas décadas, considerando o marco institucional da LDB 9394/96, também não foram ou não têm sido suficientes para garantir o sucesso de todos os alunos, segundo os resultados das avaliações desenvolvidas por organismos governamentais e não-governamentais. Tais avalições revelam que a maioria dos estudantes brasileiros tem tido desempenho muito inferior ao esperado. Entre essas políticas incluem as leis 11.114/05, que tornou o ensino obrigatório a partir dos seis anos de idade e 11.274/06, que estendeu a duração do Ensino Fundamental para nove anos, mantendo o início aos seis anos. A implantação do Ensino Fundamental de nove anos também significou, para os sistemas educacionais, a expansão do Ciclo de alfabetização para três anos, ou seja, os três primeiros anos do Ensino Fundamental. No entanto, os dados indicam que um número significativo de crianças não é alfabetizada nesse período. Das crianças matriculadas no terceiro ano da rede pública brasileira, em 2013, 11,9% foram reprovadas e 19,9% tinham distorção idade/série (BRASIL, 2014). A redução da taxa de reprovação no Ciclo de alfabetização, a partir do ano de 2010, ocorre em função de as Diretrizes Curriculares Nacionais para o Ensino Fundamental de nove anos não permitirem a retenção de alunos do primeiro e segundo anos. As taxas do fracasso Língu@ Nostr@, Vitória da Conquista, v. 8, n. 1, p. 405-419, jan-julho. 2020. 408 Lingu@ Nostr@ - Revista Virtual de Estudos de Gramática e Linguística ISSN 2317-2320 registradas nesses anos são provenientes da evasão escolar. Em 2013, a taxa de evasão no primeiro ano foi de 1,4% e de 3,6% no terceiro ano. (BRASIL, 2013). Para verificar a qualidade e a eficiência do Ciclo de alfabetização das escolas públicas, o Ministério da Educação (MEC) criou a Avaliação Nacional de Alfabetização (ANA) pela Portaria MEC nº 482, de 7 de junho de 2013. De acordo com os termos desta Portaria, em seu Artigo 4, os objetivos da ANA são: I - avaliar a qualidade, a equidade e a eficiência (incluindo condições de oferta) do Ciclo de alfabetização das redes públicas; e II - produzir informações sistemáticas sobre as unidades escolares, para que cada unidade receba o resultado global. Os resultados da primeira avaliação, publicada em 2014, denunciam que 54% dos estudantes do terceiro ano têm desempenho insuficiente em leitura, interpretação de textos e operações matemáticas básicas. Nessa avaliação, os estudantes tinham de localizar informação específica, explícita em textos simples. Apenas 11% deles conseguiram oferecer respostas de forma satisfatória. Entre os 46% dos estudantes que leem e interpretam um texto de forma adequada se encontram aqueles que estão no nível apropriado e aqueles que estão no nível desejado. Os dados apresentados aqui sobre a alfabetização no cenário educacional brasileiro revelam que o país ainda tem que enfrentar vários desafios para que seus estudantes alcancem um nível satisfatório em relação à leitura e à escrita. Para Soares (2007) os baixos índices de desempenho dos alunos no processo de alfabetização resultam de diferentes perspectivas do processo de alfabetização, de diferentes áreas do conhecimento (psicologia, linguística e pedagogia), cada uma das quais trata o tema de forma independente. Em segundo lugar, são dados que, exclusivamente, buscam a explicação do problema ora no aluno, ora no contexto cultural do aluno, ora no professor, ora no método, ora no material didático, ora finalmente no próprio meio, o código escrito (a questão das relações entre o sistema fonológico e o sistema ortográfico da língua portuguesa). Garantir a aprendizagem da leitura e da escrita a todos os que se matriculam na escola pública brasileira é um grande desafio. A falta de professores com capacitação técnica é um dos fatores que impedem essa realização, segundo a posição de Cagliari (2006). Para ele, o professor alfabetizador, que tem uma boa preparação para o exercício de sua função, sabe avaliar se um método é bom ou não. Além das escolas de formação professores formarem mal os professores alfabetizadores, Cagliari (2006), atribui a culpa também aos pacotes educacionais e à atitude conformista do próprio professor. Como resultado, o professor Língu@ Nostr@, Vitória da Conquista, v. 8, n. 1, p. 405-419, jan-julho. 2020. 409 Lingu@ Nostr@ - Revista Virtual de Estudos de Gramática e Linguística ISSN 2317-2320 permanece com suas práticas pedagógicas medíocres, que não têm contribuído para a aprendizagem de forma mais significativa dos alunos. A discussão sobre o fracasso escolar no Brasil, referente ao aprendizado da leitura e da escrita, ocorreu em dois momentos: no primeiro momento, até 1996, o discurso girou em torno do alto índice de reprovação e evasão dos alunos e, no segundo momento, com a instituição da nova LDB 9394/1996, o discurso gira em torno da não aprendizagem dos alunos. A partir de então, amplia-se o acesso das crianças brasileiras ao processo de escolarização, embora carecem de condições de permanência e sucesso escolar. (MORTATTI, 2004). O município de Vitória da Conquista ficou nacionalmente conhecido, de forma negativa, por ter tido o pior desempenho educacional, segundo o Índice de Desenvolvimento da Educação Básica (IDEB), no ano de 2009, quando atingiu apenas 2,8. Esse resultado denunciou o quanto é baixo o nível de desempenho dos alunos com o mínimo de cinco anos de escolaridade. Cabe lembrar, no entanto, que os resultados anteriores, a contar de 2005 e dos posteriores, 2011 e 2013, o município ficou abaixo da meta projetada, embora tenha tido um resultado mais significativo. Conforme os trabalhos de Soares (1985), Morais (2010), entre outros, o problema do fracasso escolar tem atingido a maioria das crianças pertencentes a setores socialmente marginalizados a ponto de muitos não aprenderem a ler e escrever. A seguir, serão abordadas as políticas e programas educacionais implementados pelo município de Vitória da Conquista entre os anos de 2002 a 2012 e suas relações com as práticas pedagógicas. Nessa relação serão discutidos os resultados ou efeitos dessas políticas para a melhoria ou não dos índices educacionais no município, considerando que todas elas justificaram o baixo desempenho educacional dos estudantes. 3. Políticas e práticas de alfabetização do/no município de Vitória da Conquista Os resultados negativos dos alunos, em relação ao seu desempenho em leitura e escrita, apontados acima, motivaram o município de Vitória da Conquista a organizar, no ano de 2002, o Ensino Fundamental (1ª a 4ª série) em Ciclos de Aprendizagem: Ciclo I (correspondendo ao 1ª e 2ª série do Ensino Fundamental e Ciclo II (3ª e 4ª série), mediante a Resolução Nº 003/02. O ponto básico dessa organização pelo município foi desenvolver o Língu@ Nostr@, Vitória da Conquista, v. 8, n. 1, p. 405-419, jan-julho. 2020. 410 Lingu@ Nostr@ - Revista Virtual de Estudos de Gramática e Linguística ISSN 2317-2320 currículo com base em competências e habilidades. Por isso, passou-se a exigir uma série de mudanças no sistema de ensino, especialmente na prática pedagógica dos professores. O debate acerca do conceito de competência amplia-se, na década de 1990, na área pedagógica, destinado ao ensino de crianças nos primeiros anos de escolaridade. O desenvolvimento de habilidades por parte de um estudante, segundo Perrenoud (1999), deve ser em torno de três anos em cada ciclo escolar. Em 2006, é instituído o Ensino Fundamental de 9 anos, por meio da Lei N° 11.274, implementando, assim, o Ciclo da alfabetização (do 1º ao 3º ano). A organização do ensino em ciclos sofreu diversas críticas por parte dos professores da rede municipal de ensino, devido à adoção do regime de progressão continuada, denominada também no município de aprovação automática, proposta por essa organização. Os fragmentos de fala a seguir, denunciam isso: “Desde a implantação do Ciclo, a SMED2 não sabe o que fazer com a educação do município. Isso bagunçou a cabeça do professor e com o aprendizado dos alunos. Ainda hoje sofremos com a imposição de propostas que, de fato, não conseguimos entender. A avaliação dos alunos foi a pior parte do Ciclo porque exigia que fosse toda qualitativa. Não tínhamos como ter um conhecimento assim dos alunos, pois o tempo era curto e tínhamos de ensinar os conteúdos. Antes dessa proposta de Ciclo, eu sabia alfabetizar meus alunos, pois era muito mais fácil. [...]. O resultado de tudo isso foi o fracasso dos alunos” (Professora Júlia. Entrevista de 9 de abril de 2014). “A aprovação automática significou o fim da educação no município. Foi a pior medida tomada pela Secretaria de Educação. [...] Nunca tínhamos visto nada parecido e não funcionou de forma significativa. A aprovação automática foi a condição que o município encontrou para resolver o problema do fracasso. Não houve, a meu ver, uma preocupação com a aprendizagem dos alunos [...] (Professora Beatriz. Entrevista de 11 de março de 2014). “A proposta é que os alunos sejam avaliados de forma contínua e cumulativa, com prevalência de aspectos qualitativos sobre aspectos quantitativos. [...] O problema é que não nos foi orientado a fazer isso. Tínhamos que preecher um parecer descritivo que, na verdade não condizia com a realidade do aluno. [...] tive que inventar muito, porque não sabia o que escrever sobre os alunos. Era uma opinião individual”. (Professora Ana. Entrevista de 11 de março de 2014). “[...] A avaliação era exigida de uma maneira que os professores não sabiam como fazer e não fomos orientados para tal. [...]. Todos os alunos do 1º e 2º anos eram aprovados, sabendo ou não” (Professora Fábia. Entrevista de 05 de abril de 2014). Evidencia-se, por meio desses fragmentos de fala, que não houve participação dos professores nesse processo de mudança de abordagem, ou seja, a proposta foi imposta pelo sistema municipal de ensino. Evidencia-se ainda que a preparação dos professores não tenha acorrido para a implementação dessa proposta, o que pode justificar a carência dos professores em relação à adoção de novas metodologias, pois não tinham conhecimento dos 2 Secretaria Municipal de Educação. Língu@ Nostr@, Vitória da Conquista, v. 8, n. 1, p. 405-419, jan-julho. 2020. 411 Lingu@ Nostr@ - Revista Virtual de Estudos de Gramática e Linguística ISSN 2317-2320 fundamentos teóricos da proposta instituída. A denúncia apresentada por meio do fragmento de fala da professora Beatriz, a seguir, parece demonstrar o objetivo da gestão municipal: “A aprovação automática foi a condição que o município encontrou para resolver o problema do fracasso. Não houve, a meu ver, uma preocupação com a aprendizagem dos alunos [...]”. (Professora Beatriz. Entrevista de 11 de março de 2014). Nessa perspectiva, concorda-se com Jacomini (2004), ao analisar a institucionalização do Ciclo na rede de ensino do Estado de São Paulo. Ela aponta três problemas que foram evidenciados pelos professores participantes desta investigação: 1) elaboração de novas propostas sem avaliar os resultados das políticas que as antecederam; 2) ausência de discussão com os professores; 3) e, o não oferecimento das condições materiais e organizacionais para que as propostas sejam implementadas a contento. As reformas escolares fracassam, novos programas não são aplicados, belas ideias são pregadas como métodos ativos, construtivismo, avaliação formativa ou pedagogia diferenciada, mas nunca são praticadas. Isso porque, na área da educação, o desvio astronômico entre o que é prescrito e o que é praticado nas condições efetivas do trabalho docente não é medido suficientemente. (PERRENOUD, 2007). Isso também foi evidenciado por meio dos fragmentos de fala das professoras, como os seguintes: “Nos últimos anos, a SMED tem exigido muito de nós. O maior problema é que ela não tem dado as condições adequadas. Ela quer que ensinamos de outra maneira, mas nós não sabemos como [...]”. (Professora Carla, Entrevista de 19 de março de 2014). “[...] do que adianta estar tudo perfeito no papel, mas na prática não funciona. Baixa as portarias, mas nós professores não temos esse conhecimento, não sabemos como se operacionaliza isso. Nós reclamamos toda reunião, mas não nos escuta, não nos orienta, mas as cobranças vêm. [...] Parece até uma perseguição a nós professores. (Professora Ana. Entrevista de 11 de março de 2014). A resistência por parte dos professores da rede municipal aumentava a cada ano em relação à organização do currículo em Ciclos de Aprendizagem. Tal resistência levou a Secretaria Municipal de Educação a declinar da ideia da expansão da organização do currículo em Ciclos de Aprendizagem para a segunda etapa do Ensino Fundamental. No entanto, por meio da Resolução nº 009/06, a rede municipal de ensino institui a organização do currículo em Ciclos de Formação Humana, revogando, assim, a Resolução nº 003/2002. Por meio dessa Resolução, o Ensino Fundamental foi instituído em três cilos, cada um com duração de três anos. Dentre os objetivos do Ciclo de Formação Humana, destacam-se: o estabelecimento do processo de formação humana, focado no pleno desenvolvimento do aluno, sua preparação Língu@ Nostr@, Vitória da Conquista, v. 8, n. 1, p. 405-419, jan-julho. 2020. 412 Lingu@ Nostr@ - Revista Virtual de Estudos de Gramática e Linguística ISSN 2317-2320 para o exercício da cidadania; o desenvolvimento da capacidade de aprender, tendo como meio básico o pleno domínio da leitura, escrita e cálculo; o desenvolvimento da capacidade de aprendizagem, levando em consideração a aquisição de competências e habilidades, bem como a formação de atitudes e valores; a continuidade do processo de aprendizagem de crianças, pré-adolescentes e adolescentes que ingressam no sistema escolar. (SMED, 2008). A organização do Ensino Fundamental em Ciclos de Formação Humana deu continuidade ao regime de Progressão Continuada nos mesmos moldes do Ciclo de Aprendizagem. O Ciclo I corresponde ao período da infância, que inclui alunos de seis a oito anos. Nas classes desse Ciclo deve-se incluir a matrícula de novos alunos, transferidos de outro regime e daqueles que não tiverem comprovação de escolaridade anterior. Com essa medida, a rede municipal de ensino registra a presença de um número significativo de crianças com distorção idade/ciclo, além de evasão e reprovação. Esses casos geralmente estão intimamente relacionados à situação socioeconômica dos alunos, uma vez que o fracasso escolar tem atingido principalmente os alunos pertencentes aos contextos desfavoráveis, economicamente. A avaliação, de acordo com a organização do Ciclo de Formação Humana, é diagnóstica, contínua, processual, participativa e emancipatória, e sua concepção deve ser construída de forma a caracterizar as observações das competências propostas. Nesse sentido, são desenvolvidas ações de reorientação para alunos com dificuldades de aprendizagem e apoio processual em relação ao desenvolvimento do aluno. Predomina, portanto, nesse processo de avaliação os aspectos qualitativos sobre os quantitativos. (SMED, 2008). No entanto, os professores não aprovaram a nova proposta implementada pelo sistema municipal de ensino, conforme os fragmentos de fala apresentados a seguir: “No Ciclo não tem sentido avaliar os alunos, pois não pode haver reprovação. Passa os alunos de qualquer jeito [...]. Avaliar acaba sendo perda de tempo”. (Professora Beatriz. Entrevista de 11 de março de 2014). “[...] Os alunos, sabendo que não serão reprovados, nem demonstram interesse pelos conteúdos porque serão aprovados de todo modo”. (Professora Elisa. Entrevista de 17 de março de 2014). De acordo com esses fragmentos de fala, evidencia-se que a avaliação seja reduzida à verificação da aprendizagem. Evidenciou-se ainda que as dificuldades apresentadas pelas professoras, em relação ao trabalho precário em sala de aula, estava relacionado à não existência de um projeto consolidado de formação continuada dos professores. As professoras continuam reivindicando a sua participação na elaboração e implementação das propostas pedagógicas. Segundo Arroyo (2011), é necessário avaliar o que os professores consideram mais importante em suas práticas para que uma proposta seja apresentada como inovadora. Na Língu@ Nostr@, Vitória da Conquista, v. 8, n. 1, p. 405-419, jan-julho. 2020. 413 Lingu@ Nostr@ - Revista Virtual de Estudos de Gramática e Linguística ISSN 2317-2320 mesma direção, Masetto (2011) acredita que um projeto, que pretende ser inovador, deve se concentrar na prática pedagógica do professor. Em 2009, como já afirmado, o município teve o seu pior Ideb e ficou entre os piores do Brasil, reafirmando, assim, que os investimentos feitos de 2004 até esse momento, não trouxeram resultados positivos. A solução apresentada pelo municío foi revogar a Resolução nº 009/06, ao reorganizar o Ensino Fundamental de nove anos, por meio da Resolução nº 002/09, em Ciclos de Aprendizagem, assim divididos: o Ciclo I, do primeiro ao terceiro ano, denominado ciclo de alfabetização; Ciclo II, quarto e quinto ano; Ciclo III, sexto e sétimo ano e Ciclo IV, oitavo e nono anos. Identificou-se que as diretrizes propostas por essa Resolução, em sua essência, são textualmente as mesmas da Resolução nº 009/06. A diferença identificada entre essas resoluções é em relação à progressão continuada: a Resolução de 002/2009 prevê a aprovação automática apenas no ciclo I, enquanto a Resolução de 009/2006 previa a aprovação automática em todos os anos de cada Ciclo. Cabe lembrar que a aprovação automática, nos dois primeiros anos de escolaridade, está prevista nas Diretrizes Nacionais do Ensino Fundamental, não sendo, portanto, prerrogativa da rede municipal de ensino de Vitória da Conquista. A Resolução nº 002/09, semelhante à Resolução nº 009/2006, parece apresentar equívocos quanto à concepção de um sistema de ensino organizado em Ciclos de aprendizagem. A questão da avaliação é um dos equívocos mais evidentes: embora o conceito e critérios de avaliação das duas resoluções sejam os mesmos, a 002/09 exclui a emissão de parecer descritivo sobre o desempenho acadêmico de cada aluno e não aborda sobre aspectos qualitativos, o que prevalecem os aspectos quantitativos, exatamente como era no sistema de seriação. Sobre essa questão, os professores assim afirmaram: “Pelo menos a avaliação voltou ao normal. Não somos obrigadas a arovar de qualquer jeito os alunos [...]”. (Professora Julia. Entrevista de 09 de abril de 2014). “Com essa nova proposta, a escola fica livre para decidir como avaliar seus alunos. Não ter que fazer parecer para cada aluno já é uma avanço. [...]”. (Professora Fábia. Entrevista de 05 de abril de 2014). “Os pareceres descritivos de cada aluno era uma grande mentira. a gente copiava [...] Aquilo não representava a verdade sobre os alunos. Era uma perda de tempo fazer aquilo e nao servia para nada pois ninguém lia”. (Professora Elisa. Entrevista de 17 de março de 2014). A adoção dessa postura em relação à avaliação parece ter sido agradável aos professores, conforme os fragmentos de fala acima. Pelo menos em relação à avaliação, podese afirmar que essa proposta, que utiliza uma terminologia de Ciclos de Aprendizagem, mas Língu@ Nostr@, Vitória da Conquista, v. 8, n. 1, p. 405-419, jan-julho. 2020. 414 Lingu@ Nostr@ - Revista Virtual de Estudos de Gramática e Linguística ISSN 2317-2320 não assumem seus fundamentos teórico-práticos. Segundo Mainardes (2009), a organização do currículo escolar em ciclos é uma política complexa que possui justificativas filosóficas, políticas, psicológicas, antropológicas e sociológicas. No caso do município de Vitória da Conquista, evidencia-se, conforme suas justificativas, que a organização do currículo em Ciclos teve a intenção apenas de reduzir as altas taxas de reprovação e evasão escolar, para amenizar o fracasso escolar. Conforme os depoimentos das professoras, constata-se que a rede municipal de ensino não tenha assumido os fundamentos teórico-práticos, de nenhuma das propostas adotadas, o que permite dizer que o sistema de ensino municipal não tenha desenvolvido práticas pedagógicas fundamentadas no modelo teórico adotado. Os resultados das avaliações divulgados no ano de 2011, apontam que o Ideb observado, da primeira etapa do Ensino Fundamental salta de 2,8 para 3,4. Embora o município tenha registrado esse avanço, continua abaixo da meta projetada (4,0). Nesse ano, o município adere ao Programa Pacto de Alfabetização com o Município, lançado pelo Governo do Estado da Bahia. Entre as ações desse Programa destacam-se a formação contínua dos professores alfabetizadores; monitoramento e avaliação; distribuição de materiais didáticos para professores e alunos; oferta de reforço escolar e organização de espaços de leitura nas escolas, preferencialmente em cada sala de aula. Embora a adesão a esse Programa tenha sido em 2011, a sua operacionalização só ocorreu a partir do ano seguinte, quando o município também adere ao Programa Nacional de Alfabetização na Idade Certa [PNAIC ], lançado pelo governo federal. O PNAIC se configura como um compromisso formal assumido pelo governo federal, pelo Distrito Federal, pelos estados e municípios, com a intenção de garantir que todas as crianças sejam alfabetizadas até os oito anos de idade. A adesão ao PNAIC implica compromisso com a alfabetização de todas as crianças em língua portuguesa e em matemática; realização das avaliações anuais universais, aplicadas pelo INEP, entre elas a ANA, com os alunos 3º ano do Ciclo da alfabetização. De acordo com o PNAIC, ser alfabetizado aos oito anos de idade significa compreender como o sistema de escrita funciona; o domínio das correspondências grafemasfonemas; a fluência da leitura e domínio das estratégias de compreensão e produção de textos escritos. Para tal, as ações do PNAIC se baseiam em quatro linhas de ação: 1. Formação presencial para professores de alfabetização e seus orientadores de estudos; 2. Material Língu@ Nostr@, Vitória da Conquista, v. 8, n. 1, p. 405-419, jan-julho. 2020. 415 Lingu@ Nostr@ - Revista Virtual de Estudos de Gramática e Linguística ISSN 2317-2320 didático, obras literárias, trabalhos de apoio educacional, jogos e tecnologias educacionais; 3. Avaliações sistemáticas e 4. Gestão, mobilização e controle social. (BRASIL, 2012). A formação continuada dos professores é a principal atividade do PNAIC que, segundo Klein e Guizzo (2017), se converteu em um instrumento importante para a busca de melhorias da qualidade da educação básica, afim de satisfazer as novas demandas por meio de cursos de capacitação para que os professores atualizem suas práticas pedagógicas. Intensificar a formação continuada dos professores fortalece os vínculos entre a teoria e a prática. As professoras consideram fundamental a formação continuada e concordam que é por meio dela que pode ocorrer o domínio dos referenciais teórico-metodológicos para se apropriarem dos novos conceitos e, consequentemente, desenvolver práticas mais significativas. No entanto, elas reclamam que é insuficiente a formação que o sistema de ensino tem oferecido. “Para nós, professoras, é muito difícil mudar a nossa prática. Temos uma prática cristalizada, baseada em nossas crenças. Não podemos inovar só com esses cursos de capacitação que a SMED oferece. Tinha de ter uma formação continuada mais sólida”. (Professora Beatriz. Entrevista de 19 de março de 2014). “Me esforço para fazer o melhor que posso, porém […]a SMED não ajuda muito com a formação que oferece. Muitas vezes, considero perda de tempo porque é sempre a mesma coisa e não ensina nada de novo. Nem pode exigir mudanças de nossa prática porque o que ensina nessa formação já é antigo”. (Professora Iris. Entrevista de 08 de abril de 2014). A partir das narrativas das professoras, pode-se constatar que o sistema municipal de educação instituiu propostas pedagógicas com fins mais políticos que pedagógicos, acreditando resolver o problema do fracasso escolar. Por meio das narrativas se verificou também que as professoras foram resistentes às propostas, primeiro por não terem participado da construção de nenhumas delas e, segundo por não terem tido conhecimento dos seus referenciais teóricos, o que as impede, de algum modo, de desenvolver novas práticas ou práticas orientadas por tais propostas. Fica evidente, desse modo, que a descontinuidade das propostas pedagógicas não foi favorável para a melhoria dos índices educacionais dos alunos. A análise realizada a partir de documentos e entrevistas sobre as relações entre as políticas e práticas pedagógicas desenvolvidas pelas professoras das classes de alfabetização na rede municipal de ensino de Vitória da Conquista e os referenciais teórico-metodológicos aponta para: a) uma preocupação mais com aspectos políticos do que com questões pedagógicas por parte do sistema de educação; b) o desconhecimento ou não domínio dos referenciais teórico-metodológicos das propostas pedagógicas implementadas pelo sistema Língu@ Nostr@, Vitória da Conquista, v. 8, n. 1, p. 405-419, jan-julho. 2020. 416 Lingu@ Nostr@ - Revista Virtual de Estudos de Gramática e Linguística ISSN 2317-2320 municipal de educação e c) as práticas pedagógicas de alfabetização contraditórias à proposta vigente, ou seja, a permanência de práticas mecânicas de ensino da leitura e da escrita. De 2002 a 2012, o sistema de ensino municipal foi marcado, conforme o que se verificou no contexto desta investigação, por propostas pedagógicas descontínuas cuja preocupação por parte da gestão municipal era mais política que pedagógica. Cada uma dessas propostas foi implementada com o objetivo de melhorar os índices educacionais, principalmente no ciclo de alfabetização. No entanto, essas propostas não promoveram as melhorias esperadas, como evidenciado no contexto dessa análise. Conclusão Neste trabalho de investigação buscou analisar as relações entre os referenciais teórico-metodológicos que fundamentam as políticas educacionais (e as propostas pedagógicas) implementadas pela rede municipal de ensino de Vitória da Conquista e as práticas pedagógicas das professoras alfabetizadoras. A alfabetização é aqui vista como um processo que vai além da simples capacidade mínima de decodificar e codificar a escrita; é entender e conceber usar os sociais da leitura e da escrita na vida cotidiana e pressupõe que haja entendimento mínimo para fazer uso dessas capacidades. Assumir essa posição é dar-se conta de que o processo de construção da linguagem escrita, devido a sua complexidade, requer muito mais que técnicas ou treino mecânico; e que a capacidade de ler e escrever depende da compreensão de como funciona a linguagem e como se usa no contexto social. Segundo Borzone, Silva y Rosemberg (2004), Morais (2012) y Soares (2015), para que a criança aprenda a linguagem escrita, é necessário o desenvolvimento de habilidades cognitivas e linguísticas. Esta posição também parte do princípio legal de que as crianças tenham direito de aprender a ler e escrever, dado que este direito tenha sido negado às crianças pobres durante muitos anos, no Brasil. É um direito de as crianças não só ingressarem na escola, como terem acesso a uma educação de qualidade que lhes proporcione situações mais autônomas, de modo que ao final do chamado ciclo de alfabetização, tenham desenvolvido habilidades de leitura e produção de textos para continuar p processo educacional de forma exitosa. Os dados construídos e analisados nesta investigação revelam aspectos importantes sobre as relações entre os referenciais teórico-metodológicas e as práticas pedagógicas das Língu@ Nostr@, Vitória da Conquista, v. 8, n. 1, p. 405-419, jan-julho. 2020. 417 Lingu@ Nostr@ - Revista Virtual de Estudos de Gramática e Linguística ISSN 2317-2320 professoras alfabetizadoras. Ao abordar esses aspectos, no processo de alfabetização, permitiu optar por referências teóricas da Linguística, Psicolinguística e Educação, a fim de manter um diálogo entre elas. Neste sentido, apresenta em seguida algumas conclusões: a) permanece mecânico o ensino da leitura e da escrita e desvinculado do círculo cultural dos alunos; b) as atividades de ensino não têm em conta o conhecimento dos alunos nem seu grau de dificuldade; c) a linguagem utilizada nas atividades de ensino privilegia apenas a padrão, desenvolvidas como se todos os alunos dominassem a leitura e a escrita e d) carência de materiais didático-pedagógicos adequados. Esta investigação, pelo menos no que se refere ao ensino da leitura e da escrita, denuncia a fragilidade das práticas de alfabetização no interior da escola pública e constata que as professoras têm muito o que aprender, todavia em relação ao trabalho com o texto, a leitura, a escrita e a análise linguística. Isto reafirma o quanto tem sido precária a formação continuada dos professores na rede municipal de ensino de Vitória da Conquista. Espera-se que a formação continuada de forma mais sólida possa contribuir com a superação do mecanicismo no processo de alfabetização; oferecer aos alunos, no que se refere à linguagem, as devidas condições de expressão de si mesmos, de possibilitar a leitura de mundo, de modo que eles organizem os conhecimentos construídos; promover a interação entre as funções cognitivas superiores dos alunos como leitores e os conteúdos expressos nos textos entre outras. Enfim, cabe ressaltar que a elaboração e implementação de uma proposta pedagógica somente fará sentido se o objetivo for discutir e repensar as referências teóricas e metodológicas e o conteúdo das áreas de conhecimento para atualizá-las, levando em consideração os avanços científicos. O não cumprimento desse objetivo promove a continuidade das práticas cristalizadas, ou seja, que valorizam o mecanismo do ensino da leitura e da escrita, apesar de, no Brasil, as teorias construtivistas apoiarem as propostas de alfabetização. Referências ARROYO, M. G. Currículo, território de disputa. Petrópolis, RJ: Vozes, 2011. BRASIL. Pacto Nacional pela Alfabetização na Idade Certa. Disponível pacto.mec.gov.br. 2012. Acesso em 05 de jan. de 2015. Língu@ Nostr@, Vitória da Conquista, v. 8, n. 1, p. 405-419, jan-julho. 2020. 418 em: Lingu@ Nostr@ - Revista Virtual de Estudos de Gramática e Linguística ISSN 2317-2320 _____. Saeb - Sistema De Avaliação da Educação Básica. Disponível em: http://www.adurrj.org.br/4poli/gruposadur/gtpe/portaria_482_7_6_13.htm, 2013. Acesso em set. de 2014. _____. Inep - Instituto Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira. Indicadores Educacionais. Brasília: INEP. Disponível em: http://portal.inep.gov.br, 2014. Acesso em 10 nov. 2018. BORZONE, A. M.; SILVA, M. L. & C. ROSEMBERG. Programa de promoción del desarrollo lingüístico y cognitivo para los jardines de infantes de la provincia de Entre Ríos. Consejo General de Educación: gobierno Entre Ríos, 2004. Disponível em: https://www.researchgate.net/profile. Acesso em 20 de nov. 2018. CAGLIARI, L. C. Alfabetização sem ba-be-bi-bo-bu. São Paulo: Scipione, 2006. JACOMINI, Márcia A. A escola e os educadores em tempo de ciclos e progressão continuada: uma análise das experiências no estado de São Paulo Educação e Pesquisa, São Paulo, v.30, n.3, set./dez. 2004. KLEIN, J. M & GUIZZO, B. S. Problematizando representações docentes nos Cadernos de formação do Programa Pacto Nacional pela Alfabetização na Idade Certa (PNAIC). Disponível em: http://dx.doi.org/10.24109/2176-6681.rbep.98i249.2763. 2017. Acesso em 12 de out. 2018. MAINARDES, J. A escola em ciclos: fundamentos e debates. São Paulo: Cortez. 2009. MASETTO, M. T. Competência pedagógica do docente universitário. São Paulo: Summus Editorial. 2011. MORAIS, A. G. Psicologia, Educação Escolar e Didáticas Específicas: refletindo sobre o ensino e a aprendizagem na alfabetização. Trabalho escrito apresentado em concurso para docente titular no Depto. de Psicologia e Orientação Educacionais da UFPE, Recife. 2010. _____. Sistema de escrita alfabética. São Paulo: Melhoramentos. 2012. MORTATTI, M. do R. L. Educação e Letramento. São Paulo: UNESP. 2004. PERRENOUD, P. Construir as competências desde a escola. Porto Alegre: Artmed. 1999. _____. A formação dos docentes no século XXI. In: PERRENOUD, P. et al (2007). As competências para ensinar no século XXI: a formação dos docentes e o desafio da avaliação. Porto Alegre: Artmed. 2007. SMED. Plano Municipal de Educação. Vitória da Conquista, Bahia. 2008. SOARES, M. As muitas facetas da alfabetização. Cad. Pesq., São Paulo. 1985. _____ . Alfabetização e Letramento: 5ª ed. São Paulo: Contexto. 2007. _____. Alfabetização no Brasil: a questão dos métodos. São Paulo: Contexto. 2015. Língu@ Nostr@, Vitória da Conquista, v. 8, n. 1, p. 405-419, jan-julho. 2020. 419 

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Revista Brasileira de Zootecnia Full-length research article Brazilian Journal of Animal Science © 2018 Sociedade Brasileira de Zootecnia ISSN 1806-9290 www.sbz.org.br R. Bras. Zootec., 47:e20170159, 2018 https://doi.org/10.1590/rbz4720170159 Non-ruminants The role of yeast culture (Saccharomyces cerevisiae) on performance, egg yolk fatty acid composition, and fecal microflora of laying hens Bülent Özsoy1*, Özlem Karadağoğlu2, Akın Yakan3, Kadir Önk4, Elif Çelik5, Tarkan Şahin6 1 Mustafa Kemal University, Veterinary Medicine Faculty, Department of Animal Nutrition and Nutritional Disease, Hatay, Turkey. Kafkas University, Kars Vocationel High School, Department of Agriculture and Animal Production, Kars, Turkey. 3 Mustafa Kemal University, Veterinary Medicine Faculty, Department of Genetic, Hatay, Turkey. 4 Kafkas University, Veterinary Medicine Faculty, Department of Animal Science, Kars, Turkey. 5 Kafkas University, Veterinary Medicine Faculty, Department of Microbiology, Kars, Turkey. 6 Kafkas University, Veterinary Medicine Faculty, Department of Animal Nutrition and Nutritional Disease, Kars, Turkey. 2 ABSTRACT - This study investigated the effects of different levels (0.05, 0.1, and 0.2%) of yeast culture supplementation on body weight, feed intake, feed conversion ratio, egg production, egg weight, egg quality traits, egg yolk fatty acid composition, and microbiological flora in feces. A total of 240 laying hens at 18-19 weeks of age were divided into four groups and fed a basal diet containing 2750 kcal/kg metabolizable energy and 16% crude protein for 16 weeks. The basal diet was supplemented with 0.05, 0.1, and 0.2% commercial yeast culture product obtained from Saccharomyces cerevisiae. The different levels of yeast culture supplementation to the diets did not statistically affect body weight change among the treatments. However, feed intake was lowest in the group fed 0.2% of yeast culture. The highest egg weights were obtained from the groups fed 0.1 and 0.2% yeast culture, when compared with control group. Regarding fatty acid composition, linolenic acid (C18:2 n6) was lowest in the group fed 0.2% yeast culture. However, yeast culture supplementation to the diet did not alter the microbial flora. Yeast culture (S. cerevisiae) supplementation to the diet of laying hens is beneficial for increasing feed intake and egg weight of laying hens without affecting the microbial flora in their digestive system. Key Words: laying hen, fatty acid, microbiological flora, yeast culture Introduction Feed additives, which are defined as substances, have long been added to animal diets to increase the utilization of feeds as well as the quantity and quality of animal products. Yeast cultures are complex fermented products containing metabolic agents produced by yeast during fermentation. Live yeast cultures contain digestive enzymes such as amylase, maltase, sucrose, lactate dehydrogenase, proteinase, polypeptides, dipeptidase, deaminase, transaminase, lipase, phospholipase, phosphatase, and phytase and provide higher digestibility (Stanley et al., 1993). Saccharomyces cerevisiae, which produces these properties, has been proven to have beneficial effects on poultry production, e.g., in reproduction, growth rate, egg production, and feed efficiency (Dawson, 1993). In addition, supplementation of yeast, yeast extracts, and yeast cultures to feed has Received: June 29, 2017 Accepted: December 5, 2017 *Corresponding author: bulent58@gmail.com Copyright © 2018 Sociedade Brasileira de Zootecnia. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. provided economic and environmental benefits in poultry diets for the past 40 years (Stone, 2007). Saccharomyces cerevisiae is known to increase the biological value of nitrogenous compounds in the digestive tract and to reduce stress-producing factors in animals. Yeast culture also helps digestion by increasing the availability of nutrients (Stanley et al., 1993). The positive effects of supplementation of yeast culture, which might also be called probiotics on the performance values of animals, have been attributed to many factors including (but not limited to) microbial flora, dose added to the feed, environmental conditions, and physical condition of the animal (Ceylan and Çiftçi, 2003). It has been shown that probiotic microorganisms reach the villi of the intestinal lumen earlier than pathogenic bacteria, thus preventing these pathogens from becoming trapped in the digestive tract (Hassanein and Soliman, 2010). In other words, probiotic microorganisms compete with pathogenic microorganisms for binding to receptors (competitor exclusion). The positive effect may also be due to the strengthening of the natural defense system and the biological regulation of intestinal microflora of the host, as well as to the direct effect of the probiotic on health or the 2 Özsoy et al. nutritional form of the probiotic (Shareef and Al-Dabbagh, 2009; Hassanein and Soliman, 2010). This study aimed to investigate the effects of the different levels (0.05, 0.1, and 0.2%) of yeast culture supplementation on feed intake (FI), feed conversion ratio (FCR), egg production, egg quality traits, egg yolk fatty acid composition, and microbiological flora in the feces of laying hens. Material and Methods A total of 240 laying hens at 18-19 weeks of age were divided into four groups (control and three trial groups) and fed for 16 weeks. Each group comprised 10 sub-groups, with each sub-group having six animals. The animals were fed a basal diet containing 2750 kcal/kg metabolizable energy and 16% crude protein (Table 1). The yeast culture (S. cerevisiae) was added to the diet at different levels (0.05, 0.1, and 0.2% body weight – BW). The BW was determined at the beginning and at the end of the study. The daily egg production of the groups was measured. The FI and FCR of animals were recorded weekly (Table 2). In the 4th, 8th, 12th, and 16th weeks of the study, the egg internal and external quality traits were determined in 10 randomly selected eggs from each group, and measurements were repeated four times. Eggs were weighed twice a week for two successive days, and average weights were recorded (Table 3). Egg yolk oil extraction was conducted using nine egg samples from each group as reported by AOAC (2000). Afterwards, methyl esters were formed with boron trifluoride. The fatty acid methyl esters were condensed under nitrogen gas and then analyzed by gas Table 1 - Composition and nutrient level of basal diet Item Ingredient (g kg−1) Corn Soybean meal Sunflower seed meal Meat and bone meal DL-methionine Limestone Dicalcium phosphate Salt Vitamin and mineral premix1 645 150 78 30 0.5 82 10 2 2.5 Calculated nutrient level Metabolizable energy (kcal kg−1) Crude protein (g kg−1) Ca (g kg−1) P (g kg−1) 2753 160 34.5 6.2 1 Provides per kg: 15,372.00 mg vitamin A; 6.28 mg vitamin E; 0.64 mg vitamin K3; 27.36 mg Mn; 89 mg Fe; 25 mg Zn; 8.76 mg Cu; 0.03 mg Co; 0.05 mg I; 0.91 mg Se. chromatography-mass spectroscopy (HP 6890/5972). The Agilent HP88 100 × 250 × 250 µm column was used in the analysis. The column starting temperature was 120 °C, the final temperature was 230 °C, and the injector and detector temperature was 250 °C. The injection speed was set to 50:1. Helium was used as a carrier gas. Fecal samples, which were collected from each group, were placed into sterile plastic tubes in the last week of the experiment. Stool samples were diluted as 10:1 in physiological saline (FTS) containing 0.9% NaCl. Subsequently, the samples were diluted in a ten-fold (log10) dilution series by using six tubes. Inoculation began from the lowest dilution rate. Plate count agar was used for general live (total mesophilic aerobic microorganism), MacConkey agar for coliform bacteria, and Sabouraud dextrose agar for yeast. Plate count agar was incubated in aerobic conditions for 48 h at 30 °C, MacConkey agar was incubated for 24 h at 37 °C for coliform bacteria count, and Sabouraud dextrose agar was incubated at 45 °C for 72 h (Arda, 1985; Arda et al., 1997). After incubation, the media were counted and the averages were taken. The mean numbers for all dilution steps were then determined, and microorganism counts were recorded for each sample according to these results. One-way ANOVA was used to determine the significance of the differences between the statistical calculations and mean values of the groups. The Duncan test was employed to determine the significance among the groups (Dawson and Trapp, 2001). Results During the experimental period, there was no side effect on the health of animals due to supplementation of yeast culture at the test concentrations added to the feed. The different levels of yeast culture supplementation to diets did not affect BW. The lowest average FI (101.96 g/day) was obtained from the group fed 0.2% yeast culture supplementation, when compared with control group (106.43 g/day) (Table 2). The highest egg production was obtained from the groups fed 0.2 (91.54%) and 0.1% supplemention (91.93%), while control group had an average egg production of 90.30% (P>0.05). Yolk diameter was lowest in the group fed 0.1% supplementation, followed by the 0.05% supplementation, control, and 0.2% supplementation, in that order. In comparison with the control and 0.05% supplementation groups, the highest Haugh units were obtained from the 0.2%and 0.1%-supplementation groups, respectively (Table 4). R. Bras. Zootec., 47:e20170159, 2018 The role of yeast culture (Saccharomyces cerevisiae) on performance, egg yolk fatty acid composition, and fecal microflora of laying... In terms of fatty acid composition (Table 5), hexadecanoic (C16:0) and oleic acids (C18:1) were the main fatty acids in the egg yolk. Myristoleic (C14:1) and hexadecanoic acid (C16:0) values were higher in the 0.1 and 0.2% yeast culture-supplementaation groups, when compared with control group (P>0.05). Polyunsaturated fatty acids (PUFA) values were numerically lower in the 0.2% yeast culture-supplementation group than in the other 3 groups. However, PUFA:saturated fatty acids (SFA) and C18:2 n6 values were significantly different between the control and experimental groups (P<0.05). The microbiological analysis of feces (Table 6) revealed that dietary supplementation of yeast culture did not influence the total bacterial number (P = 0.50). The number of coliform bacteria was lowest (1.35±0.59 log10 cfu/g) in the 0.2% yeast culture-supplementation group (P = 0.89). Table 2 - Effects of yeast culture on laying hen performance Control group Initial body weight (g) Final body weight (g) Egg production/hen/day (%) Feed intake (g/day per hen) Feed conversion ratio (g feed/g egg) Experimental group 1 1757±19.12 1720±17.86 90.30±0.60 106.43±1.17a 2.03±0.03 1740±18.20 1707±16.90 90.93±0.52 105.23±0.85a 1.99±0.03 Experimental group 2 1758±14.34 1750±19.36 91.54±0.53 104.82±1.05ab 1.97±0.02 Experimental group 3 1761±8.53 1756±21.23 91.35±0.43 101.96±1.15b 1.94±0.07 P-value 0.812 0.211 0.35 0.03 0.07 Control group: basal diet; Experiemental group 1: basal diet + 0.05% yeast culture; Experimental group 2: basal diet + 0.1% yeast culture; Experimental group 3: basal diet + 0.2% yeast culture. a-b - Means within a row with different letters are different at P<0.05. Table 3 - Weekly egg weights (g) Week Control group Experimental group 1 Experimental group 2 Experimental group 3 P-value 1-2 56.97±0.51 n = 55 57.70±0.46 n = 55 58.78±0.51 n = 58 58.37±0.59 n = 56 57.67±0.71b n = 51 57.97±0.62b n = 53 58.84±0.61b n = 54 58.85±0.59b n = 51 58.15±0.20b n = 433 56.83±0.44 n = 55 57.69±0.46 n = 58 58.52±0.44 n = 60 57.86±0.51 n = 58 58.28±0.64ab n = 53 58.77±0.57ab n = 52 59.89±0.57ab n = 55 58.16±0.55b n = 54 58.24±0.19b n = 445 57.38±0.51 n = 53 58.49±0.54 n = 53 58.79±0.56 n = 57 60.19±0.65a n = 58 58.55±0.54ab n = 56 59.80±0.65a n = 56 60.96±0.53a n = 56 60.47±0.66a n = 57 59.35±0.21a n = 446 56.34±0.53 n = 51 58.58±0.62 n = 53 59.17±0.53 n = 50 60.36±0.54a n = 57 59.97±0.55a n = 58 60.44±0.53a n = 56 60.51±0.46a n = 56 60.96±0.47a n = 56 59.59±0.19a n = 437 0.54 3-4 5-6 7-8 9-10 11-12 13-14 15-16 All periods 0.44 0.85 0.003 0.052 0.019 0.041 0.002 <0.00 Control group: basal diet; Experiemental group 1: basal diet + 0.05% yeast culture; Experimental group 2: basal diet + 0.1% yeast culture; Experimental group 3: basal diet + 0.2% yeast culture. a-b - Means within a row with different letters are different at P<0.05. Table 4 - Effects of yeast culture on egg quality traits of laying hens Egg weight (g) Shape index (%) Shell weight (g) Albumen weight (g) Yolk weight (g) Shell weight percentage (%) Albumen weight percentage (%) Yolk weight percentage (%) Yolk diameter (mm) Yolk height (mm) Haugh unit Control group Experimental group 1 Experimental group 2 Experimental group 3 P-value 60.07±0.69 78.46±0.58 5.94±0.09 39.72±0.62 14.41±0.17 9.90±0.13 66.04±0.36 24.06±0.35 39.23±0.31ab 21.49±0.28 79.86±0.30b 58.85±0.66 77.28±0.43 5.81±0.13 38.79±0.61 14.25±0.24 9.87±0.19 65.85±0.49 24.28±0.44 38.86±0.43ab 21.31±0.28 79.71±0.31b 59.66±0.73 77.81±0.36 6.05±0.11 39.18±0.67 14.44±0.21 10.14±0.16 65.58±0.45 24.28±0.41 38.08±0.41b 21.25±0.26 80.49±0.37ab 59.78±0.63 78.02±0.59 6.08±0.09 37.62±1.12 16.08±1.04 10.19±0.18 62.91±1.69 26.90±1.67 40.01±0.43a 21.48±0.35 81.18±0.29a 0.62 0.42 0.62 0.29 0.07 0.38 0.06 0.08 0.01 0.92 0.005 Control group: basal diet; Experiemental group 1: basal diet + 0.05% yeast culture; Experimental group 2: basal diet + 0.1% yeast culture; Experimental group 3: basal diet + 0.2% yeast culture. a-b - Means within a row with different letters are different at P<0.05. R. Bras. Zootec., 47:e20170159, 2018 4 Özsoy et al. Table 5 - Effects of dietary supplementation of yeast culture on yolk fatty acids (% of total methyl esters of fatty acids) of laying hens Fatty acid (%) Control group Experimental group 1 C14:0 C14:1 C:16:0 C16:1 C17:0 C17:1 C18:0 C18:1 C18:2 n6 C18:3 n6 C18:3 n3 C20:0 C20:1 C20:2 n6 C20:3 n3 C20:4 n6 C22:6 n3 C24:0 SFA MUFA PUFA UFA PUFA:SFA n6 n3 n6:n3 Nutritive value1 Atherogenic index2 Thrombogenic index3 0.31±0.015 0.014±0.004 23.96±0.44 2.97±0.15 0.18±0.04 0.22±0.046 8.87±0.23 41.07±0.69 18.09±.54ab 0.51±0.044 0.88±0.040 0.075±0.017 0.34±0.045 0.21±0.014 0.19±0.018 1.44±0.083 0.54±0.042 0.12±0.013 33.54±0.39 44.61±0.64 21.86±0.66 66.46±0.39 0.65±0.02ab 20.24±0.59 1.61±0.08 12.65±0.37 2.09±0.06 0.15±0.005 0.57±0.01 0.31±0.016 0.015±0.005 24.14±0.35 3.07±0.16 0.20±0.015 0.15±0.018 8.24±0.23 40.72±0.42 19.09±0.37a 0.43±0.043 0.97±0.035 0.061±0.006 0.27±0.016 0.25±0.024 0.19±0.011 1.28±0.10 0.46±0.057 0.11±0.005 33.10±0.39 44.22±0.46 22.67±0.49 66.89±0.39 0.69±0.02a 21.06±0.43 1.61±0.07 13.16±0.47 2.03±0.04 0.14±0.003 0.57±0.009 Experimental group 2 0.32±0.022 0.023±0.005 24.07±0.34 3.15±0.12 0.17±0.018 0.13±0.008 8.30±0.20 41.00±0.58 18.81±0.37a 0.48±0.039 0.94±0.048 0.091±0.019 0.26±0.019 0.21±0.021 0.17±0.011 1.24±0.09 0.47±0.041 0.14±0.018 33.12±0.40 44.56±0.66 22.32±0.48 66.88±0.40 0.67±0.02a 20.74±0.43 1.59±0.06 13.14±0.38 2.05±0.04 0.14±0.004 0.56±0.01 Experimental group 3 P-value 0.30±0.020 0.029±0.002 24.79±0.32 3.27±0.11 0.26±0.062 0.18±0.030 8.47±0.16 41.98±0.82 16.80±0.79b 0.45±0.028 0.84±0.058 0.12±0.025 0.29±0.04 0.21±0.030 0.19±0.01 1.27±0.03 0.47±0.021 0.10±0.007 34.04±0.27 45.73±0.89 20.23±0.87 65.96±0.27 0.59±0.03b 18.73±0.81 1.50±0.06 12.50±0.17 20.04±0.05 0.15±0.003 0.58±0.01 0.96 0.09 0.41 0.48 0.41 0.19 0.15 0.55 0.03 0.56 0.23 0.18 0.3 0.45 0.77 0.33 0.59 0.10 0.25 0.44 0.06 0.25 0.03 0.07 0.58 0.47 0.83 0.23 0.50 Control group: basal diet; Experiemental group 1: basal diet + 0.05% yeast culture; Experimental group 2: basal diet + 0.1% yeast culture; Experimental group 3: basal diet + 0.2% yeast culture. MUFA - monounsaturated fatty acids; PUFA - polyunsaturated fatty acids; UFA - unsaturated fatty acids; SFA - saturated fatty acids. 1 (C18:0 + C18:1)/C16:0. 2 (4*C14:0) + C18:0)/UFA. 3 (C14:0 + C16:0 + C18:0)/(0.5*C18:1) + (0.5*MUFA) + (0.5*n6) + (3*n3) + (n3/n6). a-b - Means within a row with different letters are different at P<0.05. Table 6 - Effects of dietary supplementation of yeast culture on fecal microflora (log10 cfu/g) of laying hens Total bacteria Coliform bacteria Yeast Control group Experimental group 1 Experimental group 2 Experimental group 3 P-value 6.32±0.57 1.71±0.58 0.15±0.043 6.13±0.99 1.79±0.29 0.36±0.15 4.85±0.99 1.80±0.39 0.23±0.09 4.78±0.97 1.35±0.59 0.29±0.05 0.50 0.89 0.46 Control group: basal diet; Experiemental group 1: basal diet + 0.05% yeast culture; Experimental group 2: basal diet + 0.1% yeast culture; Experimental group 3: basal diet + 0.2% yeast culture. Non-significant differences P>0.05. Discussion Studies on laying hens reported that addition of yeast culture or yeast metabolites did not affect BW (Yousefi and Karkoodi, 2007; Yalçın et al., 2012; Yalçın et al., 2014; Hassanein and Soliman, 2010). Similarly, our results also did not show any difference in the BW of laying hens. On the other hand, some studies indicated that yeast culture supplementation to broiler diets positively affected their BW (Shareef and Al-Dabbagh, 2009; Fasina and Thanissery, 2011; Fathi et al., 2012), which could be attributed to the strains of yeast and animal species, among other factors. Although yeast culture supplementation had no statistical effect on egg production, a numerical increase was observed. Similar results were recorded for yeast or yeast metabolite supplementation to laying hen diets in many studies (Ayanvale et al., 2006; Asli et al., 2007; Yousefi and Karkoodi, 2007; Yalçin et al., 2008; Yalçın et al., 2012; Gül et al., 2013; Sacakli et al., 2013). On the other hand, Hassanein and Soliman (2010) observed an increase in egg production in laying hens by using yeast culture supplementation at much higher concentrations (0.4, 0.8, 1.2, and 1.6%). The different levels of yeast culture supplementation to diets significantly affected FI (P<0.05). Consistent with our R. Bras. Zootec., 47:e20170159, 2018 The role of yeast culture (Saccharomyces cerevisiae) on performance, egg yolk fatty acid composition, and fecal microflora of laying... findings, yeast culture supplementation positively affected FCR of broilers (Onifade and Babatunde 1996; Miazzo et al., 2005; Shareef and Al-Dabbagh, 2009) and laying hens (El-Ella et al., 1996; Katoch et al., 2003; Yalçın et al., 2012; Gül et al., 2013; Yalçın et al., 2015). Conversely, FI and FCR was not improved by yeast culture supplementation in laying hens (Nursoy et al., 2004; Asli et al., 2007; Yousefi and Karkoodi, 2007; Hassanein and Soliman, 2010; Sacakli et al., 2013), in laying quail (Önol et al., 2003), and in broiler turkeys (Özsoy and Yalçın, 2011). The highest egg weight was observed in hens fed 0.1 and 0.2% yeast culture during the experimental period, and this difference was statistically significant (P<0.05). Our findings corroborate previous studies (Ayanvale et al., 2006; Yalçin et al., 2008; Yalçın et al., 2015; Zhong et al., 2016), but disagree with others (Nursoy et al., 2004; Asli et al., 2007; Hassanein and Soliman, 2010; Gül et al., 2013; Sacakli et al., 2013). No statistical difference was observed for shape index, shell weight, albumen weight, or yolk height. These results are consistent with those of other studies (Asli et al., 2007; Hassanein and Soliman, 2010; Yalçın et al., 2012; Yalçın et al., 2014; Yalçın et al., 2015). Yolk weight (P<0.07), yolk weight percentage (P<0.08), and yolk diameter (P<0.01) were the highest in the group fed 0.2% yeast culture supplementation. The lowest albumen weight percentage was also found in the third trial group (P<0.06). Haugh unit was calculated as 79.86, 79.71, 80.49, and 81.18 in the control and the three trial groups, respectively. The best Haugh unit was found in the third trial group (P<0.005). Similarly, previous studies (Ayanwale et al., 2006; Yousefi and Karkoodi, 2007; Asli et al., 2007; Zhong et al., 2016) reported that egg yolk weight was increased by the supplementation of yeast culture. At the end of the experiment, egg yolk fatty acids C14-C24 were determined. The C18:2 n6 values were statistically different between the control and experimental groups (P<0.05). In addition, PUFA (P = 0.06), PUFA/SFA (P<0.03), and n6 (P = 0.06) values were different between control and experimental groups. Yalçın et al. (2010) similarly reported that yeast autolysate supplementation decreased monounsaturated fatty acids (MUFA) and C18:1, but increased C14:0, C14:1, C20:0, C20:3n-3, and C20:5n-3. Yalçın et al. (2012) also reported that only C18:1 and MUFA levels increased, and the other fatty acid parameters were not affected by yeast culture supplementation. The positive effects of yeast culture supplementation on the natural defense system of the host animal and on the biological regulation of its intestinal microflora and the 5 direct effect of the probiotic on health or the nutritional form of the probiotic have been previously reported (Shareef and Al-Dabbagh, 2009; Hassanein and Soliman, 2010). In the current study, total microbial counts, coliform bacteria, and yeast values were not altered in stool samples during the last week of the study. The lowest coliform bacteria value was found in the group fed 0.2% yeast culture supplementation. Fecal yeast count was higher in the yeast culture groups than in the control group. Gül et al. (2013) reported that the number of total bacteria was decreased, but the number of total yeast was increased in the intestine by yeast supplementation to laying hen diets. Another study (Hassanein and Soliman, 2010) reported that different levels (0.4, 0.8, 1.2, and 1.6%) of yeast supplementation to laying hen diets significantly reduced the number of total bacteria, especially in the 1.6% supplementation group. However, Wang et al. (2015) found that yeast supplementation increased the total number of bacteria in the intestine of laying hens. Conclusions Yeast culture supplementation, as a probiotic to laying hen diets, does not cause any health problems. It is thus suggested that the different levels of yeast supplementation (S. cerevisiae) in laying hen diets could increase the productivity of laying hens in terms of feed intake, egg weight, and egg Haugh unit. Acknowledgments The present study was supported by the Scientific Research Project Unit of Kafkas University, Turkey (Project Number: BAP 2016 TS 19). References AOAC - Association of Official Analytical Chemistry. 2000. Official methods of analysis. 17th ed. AOAC International, Arlington, VA. Arda, M. 1985. Bakterilerin idendifikasyonları. p.266-271. In: Genel mikrobiyoloji. 3rd ed. Arda, M., ed. Ankara Üniversitesi Veteriner Fakültesi Dergisi, Ankara. Arda, M.; Minbay, A.; Leloğlu, N.; Aydın, N.; Kahraman, M.; Akay, Ö.; Ilgaz. A; İzgür, M. and Diker, K. S. 1997. Fakültatif anaerobik gram negatif çomaklar. p.45-49. In: Özel mikrobiyoloji. 4th ed. Arda, M., ed. Medisan Yayınları, Ankara. Asli, M. M.; Hosseini, S. A.; Lotfollahian, H. and Shariatmadari, F. 2007. Effect of probiotics, yeast, vitamin E and vitamin C supplements on performance and immune response of laying hen during high environmental temperature. International Journal of Poultry Science 6(12):895-900. Ayanwale, B. A.; Kpe, M. and Ayanwale, V. A. 2006. The effect of supplementing Saccharomyces cerevisiae in the diets on egg R. Bras. Zootec., 47:e20170159, 2018 6 Özsoy et al. laying and egg quality characteristics of pullets. International Journal of Poultry Science 5(8):759-763. system in broiler turkeys. Ankara Üniversitesi Veteriner Fakültesi Dergisi 58:117-122. Ceylan, N. and Çiftçi, İ. 2003. Büyütme faktörü antibiyotiklere alternatif yem katkılarının etlik piliçlerde besi performansı ve bağırsak mikroflorası üzerine etkileri. Turk Journal Veterinary Animal Science 27:727-733. Sacakli, P.; Ergun, A.; Koksal, B. H; Ozsoy, B. and Cantekin, Z. 2013. Effects of inactivated brewer’s yeast (Saccharomyces cereviciae) on egg production, serum antibody titres and cholesterol levels in laying hens. Veterinarija ir Zootechnika 61:53-60. Dawson, B. and Trapp, R. G. 2001. Basic and clinical biostatistics. 3rd ed. Lange Medical Books/McGraw-Hill Medical Publishing Division, New York. Shareef, A. M. and Al-Dabbagh, A. S. A. 2009. Effect of probiotic (Saccharomyces cerevisiae) on performance of broiler chicks. Iraqi Journal Veterianry Science 23(Suppl I):23-29. Dawson, K. A. 1993. Current and future role of yeast culture in animal production: A review of research over the last seven years. p.169-171. In: Biotechnology in the feed industry. Proceedings of the 9th Annual Symposium. Alltech Technical Publications, Nicholasville, KY. Stanley, V. G.; Ojo, R.; Woldesenbet, S.; Hutchinson, D. H. and Kubena, L. 1993. The use of Saccharomyces cerevisiae to suppress the effects of aflatoxicosis in broiler chicks. Poultry Science 72(10):1867-1872. El-Ella, M. A. A.; Attiae, M. Y.; El-Nagmy, K. Y. and Radwan, M. A. H. 1996. The productive performance of layings fed diets supplemented with some commercial feed additives. Egyptian Journal Animal Production Suppl Issue. NoV. 33:423-431. Fasina, Y. O. and Thanissery, R. R. 2011. Comparative efficacy of a yeast product and bacitracin methylene disalicylate in enhancing early growth and intestinal maturation in broiler chicks from breeder hens of different ages. Poultry Science 90(5):1067-1073. Fathi, M. M.; Al-Mansour. S.; Al-Homidan. A.; Al-Khalaf, A. and Al-Damegh, M. 2012. Effect of yeast culture supplementation on carcass yield and humoral immune response of broiler chicks. Veterinary World 5:651-657. Gül, M.; Yörük, M. A.; Sağlam, Y. S. and Aksu, T. 2013. Yumurta tavuğu rasyonlarına maya (Saccharomyces cerevisiae) ve Enterococcus faecium katkılarının performans, yumurta kalite kriterleri ve barsak mikroflorası üzerine etkileri. Atatürk Üniversitesi Veteriner Bilimleri Dergisi 8(2):137-144. Hassanein, S. M. and Soliman, N. K. 2010. Effect of probiotic (Saccharomyces cerevisiae) adding to diets on intestinal microflora and performance of Hy-Line layings hens. Journal of American Science 6(11):159-169. Katoch, S.; Kaıstha, M.; Sharma, K. S. and Kumari, M. 2003. Biological performance of chicken fed newly isolated probiotics. Indian Journal Animal Science 73:1271-1273. Miazzo, R. D.; Peralta, M. F. and Picco, M. 2005. Perfomance productiva y calidad de la canal en broilers que recibieron levadura de cerveza (S. cerevisiae). Redvet 6:1-9. Available at: <http://www. veterinaria.org/revistas/redvet>. Accessed on: April 5, 2017. Nursoy, H.; Kaplan, O.; Oğuz, M. N.; and Yılmaz, O. 2004. Effects of varying levels of live yeast culture on yield and some parameters in laying hen diets. Indian Veterinary Journal 81:59-62. Onifade, A. A. and Babatunde, G. M. 1996. Supplemental value of dried yeast in a high-fibre diet for broiler chicks. Animal Feed Science and Technology 62:91-96. Önol, A. G.; Sarı, M.; Oğuz, F. M.; Gülcan, B. and Erbaş G. 2003. Sürekli sıcak stresinde bulunan yumurta dönemindeki bıldırcın rasyonlarına probiyotik katkısının bazı verim ve kan parametreleri üzerine etkisi. Türk Journal Animal Science 27:1397-1402. Özsoy, B. and Yalçın, S. 2011. The effects of dietary supplementation of yeast culture on performance, blood parameters and immune Stone, C. W. 2007. Yeast products in the feed ındustry a practical guide for feed professionals. Available at: <http://www.diamondv. com/products/yc.html>. Accessed on: May 8, 2017. Wang, H. T.; Shih, W. Y.; Chen, S. W. and Wang, S. Y. 2015. Effect of yeast with bacteriocin from rumen bacteria on laying performance, blood biochemistry, faecal microbiota and egg quality of laying hens. Journal of Animal Physiology and Animal Nutrition 99(6):1105-1115. Yalçin, S.; Özsoy, B.; Erol, H. and Yalçin, S. 2008. Yeast culture supplementation to laying hen diets containing soybean meal or sunflower seed meal and its effect on performance, egg quality traits and blood chemistry. Journal of Applied Poultry Research 17(2):229-236. Yalçın, S.; Yalçın, S.; Çakın, K.; Eltan, Ö. and Dağaşan. L. 2010. Effects of dietary yeast autolysate (Saccharomyces cerevisiae) on performance, egg traits, egg cholesterol content, egg yolk fatty acid composition and humoral immune response of laying hens. Journal of the Science of Food and Agriculture 90(10):1695-1701. Yalçın, S.; Uzunoğlu, K.; Duyum, H. M. and Eltan, Ö. 2012. Effects of dietary yeast autolysate (Saccharomyces cerevisiae) and black cumin seed (Nigella sativa L.) on performance, egg traits, some blood characteristics and antibody production of laying hens. Livestock Science 145(1):13-20. Yalçın, S.; Yalçın, S.; Onbaşılar, İ.; Eser, H. and Şahin, A. 2014. Effects of dietary yeast cell wall on performance, egg quality and humoral immune response in laying hens. Ankara Üniversitesi Veteriner Fakültesi Dergisi 61:289-294. Yalçın, S.; Yalçın, S.; Şahin, A.; Duyum, H. M.; Çalık, A. and Gümüş, H. 2015. Effects of dietary ınactive yeast and live yeast on performance, egg quality traits, some blood parameters and antibody production to SRBC of laying hens. Kafkas Üniversitesi Veteriner Fakültesi Dergisi 21(3):345-350. Yousefi, M. and Karkoodi, K. 2007. Effect of probiotic Thepax® and Saccharomyces cerevisiae supplementation on performance and egg quality of laying hens. International Journal of Poultry Science 6:52-54. Zhong, S.; Liu, H.; Zhang, H.; Han, T.; Jia, H. and Xie, Y. 2016. Effects of Kluyveromyces marxianus isolated from Tibetan mushrooms on the plasma lipids, egg cholesterol level, egg quality and ıntestinal health of laying hens. Revista Brasileira de Ciência Avícola 18:261-268. R. Bras. Zootec., 47:e20170159, 2018 

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ACADEMIC RESEARCH IN MODERN SCIENCE International scientific-online conference MODERN PEDAGOGICAL TECHNOLOGIES ENSURING THE QUALITY OF TEACHING IN ENGLISH LESSONS IN ELEMENTARY GRADES Mirkamilova Naima Sadiliyevna English teacher school 328 mirkamilovakamila5@gmail.com https://doi.org/10.5281/zenodo.5889345 Annotation: Today, the focus is on the student, his personality, and his unique inner world. Therefore, the main goal of a modern teacher is to choose methods and forms of organizing students' educational activities that optimally correspond to the set goal of personal development. In recent years, the issue of the use of new information technologies in secondary schools has been increasingly raised. These are not only new technical means, but also new forms and methods of teaching, a new approach to the learning process. Keywords: psychological development, story-role-playing game, selforganization, intensive methodology Based on the peculiarities of the psychological development of younger schoolchildren, one of which is the predominance of the emotional sphere over the intellectual one, I chose game technology as the leading pedagogical technology in early English language teaching. In particular, as a leading educational activity – a story-role-playing game. In the story-role-playing game, the impressions of a junior schoolboy are constantly replenished, clarified, and qualitatively changed. The knowledge he has, thanks to their practically effective reproduction in the plot-role-playing game, becomes more durable, more conscious. Freedom, independence, self-organization and creativity of children in the story-role-playing game are manifested with special completeness. The creation of a unified system of scheduled and extracurricular work on the subject is the main task of the educational process. Only under the condition of an organic combination of regular and extracurricular work, a foreign language can make a significant contribution to the formation and development of a child's personality. The initial training course is divided into three stages: I. "Let's play English". Students in a playful way master the main types of speech activity - speaking, listening, get acquainted with the letters and sounds of the English alphabet, learn to read the first words. II. "Let's read and write English". At this stage, all types of speech activity are being developed, but special attention is paid to reading and writing. 106 ACADEMIC RESEARCH IN MODERN SCIENCE International scientific-online conference III. "Let's enjoy your English". At this stage, there is a further development of students' communicative competencies, as well as systematization and selfimprovement of the acquired knowledge and skills. In an effort to update the content and introduce new approaches in teaching a foreign language, focused on the development of pupils' communicative competence based on game technologies. Elements of intensive methodology, various forms of play activity, dramatization, dramatization contribute to the intensification of the learning process and ensure a high level of student learning. In my classes, I rely on the methods which, from my point of view, successfully combine all learning goals, take into account the psychological characteristics of children of early age. The program allows you to make learning understandable and joyful. The child learns by playing. To do this, interesting tasks are set for students in the course of training. The obvious advantage of this program is the presentation of lexical and grammatical material in a playful way, which is very important for children. Tired of serious educational material in other subjects, the school children easily and actively perceive problematic stories interesting to them in English lessons, plunge into the world of fairy tales. All this contributes to the emergence of a positive motivation for the subject English, and also encourages him to participate in activities in the classroom, at the same time develops the child himself. When school children have already mastered the basic skills of reading and writing in their native language, he has formed basic general educational skills, so he is ready for a more serious assimilation of more complex, not always corresponding to the equivalents of their native language, material in English. To combine programs, grades 2, 3, 4, I use such techniques and tools that allow me to build a lesson as an integral communication system: game situations like "fairy tales", interesting stories, the same actors familiar from other grades. The pupil must understand the meaning of everything that he has to reproduce, including grammatical constructions. Already from the first stage of learning English, I try to explain grammatical phenomena, but I do it with the help of game techniques, where dolls are also assistants. This is the formation of the plural of nouns, the negative structure with the verb can; the use of have (has, etc.). A young child loves to play very much, so one of the effective teaching methods is the use of various plots (for example, "Journey to the land of the Four Fairies", "Magic City"), stories (secrets, secrets of letters, the eternal adventures of Simon), game situations (the appearance of Karabas, Master Lomaster, Santa Claus). The main characters of my game plots in the lessons are dolls and toys. Dolls are my best assistants in English lessons: Alice, Simon, Pinocchio, Malvina, Dunno, etc. These 107 ACADEMIC RESEARCH IN MODERN SCIENCE International scientific-online conference dolls are present from the first lessons, they talk about the countries of the language being studied, their families, friends, hobbies, teach children, enter into an argument with them, travel, arrange holidays. These dolls have their own biographies, a certain character, their own life stories. So, Alice and Simon are present at almost all lessons, Pinocchio sells us goods in the magic store, and is also a guide to school to Malvina. Malvina teaches us the letters and sounds of the English alphabet, tells us about her school. Dunno appears in those moments when we do not know something, we are experiencing difficulties, but together we find a way out of a difficult situation. References Kama Einhorn. ESL Activities and Mini-Books for Every Classroom. Scholastic Publishing. New York. 2011 Stocker D. & Stocker J. Children's ESL Curriculum: Learning English With Laughter. Longman. 2013 108 

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Typifying Mechanisms for Gender Digital Equity in Latin America Gabriela Marín-Raventós, Marta Calderón-Campos To cite this version: Gabriela Marín-Raventós, Marta Calderón-Campos. Typifying Mechanisms for Gender Digital Equity in Latin America. 6th IFIP World Information Technology Forum (WITFOR), Sep 2016, San José, Costa Rica. pp.159-170, �10.1007/978-3-319-44447-5_15�. �hal-01429744� HAL Id: hal-01429744 https://inria.hal.science/hal-01429744 Submitted on 9 Jan 2017 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 Typifying Mechanisms for Gender Digital Equity in Latin America Gabriela Marín-Raventós1, Marta Calderón-Campos2 1 CITIC-ECCI, Universidad de Costa Rica gabriela.marin@ucr.ac.cr 2 ECCI, Universidad de Costa Rica marta.calderon@ucr.ac.cr Abstract. Gender digital equity in Latin America and the Caribbean is our goal. We analyze gender specific structural inequalities that constitute barriers for women´s access to ICT: such as education, traditional cultural beliefs and practices, and economic inequality. We argue also that male-dominated ICT design and implementation (functionality, content, and human-computer interaction) can inhibit gender digital equity. We present a model to typify the existing mechanisms for reaching gender digital equity and propose a new mechanism: creating gender consciousness in ICT design and construction, and attracting women to the ICT field. The proposed model reflects the four mechanisms and depicts the possible means through which they can be implemented. Moreover, the model appoints actors capable of and responsible for such means. Recommendations for the ICT community regarding gender digital equity are also included. Keywords: ICT and Gender · Inequity · Digital Divide · Digital Equity 1 Introduction Latin America and the Caribbean is a middle-income region, with the majority of its 42 countries and territories belonging to that category. It is a heterogeneous region, ranging from low income countries, such as Honduras, Nicaragua, Guatemala, Paraguay, Bolivia, and Dominican Republic to higher income, and regarded as more developed countries, such as Chile, Mexico, Argentina, and Brazil [1]. According to the Socio-Economic Database for Latin America and the Caribbean (SEDLAC), 34.3% of the Latin American population is middle class, with income between $10 a day and $50 a day; 25.3% is still under the poverty line of $4 a day [1]. In last decades, many countries in Latin America and the Caribbean have made a big effort to close their gender gaps [2]: poverty has been reduced to lower levels, more girls are in school, child mortality has dropped, and diseases are being fought. However, gender issues remain and reduction in gender gaps does not mean that women´s empowerment has been reached [2]. In particular gender digital divide is still prevalent in the Region. Digital divide is “an inequality in the power to communicate and to process information digitally” [3]. In most existing published research about digital divide, the word gender refers to sex, that is, to biological women and men, since information about gender identity is not available [3]. In our research, we adopt this position. In the next section we analyze general gender gaps in Latin America and the Caribbean. In section 3, numbers and aspects of the Region´s digital divide are presented. Causes of gender digital divide are presented in section 4, with the objective of trying to identify means for obtaining gender digital equity. Understanding ICTs and their potential for the empowerment of women is necessary in order to overcome the gender digital divide. Section 5 describes some different types of projects that have been implemented throughout the region to overcome gender digital divide. Our main contribution is in Section 6 where we present a model to typify the existing three mechanisms for reaching gender digital equity and propose a new mechanism: creating gender consciousness in ICT design and construction, and attracting women to the ICT field. Finally, Section 7 includes the conclusions and recommendations for the ICT community, which includes government, academia, non-profit organizations, and industry, as a guide for them to design and implement solutions for each of the four mechanism categories mentioned above, in order to contribute to reach gender digital equality and equity. 2 General Gender Gaps in the Region Figure 1 shows that women are still underrepresented in the best-paid professions and receive significantly lower salaries than men. The average gender earning gap between men and women is 58% in the region. Engineering, including computer-related professions, is the field in which female representation reaches the lowest percentage (17%), and the second highest gender earning gap (76%). The World Economic Forum introduced the Global Gender Gap Index (GGGI) in 2006 for capturing the magnitude of gender-based differences [2]. Three concepts are important for understanding the GGGI. The first is that it measures gaps rather that levels of attainment. The second is that it measures outcome variables instead of input variables. This means that variables such as rights, culture or customs, considered inputs, are not reflected in the GGGI. Output variables are related to basic rights, such as education and health. The third concept is that the index ranks countries by gender equity rather than by women´s empowerment. The GGGI considers four categories: Economic Participation and Opportunity, Educational Attainment, Health and Survival, and Political Empowerment. According to the World Economic Forum, Latin America and the Caribbean have closed the gender gap by 70%. Since 2006, this region “has shown the most improvement on the overall GGGI and second-most improvement on both the Economic Participation and Opportunity and Political Empowerment sub-indexes” [2]. Fig. 1. 10 TOP-Paying Occupations in Latin America, CIRCA 2007. (Source: [4]) However, according to the Inter-American Development Bank [5], even with more education than men, women are still concentrated in lower-paid occupations. When comparing men with women of the same age and educational level, men earn 17 percent more than women. Changing household roles and stereotypes is essential to attaining gender equity in the labor market. Women´s reality is still difficult. Too many women still die in childbirth. Employment, salary and decision making gaps still exist. More needs to be done in order to enhance access to education, job opportunities, and reproductive health services. 3 Digital Divide in Latin America and the Caribbean The digital divide is rooted in the very issues that constrain Latin America’s overall economic development: income inequality, lack of infrastructure, and still-nascent technological knowledge base [6]. Note that income inequality is not necessarily related to a country´s wealth. Figure 2 depicts some statistics that highlight the heterogeneity in Latin America´s income inequality. Uruguay ranks as the most equitable country in Latin America, with the wealthiest 20 percent receiving 8 times more than the poorest 20 percent. Venezuela comes in at number two. The United States takes eleventh place -- behind Costa Rica and ahead of Colombia, and several spots below its southern neighbor Mexico. Brasil, Guatemala, and Honduras are regarded as the countries with highest inequality, and therefore, more susceptible to digital divide. Information and communication technologies (ICT) face constraints in the region, such as a poor legal framework for the development of the ICT sector, heavy administrative burdens, almost non-existing government prioritization for ICT development, low Internet penetration, rates and pervasive brain drain which undermines the potential for faster growth of the economies’ ICT sectors [8]. Uruguay Venezuela El Salvador Peru Ecuador Mexico Nicaragua Chile Argentina Costa Rica United States Colombia Panama Dominican Republic Bolivia Paraguay Brasil Guatemala Honduras Inequality index: the annual income of the richest 20% divided by the annual income of the poorest 20% 0 5 10 15 20 25 30 Fig. 2. Inequality in the Americas (Source: [7]) Despite the digital divide has been closing in Latin America and the Caribbean, improvement has been lower amongst women, youth, indigenous peoples, afrodescendants, and rural populations [9]. In order to close this gap, it is necessary to understand the problem of the digital divide. The most difficult barrier to overcome is not related to access (telecommunication infrastructure and dissemination of artifacts), but to use [10]. Opportunities created by technological innovations depend on the use made of them and how they affect the professional development and life of people. The Economic Commission for Latin America and the Caribbean (ECLAC) [11] points out that there is a lag in the ability to adopt and disseminate information technology and systems innovation in Latin America and the Caribbean. They point out that this panorama is even more discouraging given the fact that in recent years, the region´s connectivity growth has been the world´s fastest. Unequal access of different social sectors to this new technology is their concern. ECLAC warns that the risk of widening the domestic digital divide is greater within the region, than the threat of increasing the gap between the region and the developed world [11]. In particular we are interested in examining how to diminish digital divide existing amongst Latin American and the Caribbean women. 4 Gender Digital Divide Gender digital divide is a reality in many developing countries. Hilbert found that women used Internet less than men, both in Latin America and Africa [3]. Other two differences between men and women, related to having or not a job and attending an educational institution, were found. In both cases, men reach higher employment and educational attainment percentages. However, when eliminating these two facts, that is, when considering only educated and working women and men, women become more active Internet users than men. Technophobic arguments, such as thinking that women have a negative attitude toward ICTs, are no longer valid [3] [12]. However, there are gender specific structural inequalities that constitute barriers for women´s access to ICTs. According to [10], they are education, traditional cultural beliefs and practices, as well as economic inequality. The gender digital divide can only be overcome reaching gender equality and gender equity. According to the International Fund for Agricultural Development, gender equality “means that women and men have equal opportunities, or life chances, to access and control socially valued goods and resources. This does not mean that the goal is that women and men become the same, but it does mean that we will work towards women's and men's equal life chances [13].” On the other hand, gender equity “means fairness of treatment for women and men, according to their respective needs. This may include equal treatment or treatment that is different but which is considered equivalent in terms of rights, benefits, obligations and opportunities [13].” We believe gender equality is the basis upon which gender equity can be sought. 5 Towards Gender Equity: Types of Projects First, we should be aware that ICTs uses are not gender neutral, even when producers´ goal is to sell technology with the least variation in order to lower costs. For example, in a study of How Women and Men Use the Internet [14], Deborah Fallows identified several gender differences: (1) Men are more interested than women en technology and are more tech savvy. (2) Moreover, significantly more men than women: maintain and fix their own computers, are more likely to try new gadgets and applications and software, and are more confident in themselves as searchers and geeks, and pursue and consume information online more aggressively. (3) Women are more enthusiastic online communicators, use email in a more robust way, and are more concerned about general criminal use of the internet than men. Understanding ICTs and their potential for the empowerment of women is necessary in order to overcome the gender digital divide. Empowering women will give them the capacity to cope with life requirements more efficiently and to transform life conditions. Initiatives for building these two capacities exist and have to be encouraged [10]. In order to build the capacity of coping with life requirements, it is necessary to provide women with the skills that will give them self confidence to use technology. Projects such as TIC-as (ICT-as), under the leadership of the cooperative Sulá Batsú [14], can help to achieve this goal, integrating more women in rural areas to ICTs. Women need to get basic knowledge for using word processors and spreadsheets and navigating in the web. They can also use technology in order to learn about other fields, such as agriculture, legislation, negotiation, marketing, and finance. This can help them to find a job, or a better job, or even, start their own business. Attracting women to ICT studies is also important because it can help women transform life conditions. ICT is not gender neutral [15]. There are differences be- tween men and women on hardware and software requirements, desired interaction modes, use contexts, and acquisition capacity. Therefore, increasing the participation of women in ICT design and development can help to promote gender digital equity, create awareness of the importance of including gender issues when designing and developing technology, destroy the public perception that ICT is a male domain, ensure a competitive workforce, and enhance the quality of women in general [16]. Initiatives such as Meninas Digitais (digital girls) in Brazil [17] and Work Intel Network (WIN) [18] aim primarily at students of high school and technological education, to motivate them to follow careers in ICT. The robotic program of the Fundación Omar Dengo in Costa Rica is designed to develop creativity and innovation skills in children creating and programming robots [19]. It is interesting to note that most initiatives to empower women and to reduce gender digital divide are women driven, and do not correspond to structured public policies. 6 Typifying Mechanisms for Reaching Gender Digital Equity Mechanisms for reaching digital equity must attack several dimensions, since it is not only an economical issue. Several mechanisms have been proposed and implemented. We present a characterization of mechanisms based on the degree of ICT appropriation that women can reach. The first category is providing women with access to technology. This seems simple, but the effect is not necessarily the expected. Women who have not received training on computers can feel frustrated. If the computer is at home and available for all the members of the family, there is no warranty that women will use it. Women could think that the computer is for her husband and kids, and that it is not worth using it [20]. Access can also be provided through public computer centers, but some women may not visit these places by themselves. If there is a charge for using a computer, women with low income can hardly afford it, and if they can, probably they will find other priorities to invest the money, forexample, to enhance the quality of living of her children. Moreover, moving to a public computer center may not be possible for some women who have to take care of other members of the family. Access to mobile telephones plays an important role, not only because they are low cost devices, but because they are for personal use (not for family use), and can help overcome the impossibility of moving to a public computer center. The second category is allowing women to learn how to use standard software tools (for example, word processing and spreadsheets). This can be a social activity, so that a group of women can learn together and support amongst themselves. However, these software tools may not be useful enough to make women enthusiastic TIC users. Additionally, as stated before, women may not be able to leave their house and move to the training center, because of distance, cost, or obligation of taking care of someone else. The third category is empowering women through enabling women to learn how to use ICTs to take control of their lives. TICs can be tools for improving life con- ditions. Women must be empowered such that they can find out how technology can help them improve their own quality of life: for example, help them control domestic expenses, sell products they produce, increase their income, find employment opportunities, or learn about topics they need to know, such as health, child care and nutrition, among others [3]. Even more importantly, ICTs can help women increase their self esteem. ICTs can become channels to reunite women groups to fight for their own rights. Evaluation methodologies are required in order to determine whether an ICT project really improves women´s social conditions and promotes a positive change at personal, institutional, community, and social levels. For example, the Gender Evaluation Methodology (GEM), developed by the Association for Progressive Communications (APC), has been used in many initiatives since 2002 [21]. GEM, created with the participation of experts on ICT development in 25 countries from Latin America, Asia, Africa, and Central and East Europe, is useful for determining what role ICTs play in changes that empower women and how this changes shift gender relations between women and men. Methodologies like GEM provide policy makers and the ICT community with feedback that can help them improve the social effects of ICT projects [21]. Moreover, when men and women share the learning experience and work together, in an environment without discrimination in which both have the same participation opportunities, men can change their opinion about women´s ability to use computers. This is very important because respect and power relations can become more favorable for women. In Latin America, more than 300 community telecenters have been installed [12]. At these places, people can learn about ICTs and how to use them to promote community development. People, from very different backgrounds, work at these telecenters. The Women´s Networking Support Programme (WNSP), of the APC, is “a network of women throughout the world committed to using technology for women’s empowerment” [22]. WNSP experiences in telecenters show that the diversity of technological solutions and innovation are promoted when men and women participate in an equitable environment [12]. Previous three categories to pursue gender digital equity have been studied and promoted in most countries. We want to highlight the importance of higher female representation in ICT related jobs as a means to enhance gender digital equity. It constitutes our forth category of mechanisms: creating gender consciousness in ICT design and construction, and attracting women to the ICT field. Women have special gender needs that will hardly be considered if they do not participate in ICT´s design and implementation. An interesting example of gender consciousness is that Hewlett-Packard (HP) discovered that quilters used HP ink-jet printers to transfer patterns onto cloth [23]. This is a female need that HP seriously considered and developed custom quilting software. This decision was taken due to the presence of a significant number of women at HP. Women in the ICT field should become aware of situations such like the shown in this example. Many women in the world are owners of small businesses which could take advantage of ICTs. Paying more attention to their needs could help ICT developers, or women themselves,find new business opportunities which would improve female living and working conditions. Women on ICT jobs would be the voice of many unheard women and would help achieve gender digital equity. Attracting women to ICT college careers and empowering them to play a proactive role in the professional field is a challenge. Numbers show that the percentage of women studying ICT careers is very low and it is even decreasing not only in our Region [24]. Consciousness on this fact has been obtained but researchers and university department authorities had not succeeded in attracting more women. Figure 3 evidence the actors, and the means they normally use, to implement the four mechanisms identified to enhance gender digital equity. Most proposals to diminish gender digital divide concentrate on the first three mechanisms. However, we believe the fourth mechanism, creating gender consciousness in ICT design and construction, can facilitate the previous three, not only in Latin America and the Caribbean, but globally. Women can design and develop ICT technologies better suited for female needs, can develop more female friendly applications and e-learning tools to promote ICT use amongst the female community, and can help empower women. Fig. 3. Mechanisms for Reaching Gender Digital Equity 7 Recommendations and Conclusions The ICT community, which includes government, academia, non-profit organizations, and industry, can contribute to design and implement solutions for each of the four mechanism categories mentioned above, in order to contribute to reach gender digital equality and equity. For the first category, the ICT community should continue developing cost efficient devices, protocols and communication technologies, in order to warranty low cost access to women. Community access centers are a source to provide access, if schedules are designed to incorporate women´s needs, and daycare facilities for children and elderly are provided. Moreover, they must foster an environment in which women can interact with someone who can solve her doubts. Mobile technology may play an important role since they avoid the need to attend community centers. They are easy to use and low cost. Government policies can provide incentives and financial sources to supply mobile technology access to women. They can impose conditions on companies that want to operate in a country, such as obliging an operator to donate telephone connections to the lowest income population. For the second category, it is important to be aware of the limitations women can face. Developing on-line training programs may be very convenient, because women would not have to leave home, and could choose when to connect and interact with the applications. E-learning is a challenging option requiring the participation of professionals from different backgrounds. Content creation is a major issue, because training applications should be easy to use and should offer options to solve doubts. Usability is a very important feature, since women value it more than men, and may not have too much time to experiment with the application [15]. For the third category, the ICT community should understand women´s needs. These are not necessarily standard, since needs could depend on the natural and social environment. An important issue is promoting innovation and entrepreneurship among women. Introducing women to electronic commerce and social networks to sell their products is an example of how this could be achieved. Another possibility is thinking about how to introduce women in the service sector, that, in general, offers better income conditions that the manufacturing sector. Telework may become helpful to overcome limitations women face when looking for a job. Women could receive necessary job training through on-line applications. Creating online content that can help women improve their living conditions must be a cooperative activity result of the interaction of the ICT community and women. Examples of key areas of content are education, health, nutrition, and child and elder care. Language used has to be understandable and adapted to the natural and social environment. Women should not be only content consumers. Introducing women to peer production in any of its two modalities, commons-based and corporate-based, could help them to introduce them in the creation of information (e.g., financial services, accounting) and culture (e.g., music) [25]. This would allow women to get an income and would help balance gender-power relations. Giving women the possibility of participating in community decisions related to ICT projects makes sense when they have understood the opportunities technology offers. Government policies can enforce female participation in decision making, but cultural aspects could neutralize the possible effects of these policies. Teaching women how they can assume an active role in the Internet governance, through commonsbased peer production, would help them to become more autonomous, because they could create services and information by and for themselves and could freely organize in non-hierarchical organizations [25]. The ICT community should also take advantage of international volunteer programs, in which experts in ICT volunteer as tutors in education programs. It would help to lower program costs. For example, from 2001 to 2013, Korea Internet Volunteers (KIT) has contributed with more than 900 volunteer teams, mostly formed by college professors and students, who have supported ICT education programs in 70 countries in Latin America, Africa and other continents [26]. Governments and nonprofit organizations should promote local volunteer programs, in which active and retired ICT experts could participate. ICT enterprises could establish social programs in which both the company and the employees contribute according to their capabilities. Companies could provide materials and ICT tools helpful to women, and employees could volunteer to work as instructors. However, the ICT community should understand that introducing ICT in a community is not sufficient to achieve positive social changes. Reviewing existing evaluating methodologies and promoting its use is a pending task. For the fourth category, creating gender consciousness in ICT design and construction, especially attracting women to the ICT field, is a pending task worldwide. Moreover, retaining them not only while they study, but also during their professional life, requires also our attention. Their presence in classrooms and working places can contribute to get higher levels of innovation and competitiveness. Women conceptualize a profession mostly based on the opinion of others and the messages they receive from the society [24] [27]. On the other hand, men make their career decision based on their personal experiences with technology, starting from toys they have played with. Therefore, attracting young women to computer-based careers requires of several changes. Young girls should have the same opportunity as boys of playing with toys that let them solve problems and design solutions. Girls should receive support from their parents and professors when they decide to study a technological college career. The ICT community can develop promoting programs showing what ICT professionals do, highlighting the opportunities that ICT offer to improve people´s lives and promoting professional development. Media can highlight the achievements of women in technological fields, which would help to change stereotypes that keep young girls away from ICT careers and would provide them with models to follow. Universities can play a more proactive role in recruiting ICT students. Both female and male professors can visit primary and high schools to talk about possible professional development paths and to resolve the doubts students could have. Some changes on Computer Science curricula can also be beneficial. Changing the program of the career introductory course from a technological approach to an ICT potential impact approach can help women to be sure they made the right decision when choosing career. A mentoring system during the first college year, in which women are empowered and motivated to express their needs and opinions and their self esteem is increased, could help retain them in the ICT field. Promoting a non-discrimination study environment and working on teams in which at least a woman is always present might help male classmates understand that they get better technological solutions when both sexes cooperate. The ICT community must become aware of the importance of designing technology considering gender differences. The field of gender human-computer interaction has contributed to understand that women and men use computers for different purposes and goals [15], due to differences in their personal interests and in the roles both sexes play within their families and communities. Physical differences (e.g., in weight, height, and strength) should also be considered, in order to avoid personal damage and fatigue. However, there are no general rules that can be applied when identifying special needs of men and women, because human beings are complex and their motivations and interests sometimes can seem contrary to what is expected. If the aforementioned mechanisms for empowering women and helping them empower themselves are effective, more Latin American women could become business owners, new needs may emerge, and ICT women may think about them more seriously than men would. It is difficult to do research to prove the possible positive impacts of (1) gender conscious technology design, and (2) greater presence of women in the ICT field, on gender digital equity. Cause effect factors on long term societal phenomena are very hard to measure. We invite the international community to try to propose mechanisms to address this issue, and help us demonstrate the impact of our proposed mechanism. Finally, to foster gender digital equity, women in ICT related jobs should not limit their activities to designing and building technology. They should also have the opportunity of participating in the definition of related policies, making sure that regulations and incentives necessary to motivate and facilitate the creation and use of applications favoring women, are created and implemented. References 1. Socio-Economic Database for Latin America and the Caribbean. Stats. http://sedlac.econo.unlp.edu.ar/eng/statistics.php 2. The Global Gender Gap Report. World Economic Forum, Switzerland (2015) 3. Hilbert, M.: Digital gender divide or technologically empowered women in developing countries? A typical case of lies, damned lies, and statistics. Women’s Studies International Forum, 34(6), 479–489 (2011) 4. Ñopo, H.: The Paradox of Girl´s Educational Attainment. In: America´s Quarterly, issue Gender Equality: Political Backrooms, Corporate Boardrooms and Classrooms. 101 – 104 (2012) 5. Ñopo, H.: New Century, Old Disparities. Gender and Ethnic Gaps in Latin America and the Caribbean. Inter-American Development Bank, Washington D.C. (2012) 6. Marín, G.: Digital Equity and Gender Issues in Latin America, 23rd IFIP World Computer Congress (WCC 2015), http://www.wcc2015.org/WORKSHOP_ON_DIGITAL_EQUITY_speakers.php 7. The Huffington Post: U.S. Income Inequality Worse Than Many Latin American Countries http://www.huffingtonpost.com/2013/01/27/us-income-inequalitywors_n_2561123.html 8. World Economic Forum: Singapore overtakes the United States in the World Economic Forum's Global Information Technology Report, http://web.worldbank.org/archive/website00818/WEB/OTHER/SINGAP-2.HTM 9. Inter-American Development Bank.: Gender Equality in LAC and the IDB, http://www.iadb.org/en/topics/gender-indigenous-peoples-and-africandescendants/gender-equality/gender,1926.html 10. Huyer, S, Sikoska, T.: Overcoming the Gender Digital Divide: Understanding ICTs and Their Potential for Empowerment of Women. Instraw Researh Paper Series No. 1, April (2003) 11. Economic Commission for Latin America and the Caribbean: Digital Divide could Widen in Latin America, http://www.cepal.org/en/pressreleases/digital-dividecould-widen-latin-america 12. Sabanes, D.: Mujeres y Nuevas Tecnologías de la Información y la Comunicación. In: Cuadernos Internacionales de Tecnología para el Desarrollo Humano 2, Spain (2004) 13. International Fund for Agricultural Development, http://www.ifad.org/gender/glossary.htm 14. Fallows, D.: How Women and Men Use the Internet. Pew Research Center Washington, http://www.pewinternet.org/2005/12/28/how-women-and-men-use-the-internet/, (2006) 15. Sula Batsú: TIC-as La ciencia nos necesita, http://sulabatsu.com/ticas/que-estic-as/ 16. Calderón, M., Marín, G.: Tablet Use Patterns and Drivers of User Satisfaction: a Gender Approach. In: Collazos, C., Liborio, A., Rusu, C. (eds) 6th Latin American Conference, CLIHC 2013, LNCS, vol. 8278, pp 71– 78. Springer, Heidelberg (2013) 17. Ashcraft, C., Eger, E., Friend, M.: Girls in IT: The Facts. National Center for Women and Information Technology (2012) 18. Program Meninas Digitais, https://sbcmt.wordpress.com/meninasdigitais/ 19. Kass, K.: How Intel Empowers their Female Work Force. United States. Intel, https://www.simply-communicate.com/case-studies/company-profile/how-intelempowers-their-female-work-force 20. Fundación Omar Dengo: Programa de robótica educativa infantil, http://www.fod.ac.cr/robotica/index.php/red-de-roboticaeducativa/10-proyectos/14-cursos-infantiles. 21. Gorano, C., Goñi, M.: TIC para el Cambio Social: Las Mujeres Protagonistas. Intendencia Municipal de Montevideo, Montevideo, Uruguay (2007) 22. Association for Progressive Communications: GEM Gender Evaluation Methodology for Internet and ICTS. http://www.genderevaluation.net/ 23. Association for Progressive Communications: Glossary, https://www.apc.org/en/glossary/term/253 24. Speizer, I.: By Adding More Women to its Workforce, HP Sees Customer Need More Clearly. Workforce, http://www.workforce.com/articles/by-adding-morewomen-to-its-workforce-hp-sees-customer-needs-more-clearly, (2004) 25. Calderón M.E., Marín, G.: En Quién o en Qué Confían las Mujeres para Pomar la Decisión de Estudiar Computación. In: Revista Novatica 231, 13–21 (2015) 26. Yochai Benkler, La Riqueza de las Redes. Icaria Editorial, Barcelona, España (2015) 27. Red de Redes Telecentros LAC, Proyectos, http://www.telecentros.org/proyectos.php?id=2 28. Adya, M., Kaiser, K.M.: Factors Influencing Girls´ Choice of Information Technology Careers. In Encyclopedia of Gender and Information Technology. Ed. Eileen M. Trauth. Hershey: IGI Global, pp. 282–288 (2006) 

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Published for SISSA by Springer Received: November 24, 2023 Revised: January 5, 2024 Accepted: January 10, 2024 Published: January 23, 2024 Elliptic genera from classical error-correcting codes a,b and Shinichiro Yahagia a Department of Physics, Faculty of Science, The University of Tokyo, Bunkyo-Ku, Tokyo 113-0033, Japan b Department of Physics, Osaka University, Machikaneyama-Cho 1-1, Toyonaka 560-0043, Japan E-mail: kawabata-koki382@g.ecc.u-tokyo.ac.jp, yahagi@hep-th.phys.s.u-tokyo.ac.jp Abstract: We consider chiral fermionic conformal field theories constructed from classical error-correcting codes and provide a systematic way of computing their elliptic genera. We exploit the U(1) current of the N = 2 superconformal algebra to obtain the U(1)-graded partition function that is invariant under the modular transformation and the spectral flow. We demonstrate our method by constructing extremal N = 2 elliptic genera from classical codes for relatively small central charges. Also, we give near-extremal elliptic genera and decompose them into N = 2 superconformal characters. Keywords: Field Theories in Lower Dimensions, Superstrings and Heterotic Strings, Scale and Conformal Symmetries ArXiv ePrint: 2308.12592 Open Access, © The Authors. Article funded by SCOAP3 . https://doi.org/10.1007/JHEP01(2024)130 JHEP01(2024)130 Kohki Kawabata Contents 1 2 Elliptic genera and weak Jacobi forms 2.1 N = 2 superconformal symmetry 2.2 Elliptic genera as weak Jacobi forms 2.3 Extremal elliptic genera 3 4 6 9 3 Elliptic genera from classical codes 3.1 Fermionic code CFTs 3.2 Constraints on U(1) current 3.3 Elliptic genera of fermionic code CFTs 3.4 Spectral flow and chiral ring 10 10 14 16 22 4 Extremal N = 2 elliptic genera 4.1 Central charge 12 4.2 Central charge 24 25 25 27 5 Other examples 5.1 n = 12 , p = 2 5.2 n = 24 , p = 3 5.3 n = 36 , p = 3 28 28 29 31 6 Discussion 33 A N = 2 superconformal characters 35 1 Introduction Recently, the construction of conformal field theories (CFTs) from error-correcting codes has been revisited and extended to fermionic CFTs with supersymmetry. It has been widely known that error-correcting codes can be exploited to construct CFTs for a long time. The program initially began with chiral bosonic CFTs constructed from classical error-correcting codes [1–3], driven by insights from the understanding of the Monstrous moonshine [4]. Afterward, via fermionization, some of their fermionic counterparts have been shown to have supersymmetry [5–8]. These pioneering developments have inspired the direct construction of supersymmetric chiral CFTs from classical ternary codes [9] and its generalization to classical p-ary codes [10].1 The chiral fermionic CFTs, termed fermionic code CFTs, have Although this paper focuses on chiral CFTs constructed using classical error-correcting codes, there is another approach that builds nonchiral CFTs from quantum error-correcting codes [11–13]. Recently, the direction has taken significant advancement in various aspects, including the modular bootstrap program [14–16] and the relation to holographic duality [17–19]. See [20–27] for other related progress. 1 –1– JHEP01(2024)130 1 Introduction n h i TrHR (−1)F q L0 − 24 , (1.1) where q = e2πiτ , τ is the torus modulus, and L0 is the zero mode of the Virasoro generators. To compute the elliptic genus, we extend the Witten index to the partition function graded by U(1) charge J0 n h i TrHR (−1)F q L0 − 24 y J0 , (1.2) where y = e2πiz and z ∈ C. Then, we obtain the following main theorem (Theorem 3.1): if a classical code C satisfies • The Construction A lattice Λ(C) is an odd self-dual lattice of rank n ∈ 12Z. • The minimum norm of characteristic vectors in Λ(C) is n/3. the graded torus partition function (1.2) of a fermionic code CFT becomes a weak Jacobi form by taking an appropriate U(1) charge J0 . To extend torus partition functions to elliptic genera, we identify the U(1) current of the N = 2 superconformal algebra in fermionic code CFTs. Since elliptic genera are torus partition functions with the insertion of U(1) current describing the R-symmetry, we need to identify the R-symmetry in fermionic code CFTs. Fermionic code CFTs consist of free chiral bosons and have U(1) currents generated by the free bosons. We take a linear combination of them and impose some constraints to describe the R-symmetry. We see that the constraints are satisfied if one determines a linear combination by a lattice vector called a characteristic vector [29, 33, 34]. The corresponding U(1)-graded partition functions show the modular invariance and spectral flow invariance for the central charge being a multiple of 12 (Proposition 3.3). Furthermore, when satisfying an additional condition, the U(1)-graded partition functions turn out to be weak Jacobi forms (Theorem 3.1). With these observations in hand, we investigate the N = 2 structure of fermionic code CFTs. One characteristic –2– JHEP01(2024)130 demonstrated their utilities in verifying conjectured constraints on superconformal indexes [9] and searching for theories with large spectral gaps [10]. The construction of fermionic code CFTs exploits the relationship between classical error-correcting codes and Euclidean lattices. Concretely, a code can specify a lattice by the method called “Construction A” [28]. Then the lattice can be regarded as a momentum lattice in a CFT and provides a set of vertex operators in the Neveu-Schwarz (NS) sector. The Ramond (R) sector can be obtained from the shadow of the lattice, which is a set of points shifted by half of a certain vector, called the characteristic vector [29]. This paper aims to compute the elliptic genera of fermionic code CFTs. In supersymmetric quantum field theories, the Witten index [30, 31] plays an essential role in characterizing a theory through a topological invariant under continuous deformations preserving supersymmetry. For N = 2 superconformal theories, one can refine the Witten index to the elliptic genus [32] by inserting the U(1) charge. The elliptic genera are invariant under the modular transformation and the spectral flow and are described as weak Jacobi forms in number theory. For a fermionic code CFT with central charge n, the Witten index can be computed by the partition function twisted by the fermion parity (−1)F in the Ramond sector HR [9, 10] 2 Elliptic genera and weak Jacobi forms In this section, we review N = 2 supersymmetric CFTs with special attention to their elliptic genera following [36, 40, 42–47]. Section 2.1 is devoted to introducing the N = 2 superconformal algebra and its spectral flow. We also review N = 2 structures such as the Ramond ground states and chiral primary fields. Noting that the elliptic genera are invariant –3– JHEP01(2024)130 of N = 2 superconformal algebra is the spectral flow connecting a periodicity condition to a different one (e.g., the anti-periodic one to the periodic one) [35]. In the Hilbert space of a fermionic code CFT, we show that the spectral flow can be regarded as a shift of a momentum lattice by a characteristic vector, and an operator acting as the spectral flow arises as a vertex operator whose momentum is a characteristic vector. Also, we consistently identify the BPS states known as chiral primary states and show that they obey a strong constraint between their conformal weights and U(1) charges [36] in fermionic code CFTs. We demonstrate our computation of elliptic genera through the construction of extremal N = 2 elliptic genera from fermionic code CFTs. The notion of extremal CFTs was introduced to explore potential holographic duals to pure AdS3 gravity [37] (see also [38, 39]). It was generalized to theories with N = 2 supersymmetry by defining extremal elliptic genera [40], with some of them realized using actual CFTs with relatively small central charges [6, 41]. However, to our best knowledge, these extremal elliptic genera have been found independently, and there has not been a unified construction yet. In this paper, we uniformly reproduce the extremal N = 2 elliptic genera with the central charges 12, 24 in [6, 41] by employing our computation with classical codes. We also construct other elliptic genera from classical codes and interpret them as near-extremal elliptic genera, which obey a weaker condition on extremality [40]. Moreover, we decompose these elliptic genera into N = 2 superconformal characters and discuss their implications. The organization of this paper is as follows. In section 2, we review N = 2 superconformal field theories and their elliptic genera. Taking the spectral flow of importance, we explain the Ramond ground states, chiral primary states, and their bounds on conformal weights and U(1) charges. Also, we introduce elliptic genera as weak Jacobi forms and define their polar regions and extremal elliptic genera, which we will use in later sections. Section 3 is the central part of the present paper. Starting with a brief review of fermionic code CFTs, we develop the computation of their elliptic genera. We obtain partition functions that return the elliptic genera for N = 2 theories. To compute elliptic genera concretely, we explain how to choose an appropriate U(1) current in fermionic CFTs constructed from p-ary codes. Also, we identify the spectral flow operator and chiral primary states in fermionic code CFTs, and we see that, especially in the ternary case (p = 3), those expressions become much simple. In section 4, we exemplify the calculation of elliptic genera by reproducing extremal elliptic genera. We deduce classical p-ary codes that yield extremal elliptic genera for small prime p and obtain fermionic code CFTs with extremal elliptic genera for central charges 12, 24. Section 5 deals with other examples of elliptic genera. Considering some classical codes, we give their elliptic genera that admit an N = 2 character decomposition and discuss their near-extremality. In section 6, we summarize the results and their implications. Appendix A aligns representations of the N = 2 algebra and their characters. under the modular transformation and spectral flow, we describe them with characterization as weak Jacobi forms in section 2.2. In section 2.3, we introduce extremal and near-extremal elliptic genera defined in [40], which we will reproduce from classical error-correcting codes in section 4 and section 5, respectively. 2.1 N = 2 superconformal symmetry Then, the supercurrents G± (z) have charge Q = ±1 under the U(1) current J(z). For an N = 2 theory with the central charge n, these operators have the following operator product expansions (OPEs): T (z)T (w) ∼ n 2 (z − T (z)G± (w) ∼ w)4 3 2 + 2 1 T (w) + ∂T (w) , 2 (z − w) z−w G± (w) + 1 ∂G± (w) , z−w (z − J(w) ∂J(w) T (z)J(w) ∼ + , (z − w)2 z−w J(z)J(w) ∼ w)2 n 3 G± (z)G± (w) ∼ 0 , J(z)G± (w) ∼ ± , (z − w)2 2 2J(w) 2T (w) + ∂J(w) 3n G+ (z)G− (w) ∼ + + . 3 2 (z − w) (z − w) z−w G± (w) , z−w (2.2) The mode expansions of the operators are T (z) = Lm , z m+2 m∈Z X G± (z) = X G± r r z r+ 32 , J(z) = X m∈Z Jm z m+1 , (2.3) where r ∈ Z + 1/2 in the Neveu-Schwarz (NS) sector and r ∈ Z in the Ramond (R) sector. Accordingly, the commutation relations of the N = 2 algebra are n [Lm , Lk ] = (m − k) Lm+k + (m3 − m) δm+k,0 , 12  ± ± Lm , Gr = (m/2 − r) Gm+r , [Lm , Jk ] = −kJm+k ,   (2.4) ± [Jm , Jk ] = (n/3) m δm+k,0 , Jm , G ± r = ±Gm+r ,   n o n 2 1 + − Gr , Gs = 2 Lr+s + (r − s)Jr+s + r − δr+s,0 , 3 4 + − − and {G+ r , Gs } = {Gr , Gs } = 0. The N = 2 superconformal algebra has the spectral flow isomorphism [35]. This connects different sectors (e.g., the NS sector and the R sector) by twisting the boundary condition of the operators by the U(1) current J(z). Using a twist parameter θ, the spectral flow operator acting on the Hilbert space H0 is denoted by Uθ : H0 → Hθ , –4– (2.5) JHEP01(2024)130 An N = 2 superconformal theory has stress tensor T (z), two supercurrents G1 (z), G2 (z), and SO(2) ∼ = U(1) current J(z) describing the R-symmetry. It is convenient to define   √ G± (z) = G1 (z) ± i G2 (z) / 2 . (2.1) where Hθ is the Hilbert space quantized on the twisted boundary condition with the parameter θ. Under the spectral flow, the Virasoro generators and the modes of the U(1) current and the supercurrents transform by ([36]) Uθ Lm Uθ−1 = Lm + θJm + n 2 θ δm,0 , 6 n θδm,0 , 3 = G− r−θ . Uθ Jm Uθ−1 = Jm + −1 Uθ G+ = G+ r Uθ r+θ , −1 Uθ G− r Uθ (2.6) Accordingly, a state |h, Q⟩ ∈ H0 with conformal weight h and U(1) charge Q transforms into the twisted state  (2.7) For θ ∈ Z + 1/2, the spectral flow connects the NS and R sectors. Then, the NS sector and the R sector are isomorphic in N = 2 superconformal theories [35]. For θ ∈ Z, the spectral flow takes NS to NS and R to R. A supersymmetric theory has a unitarity bound on the conformal weight in the R sector. From the N = 2 algebra, we have the relation n o + G− = 2L0 − n/12 . 0 , G0 (2.8) Since the left-hand side is a positive operator, any state in the R sector must satisfy the bound h ≥ n/24. One can show that the bound is saturated (h = n/24) if and only if a state satisfies the condition G± m |ϕ̃⟩ = 0 , (m ≥ 0) . (2.9) To see this, suppose |ϕ̃⟩ satisfies h = n/24, which implies that n 2 o − + + ⟨ϕ̃| G− 0 , G0 |ϕ̃⟩ = 0 = G0 |ϕ̃⟩ + G0 |ϕ̃⟩ 2 . (2.10) − Hence we have G+ 0 |ϕ̃⟩ = G0 |ϕ̃⟩ = 0. Note that the operators Jm (m > 0), which lower the L0 eigenvalue, must annihilate a state |ϕ̃⟩ saturating h = n/24. Otherwise, the unitarity bound does not hold. Therefore, using the N = 2 commutation relation h i ± Jm , G± 0 = ±Gm , (2.11) we obtain G± m |ϕ̃⟩ = 0 for m ≥ 0. These states have the lowest energy in the R sector, so they are called the Ramond ground states. Since the NS and R sectors are isomorphic, there exist states in the NS sector, which are connected to the Ramond ground states by the spectral flow. Let us consider the spectral flow with θ = −1/2. Then, the supercurrents transform into −1 + U−1/2 G+ r U−1/2 = Gr−1/2 , −1 − U−1/2 G− r U−1/2 = Gr+1/2 . (2.12) Using these relations, the condition (2.9) for the Ramond ground states becomes   −1 ± U−1/2 G± m U−1/2 U−1/2 |ϕ̃⟩ = Gm∓1/2 |ϕ⟩ = 0 –5– (m ≥ 0) , (2.13) JHEP01(2024)130 n n Uθ |h, Q⟩ = h − θQ + θ2 , Q − θ ∈ Hθ . 6 3 where |ϕ⟩ = U−1/2 |ϕ̃⟩ is in the NS sector. Hence, the resulting state |ϕ⟩ satisfies the conditions G+ −1/2 |ϕ⟩ = 0 , + G− m+1/2 |ϕ⟩ = Gm+1/2 |ϕ⟩ = 0 (m ≥ 0) . (2.14) n o + G− 1/2 , G−1/2 |ϕ⟩ = (2L0 − J0 ) |ϕ⟩ . (2.15) As the left-hand side is a positive operator, any state in the NS sector satisfies the bound h≥ Q . 2 (2.16) Chiral primary states saturate the bound and have the conformal weight h = Q/2. Similarly, an anti-chiral primary state with charge Q has the conformal weight h = −Q/2. Additionally, we can show that any state with h = Q/2 is both chiral and primary. We can also obtain other bounds on chiral primary states from the N = 2 algebra n o + G− 3/2 , G−3/2 = 2L0 − 3J0 + 2n/3 . (2.17) Since the left-hand side is a positive operator and any chiral primary state satisfies h = Q/2, we have the inequality (2.18) h ≤ n/6 . Via the state-operator isomorphism, the operators corresponding to chiral primary states are called chiral primary fields. Using the inequalities (2.16) and (2.18), it is straightforward to see that the OPEs between chiral primary fields do not have singular terms and are welldefined without regularizations [36]. Such OPEs are closed in chiral primary fields. Hence, the space of chiral primary fields is called a chiral ring with the product structure by the operator product. In section 3.4, we identify the spectral flow operator, the Ramond grounds states, and the chiral ring in fermionic CFTs constructed from classical error-correcting codes. 2.2 Elliptic genera as weak Jacobi forms Let us consider a supersymmetric CFT with the central charge n = 6m (m ∈ Z) on the torus with the modulus τ = τ1 + iτ2 . The Witten index is defined by the Ramond sector partition function with the insertion of the fermion parity (−1)F n h n i TrHR (−1)F q L0 − 24 q̄ L̄0 − 24 , –6– (2.19) JHEP01(2024)130 These states are called chiral primary states [36], which can be obtained from the Ramond ground states by the spectral flow with θ = −1/2. In the opposite direction, the spectral flow with θ = 1/2 transforms chiral primary states into the Ramond ground states. Similarly, anti-chiral primary states are defined by only replacing G+ with G− in (2.14) and they are mapped to the Ramond ground states by the spectral flow θ = −1/2. Chiral primary states are subject to a strong constraint on the conformal weight and the U(1) charge. From the N = 2 algebra, we have the relation where q = e2πiτ . For an N = 2 superconformal theory, the Witten index can be refined to an elliptic genus using the U(1) current J(z) of the N = 2 algebra.2 Following [49], we define the elliptic genus by n h n i ZEG (τ, z) = TrHR (−1)F q L0 − 24 q̄ L̄0 − 24 y J0 , (2.20) where J0 is the zero mode of J(z) and y = e2πiz with z ∈ C. If the left- and right-moving R charges (Q, Q̄) satisfy Q − Q̄ ∈ Z for any state, we can define the fermion parity F = FL + FR in terms of the U(1) current [36, 49, 50] (2.21) In this paper, we restrict our attention to theories with integral U(1) charges. Then, the modularity properties together with the spectral flow invariance and the unitarity bound in the R sector can be summarized that the elliptic genus ZEG (τ, z) is a weak Jacobi form of weight 0 and index m [43]. Definition ([51]) A weak Jacobi form ϕ(τ, z) of weight w and index m ∈ Z satisfies the transformation laws aτ + b z ϕ , cτ + d cτ + d   2 cz w 2πim cτ +d = (cτ + d) e ϕ(τ, z + lτ + l′ ) = e−2πim(l 2 τ +2lz) a b c d ϕ(τ, z) , ! ∈ SL(2, Z) , l, l′ ∈ Z . ϕ(τ, z) , (2.22) (2.23) where q = e2πiτ , y = e2πiz and has a Fourier expansion in terms of the integer coefficients c(n, l) ϕ(τ, z) = X (2.24) c(n, l) q n y l , n≥0,l∈Z with c(n, l) = (−1)w c(n, −l). The modular transformation (2.22) is generated by the T and S transformations: T : (τ, z) → (τ + 1, z) , ϕ(τ + 1, z) = ϕ(τ, z) , 1 z S : (τ, z) → − , τ τ 1 z ϕ − , τ τ    ,  11 01 2 w 2πim zτ =τ e ϕ(τ, z) , ! 0 −1 1 0 (2.25) , ! , (2.26) where the matrices are in SL(2, Z). The shift of z (2.23) is generated by (τ, z) → (τ, z + 1) , (τ, z) → (τ, z + τ ) , ϕ(τ, z + 1) = ϕ(τ, z) , ϕ(τ, z + τ ) = e −2πim(τ +2z) (2.27) ϕ(τ, z) . (2.28) These generators are not independent since the shift by τ (2.28) can be derived by combining the S transformation (2.26) and the shift by integer (2.27). In addition, if ϕ(τ, z) can be 2 For another refinement called a mod-2 elliptic genus, see e.g. [48]. –7– JHEP01(2024)130 (−1)F = exp [iπ(J0 − J¯0 )] written as (2.24), then the T transformation (2.25) and (2.27) are automatically satisfied. The coefficients condition c(n, l) = (−1)w c(n, −l) can be derived from S 2 , i.e., (τ, z) → (τ, −z). Thus, to prove that ϕ(τ, z) is a weak Jacobi form, it is enough to check the S transformation (2.26) and the Fourier expansion (2.24). A weak Jacobi form ϕ(τ, z) can be decomposed into a set of vector-valued modular forms hµ (τ ) and theta functions θm,µ (τ, z) with µ ∈ Z/2mZ: ϕ(τ, z) = hµ (τ ) θm,µ (τ, z) . X (2.29) µ ∈ Z/2mZ hµ (τ ) = cµ (n) q n/4m , X θm,µ (τ, z) = X ql 2 /4m yl , (2.30) l∈Z l = µ mod 2m n = −µ2 mod 4m 2 with cµ (n) = (−1)2ml c( n+l 4m , l) and l = µ mod 2m. There has been an attempt to construct a Jacobi form from a prescribed set of coefficients [46] (one can refer to [52] as math literature). For our purposes, it is more convenient to use the following expression. A weak Jacobi form of even weight can also be expressed as a polynomial consisting of four generators ([40, 51]) E4 (τ ) , E6 (τ ) , ϕ0,1 (τ, z) , ϕ−2,1 (τ, z) (2.31) (w, m) = (4, 0) , (6, 0) , (0, 1) , (−2, 1) . (2.32) of weights and indices Here, E4 and E6 are the Eisenstein series E4 (τ ) = 1 + 240 E6 (τ ) = 1 − 504 ∞ X n3 q n 1 − qn n=1 ∞ X n5 q n n=1 1 − qn = 1 + 240q + 2160q 2 + O(q 3 ) , (2.33) = 1 − 504q − 16632q + O(q ) , 2 3 and ϕ0,1 and ϕ−2,1 are given by ϕ0,1 (τ, z) = 4 θ2 (τ, z)2 θ3 (τ, z)2 θ4 (τ, z)2 + + θ2 (τ, 0)2 θ3 (τ, 0)2 θ4 (τ, 0)2 ! = y + 10 + y −1 + (10y 2 − 64y + 108 − 64y −1 + 10y −2 )q + O(q 2 ) , θ1 (τ, z)2 ϕ−2,1 (τ, z) = η(τ )6 (2.34) = y − 2 + y −1 + (−2y 2 + 8y − 12 + 8y −1 − 2y −2 )q + O(q 2 ) . More explicitly, a weak Jacobi form ϕ(τ, z) of weight w ∈ 2Z and index m ∈ Z can be written as ϕ(τ, z) = X cq,r,s,t (E4 )q (E6 )r (ϕ0,1 )s (ϕ−2,1 )t , cq,r,s,t ∈ R (2.35) where the sum is taken for q, r, s, t ∈ Z≥0 such that 4q + 6r − 2t = w and s + t = m. When w = 0, the number of terms is round((m + 3)2 /12) where round(x) = ⌊x + 12 ⌋. –8– JHEP01(2024)130 where According to [40], the polar region of index m is defined by n o P (m) = (n, l) ∈ Z2 | 1 ≤ l ≤ m , 0 ≤ n , p := 4mn − l2 < 0 . (2.36) The term q n y l such that (n, l) ∈ P (m) is called the polar term. For example, the polar terms of small even m are m = 2 : y2, y m = 4 : y ,y ,y ,y 4 3 2 5 4 3 2 6 5 (2.37) · · · 4 terms, (2.38) · · · 8 terms. (2.39) A weak Jacobi form ϕ(τ, z) of weight 0 and index m is uniquely determined by the coefficients of the polar terms since the number of the polar terms of index m is greater than or equal to the number of the independent terms in (2.35) [40, 53]. 2.3 Extremal elliptic genera The NS vacuum irreducible character of the N = 2 algebra at the central charge n = 6m is n h L0 − 24 2πizJ0 ch(m) e vac (τ, z) = TrV0 q i 1 =q −m 4 1 ∞ Y (1 + yq k+ 2 )(1 + y −1 q k+ 2 ) (1 − q) (1 − q k )2 k=1 , (2.40) where V0 denotes the vacuum Hilbert space. Using spectral flow, we get the corresponding terms for the R sector as SFθ ch(m) vac X θ∈ 12 +Z := X  1 τ, z + 2  n h 1 TrV0 Uθ q L0 − 24 e2πi(z+ 2 )J0 Uθ−1 i (2.41) θ∈ 12 +Z = (−1)m (1 − q) y m ∞ Y (1 − yq k+1 )(1 − y −1 q k ) (1 − q k )2 k=1 + (Non-polar terms) . Note that only SF1/2 ch(m) vac is explicitly written since all terms from θ ̸= 1/2 are non-polar. According to [40], we define the extremal elliptic genus as the “closest” weak Jacobi form to SF1/2 ch(m) vac . More precisely, we call a weak Jacobi form ϕ(τ, z) of weight 0 and index m as the extremal elliptic genus when the coefficients of the polar terms are equal to SF1/2 ch(m) vac . (m) The polar terms of SF1/2 chvac at small even m are m = 2 : y 2 + 0y , (2.42) m = 4 : y 4 + 0(y 3 + y 2 + y) , (2.43) m = 6 : y + 0(y + y + y + y + y) + q(y − y ) 6 5 4 3 2 6 5 (2.44) where the polar terms with coefficients 0 are also shown. As already mentioned, we can uniquely determine the weak Jacobi form from these polar terms. For example, at m = 2, a –9– JHEP01(2024)130 m = 6 : y , y , y , y , y , y, qy , qy 6 · · · 2 terms, weak Jacobi form of weight 0 can be decomposed into ϕ20,1 and ϕ2−2,1 E4 as (2.35), and (2.42) is satisfied when 1 5 (m=2) Zext (τ, z) = ϕ20,1 + ϕ2−2,1 E4 6 6 (2.45) = y 2 + 22 + y −2 + O(q) . Similarly, at m = 4, (2.43) is satisfied when (m=4) Zext 1 4 1 11 67 4 ϕ + ϕ2 ϕ2 E4 + ϕ0,1 ϕ3−2,1 E6 + ϕ E2 432 0,1 8 0,1 −2,1 27 144 −2,1 4 = y 4 + 46 + y −4 + O(q) . (τ, z) = (2.46) n o P (m) = (n, l) ∈ Z2 | 1 ≤ l ≤ m , 0 ≤ n , 4mn − l2 ≤ −β . (2.47) Following [40], we call a weak Jacobi form ϕ(τ, z) of weight 0 and index m as a β-extremal elliptic genus when the coefficients of the terms in the β-truncated polar region are equal to SF1/2 ch(m) vac . Note that a β-extremal elliptic genus is not unique for general β. 3 Elliptic genera from classical codes In this section, we provide a way of computing the elliptic genera of fermionic code CFTs. Section 3.1 briefly reviews the construction of fermionic CFTs from classical p-ary codes. Choosing the U(1) current consistent with the N = 2 algebra from fermionic code CFTs in section 3.2, we obtain the U(1)-graded partition function that is invariant under the modular transformation and the spectral flow in section 3.3. Also, we identify the spectral flow operator and chiral ring of fermionic code CFTs in section 3.4. 3.1 Fermionic code CFTs In this subsection, we review the construction of fermionic CFTs from classical codes over finite fields Fp = {0, 1, · · · , p − 1} of prime order p [10] (see [9] for the ternary case (p = 3)). A p-ary linear code of length n can be defined as a subspace C of Fnp and the dimension of C is denoted by k ≤ n (see e.g. [29, 54–60] for more details). It is convenient to introduce a generator matrix G to define a linear code. A generator matrix G is a k × n matrix of rank k and generates a linear code C= n c ∈ Fnp c = x G , x ∈ Fkp o (3.1) . The inner product between elements v = (v1 , · · · , vn ), w = (w1 , · · · , wn ) ∈ Fnp is given by P the standard Euclidean product v · w = ni=1 vi wi ∈ R. Associated with the inner product, we define the dual code of C by C⊥ = n v ∈ Fnp c · v = 0 – 10 – mod p , c ∈ C o . (3.2) JHEP01(2024)130 Note that such a weak Jacobi form may not exist for general m, and even if it does, there may be no actual CFT that realizes the extremal elliptic genus. In fact, at m = 6, we cannot get (2.44) from (2.35) with any coefficients, which means the non-existence of the extremal elliptic genus. Therefore, a relaxed definition of near-extremal elliptic genus is also useful. The βtruncated polar region for β > 0 is defined by We call C self-orthogonal if C ⊂ C ⊥ . Then, the dimension of C must be k ≤ n/2. Also, we call C self-dual if C = C ⊥ . In this case, C must satisfy k = n/2, then n ∈ 2Z. For the binary case (p = 2), we can further classify self-orthogonal codes into two classes. A binary linear code C is doubly-even if any c ∈ C satisfies c · c ∈ 4Z. On the other hand, C is called singly-even if there exists an element c ∈ C such that c · c ∈ 4Z + 2. A linear code C ⊂ Fnp can construct a Euclidean lattice via Construction A [28, 29, 60] ( Λ(C) = c + pm ∈ Rn c ∈ C, m ∈ Zn √ p ) , (3.3) Λ ∗ = λ ′ ∈ Rn | λ · λ ′ ∈ Z , λ ∈ Λ . (3.4)  A lattice is called integral if Λ ⊂ Λ∗ and self-dual if Λ = Λ∗ . Additionally, for an integral lattice Λ, we call Λ even if any element λ ∈ Λ has an even norm λ · λ ∈ 2Z, and odd if there exists an element λ ∈ Λ such that λ · λ ∈ 2Z + 1. The Construction A lattice Λ(C) inherits the property of a linear code C. The following propositions are essential to constructing fermionic code CFTs. Proposition 3.1 ([10]) For an odd prime p, the Construction A lattice Λ(C) is odd self-dual if and only if a linear code C ⊂ Fnp is self-dual. Proposition 3.2 ([10]) For p = 2, the Construction A lattice Λ(C) is odd self-dual if and only if a linear code C ⊂ Fn2 is singly-even self-dual. These propositions guarantee that odd self-dual lattices can be constructed from self-dual codes for an odd prime p and singly-even self-dual codes for the binary case (p = 2). In what follows, we focus on such classes of linear codes and the corresponding odd self-dual lattices to yield fermionic CFTs. Specifically, we define the NS sector of a fermionic code CFT by using the Construction A lattice Λ(C), and the R sector by the shadow of Λ(C). To introduce the shadow of a lattice, consider an element χ ∈ Λ(C) ⊂ Rn such that χ·λ=λ·λ mod 2 , (3.5) for any λ ∈ Λ(C). Such an element χ ∈ Λ(C) is called characteristic [29, 33, 34]. A characteristic vector always exists for an odd self-dual lattice and the choice of a characteristic vector is not unique to a lattice. It is known that all the characteristic vectors have norm χ · χ = n mod 8 [33, 61]. Since self-dual codes C ⊂ Fnp exist only for n ∈ 2Z, characteristic vectors of the Construction A lattice Λ(C) have even norms: χ · χ ∈ 2Z , χ ∈ Λ(C) . (3.6) Using a characteristic vector χ ∈ Λ(C), we can define the shadow of Λ(C) by S(Λ(C)) = Λ(C) + χ χ = λ+ λ ∈ Λ(C) . 2 2  – 11 –  (3.7) JHEP01(2024)130 which has the standard Euclidean inner product denoted by ·. As in classical codes, the dual lattice of Λ ⊂ Rn is The shadow of a lattice is not a lattice since it is not closed under addition. While there are several choices for a characteristic vector, the shadow itself does not depend on the choice of a characteristic vector χ ∈ Λ(C). We construct the NS sector of a fermionic CFT from the Construction A lattice using the lattice construction of chiral CFTs (see e.g. [62, 63]).3 Let X(z) be an n-dimensional chiral boson. We fix the normalization by the OPE X(z)X(w) ∼ − log(z − w). To define the NS sector, we associate an element λ ∈ Λ(C) of the Construction A lattice to the vertex operator Vλ (z) = : eiλ·X(z) : , λ ∈ Λ(C) , (3.8) Li (z) = i ∂X i (z) , T (z) = − 1 : ∂X(z) · ∂X(z) : , 2 Vλ (z) = : eiλ·X(z) : . (3.9) Via the state-operator isomorphism, vertex operators Vλ (z) are mapped to momentum states |λ⟩, which are eigenstates of the Virasoro generator L0 with eigenvalue h = λ2 /2. Combining with the excitation by the bosonic oscillators αki , the NS sector of a fermionic code CFT is n i1 ir HNS (C) = α−k · · · α−k |λ⟩ r 1 o λ ∈ Λ(C) , r ∈ Z≥0 , (3.10) where the oscillators are in negative modes (k1 , · · · , kr > 0). The conformal weight of P i1 ir α−k · · · α−k |λ⟩ is h = λ2 /2 + rj=1 kj , which is an integer if λ2 ∈ 2Z and a half-integer r 1 if λ2 ∈ 2Z + 1. From the spin-statics theorem, such a state is bosonic when λ2 ∈ 2Z and fermionic when λ2 ∈ 2Z + 1. Thus, we can define the fermion parity in the NS sector by (−1)F = (−1)χ·λ , (3.11) where χ ∈ Λ(C) is a characteristic vector satisfying (3.5). This motivates us to divide the Construction A lattice into two parts: Λ(C) = Λ0 ∪ Λ2 where Λ0 = {λ ∈ Λ(C) | χ · λ = 0 mod 2} , Λ2 = {λ ∈ Λ(C) | χ · λ = 1 mod 2} . (3.12) On the other hand, the R sector can be defined by associating each element in the shadow S(Λ(C)) to the vertex operator χ χ Vλ+ χ (z) = : ei(λ+ 2 )·X(z) : , λ + ∈ S(Λ(C)) . (3.13) 2 2 The R sector of a fermionic code CFT is    χ χ i1 ir HR (C) = α−k · · · α λ + λ + ∈ S(Λ(C)) , r ∈ Z (3.14) ≥0 , −kr 1 2 2 where the bosonic oscillators are in negative modes (k1 , · · · , kr > 0). In the R sector, the fermion parity is well-defined only up to an overall factor. In analogy with the NS sector (3.11), i1 ir we give the fermion parity of α−k · · · α−k λ + χ2 ∈ HR (C) by r 1 χ (−1)F = (−1)χ·(λ+ 2 ) . (3.15) Recently, the classification program of chiral fermionic CFTs has developed. Up to now, the classification has been completed for central charges less than or equal to 24. See [64–66]. 3 – 12 – JHEP01(2024)130 where we omit the cocycle factor because it does not matter for our purpose. The fundamental operators in a fermionic code CFT can be given by Note that χ · (λ + χ2 ) is an integer from (3.6). Thus, we can naturally divide the shadow S(Λ(C)) into two parts: S(Λ(C)) = Λ1 ∪ Λ3 where χ Λ1 = λ + ∈ S(Λ(C)) χ · λ + 2   χ Λ3 = λ + ∈ S(Λ(C)) χ · λ + 2  χ =0 2  χ =1 2   mod 2  mod 2  , (3.16) . Let us place a fermionic code CFT on the torus with the modulus τ = τ1 + iτ2 . Then, the torus has the spacial and timelike cycles timelike : w ∼ w + 2πτ , (3.17) where w is the cylindrical coordinate. Depending on the periodicity (correspondingly, the choice of spin structures), we have the four partition functions n h i ZNS (τ ; Λ(C)) = TrHNS q L0 − 24 = h F ZN fS (τ ; Λ(C)) = TrHNS (−1) q h ZR (τ ; Λ(C)) = TrHR q n L0 − 24 i 1 η(τ )n n L0 − 24 1 η(τ )n 2 λ ∈ Λ(C) q 1 (λ+ χ )2 2 2 (3.18) , λ ∈ Λ(C) i 1 η(τ )n 1 Here, η(τ ) is the Dedekind eta function η(τ ) = q 24 can be collectively denoted by 1 η(τ )n 1 (−1)χ·λ q 2 λ , X X F L0 − 24 ZR = e (τ ; Λ(C)) = TrHR (−1) q Z α,β (τ ; Λ(C)) = 2 q 2λ , λ ∈ Λ(C) = 1 = η(τ )n n h i 1 X X χ 1 χ 2 (−1)χ·(λ+ 2 ) q 2 (λ+ 2 ) . λ ∈ Λ(C) Q∞ m=1 (1 − q χ m ). 1 These partition functions χ 2 (−1)βχ·(λ+α 2 ) q 2 (λ+α 2 ) (3.19) R : (1, 0) , (3.20) X λ ∈ Λ(C) where (α, β) corresponds to each sector by NS : (0, 0) , ′ f : (0, 1) , NS e : (1, 1) . R ′ Note that Z α,β (τ ; Λ(C)) = Z α ,β (τ ; Λ(C)) for integers α ≡ α′ , β ≡ β ′ mod 2. On the torus, any modular transformation is generated by the modular S transformation: τ → −1/τ and the modular T transformation: τ → τ + 1. Under these modular transformations, the partition functions behave as Z α,β (τ + 1; Λ(C)) = eiπ(3α 2 −1) n 12 n Z α,α+β+1 (τ ; Λ(C)) , Z α,β (−1/τ ; Λ(C)) = (−1)αβ 2 Z β,α (τ ; Λ(C)) . (3.21) The phases caused by the modular transformations can be canceled by coupling with 2n right-moving Majorana-Weyl fermions. Then, the partition functions of a fermionic code CFT show the expected modular transformation laws. – 13 – JHEP01(2024)130 spacial : w ∼ w + 2π , 3.2 Constraints on U(1) current Li (z) = i ∂X i (z) , (i = 1, 2, · · · , n) . (3.22) Let us consider a linear combination of these U(1) currents and denote it by J(z) = n X ai Li (z) , (3.23) i=1 where a = (a1 , a2 , · · · , an ) ∈ Rn . Although J(z) gives a U(1) current in a fermionic code CFT, such a choice of the coefficients does not always realize the R-symmetry. We figure out some conditions for the U(1) current to describe the R-symmetry and explicitly construct the U(1) current J(z) satisfying those constraints. The first constraint comes from the consistency with the N = 2 superconformal algebra (2.2). Since we have the OPE between a pair of the current J(z) J(z)J(w) ∼ a·a , (z − w)2 (3.24) we require J(z) to satisfy the norm condition a·a= n , 3 (3.25) where n is the central charge. Another condition comes from the relationship (2.21) between the U(1) charge Q and the fermion parity (−1)F . As shown in the previous section, both the NS and R sectors of a fermionic code CFT are built out of the vertex operators ( Vκ (z) = : eiκ·X(z) : , κ∈ Λ(C) (NS sector) S(Λ(C)) (R sector) . (3.26) Taking the OPE between the U(1) current J(z) and a vertex operator Vκ (w), we obtain J(z) Vκ (w) ∼ – 14 – a·κ . z−w (3.27) JHEP01(2024)130 We have introduced chiral fermionic CFTs constructed from classical codes using Euclidean lattices. It has been known that all the fermionic code CFTs have N = 2 supersymmetry in the ternary case (p = 3) [9]. Also, there is strong evidence for the presence of supersymmetry in a class of fermionic code CFTs in other cases (p ̸= 3) [10]. For an N = 2 supersymmetric theory, the elliptic genus can be computed by the partition function with the U(1) charge inserted. Here, the corresponding U(1) current must be consistent with the N = 2 algebra and describes the R-symmetry. We need to identify the U(1) current in fermionic code CFTs to compute their elliptic genera. This section aims to find the U(1) current by solving constraints for a U(1) current to realize the R-symmetry. By construction, a fermionic code CFT with the central charge n always has the following U(1) currents: Thus, each vertex operator Vκ (z) has the charge Q = a · κ with respect to the U(1) current J(z). On the other hand, combining (3.11) and (3.15), the fermion number of Vκ (z) is F =χ·κ mod 2 , (3.28) eπi Q = (−1)F , (3.29) which results in Q = F mod 2. From (3.27) and (3.28), the U(1) charge of Vκ (z) is given by Q = a · κ and the fermion number is F = χ · κ mod 2. Therefore, we obtain an additional constraint on the coefficients a a·κ=χ·κ mod 2 , (3.30) where κ ∈ Λ(C) for the NS sector and κ ∈ S(Λ(C)) for the R sector. In the NS sector, the constraint requires the coefficients a ∈ Rn to satisfy a·λ=χ·λ=λ·λ mod 2 λ ∈ Λ(C) , (3.31) where we used the definition of a characteristic vector χ ∈ Λ(C). This requires a ∈ Rn to be a characteristic vector of Λ(C). Since characteristic vectors constitute a coset of 2Λ(C) in Λ(C): χ + 2Λ(C) = 2S(Λ(C)) [61], the vector a can be written as a = χ + 2λ′ , λ′ ∈ Λ(C) . (3.32) On the other hand, a vertex operator in the R sector takes the form Vλ+ χ (z) where 2 λ ∈ Λ(C). In this case, the constraint becomes χ a· λ+ 2   χ =χ· λ+ 2   mod 2 , λ ∈ Λ(C) . (3.33) Assuming the constraint (3.32) in the NS sector, the constraint coming from the R sector returns χ · λ′ = 0 mod 2, from which we conclude a = χ + 2λ′ , λ ′ ∈ Λ0 . (3.34) Note that χ is a characteristic vector that fixes the fermion parity. From the above discussion, we essentially have two constraints (3.25) and (3.34) on the choice of the U(1) current. Now we propose to define the U(1) current J(z) by taking λ′ = 0 ∈ Λ0 . Accordingly, we fix the coefficients a ∈ Rn by taking the same vector as χ: a = χ. – 15 – (3.35) JHEP01(2024)130 where χ ∈ Λ(C) is a characteristic vector of Λ(C). Let us assume that the U(1) charge Q of each state is always integral (we check the consistency soon in this subsection). From (2.21), the fermion parity (−1)F is the Z2 subgroup of the R-symmetry in N = 2 theories. Then, the fermion number F and the U(1) charge Q must be related by Then, to meet the constraints, we only need to choose a characteristic vector χ ∈ Λ(C) satisfying χ · χ = n/3. The corresponding U(1) current is J(z) = n X χi Li (z) . (3.36) i=1 The Construction A lattice Λ(C) ⊂ Rn does not necessarily have a characteristic vector χ ∈ Λ(C) with χ · χ = n/3. Even if present, such a characteristic vector is not unique to Λ(C) in general. For later convenience, we define a set of characteristic vectors with norm n/3 by (3.37) Note that this set becomes empty unless n ∈ 12Z since χ · χ = n mod 8. Hence, we only have to specify an element χ ∈ Xn/3 to define the U(1) current J(z). We will see that, for the choice of the U(1) current (3.36), the U(1)-graded partition function exhibits the expected modular invariance together with the spectral flow invariance. Once we pick up a characteristic vector χ ∈ Xn/3 for defining the fermion parity, the conditions on the U(1) current are satisfied by choosing the coefficients a ∈ Rn according to (3.35). For the choice of J(z), a vertex operator Vλ (z) in the NS sector has the U(1)
charge Q = χ · λ and Vλ+ χ (z) in the R sector has the U(1) charge Q = χ · λ + χ2 . These 2 U(1) charges are integral because all the characteristic vectors of the Construction A lattice have even norms χ · χ ∈ 2Z as in (3.6). This is consistent with the assumption of (2.21) that we have used to determine the U(1) current J(z). Using the fact that the bosonic oscillators are neutral with respect to the U(1) current i1 ir J(z), the state-operator isomorphism tells us that an NS state α−k · · · α−k |λ⟩ ∈ HNS (C) r 1 has the following conformal weight and U(1) charge: h= r λ2 X + kj , 2 j=1 Q = χ · λ, (3.38) i1 ir On the other hand, a Ramond state α−k · · · α−k |λ + χ2 ⟩ ∈ HR (C) has r 1 h= 1 χ λ+ 2 2  2 + r X  kj , j=1 Q=χ· λ+ χ 2  . (3.39) Here, kj (j = 1, 2, · · · , r) are positive integers. The next section reveals that the Ramond partition function with the corresponding U(1) charge inserted shows the same modular transformation law as weak Jacobi forms. 3.3 Elliptic genera of fermionic code CFTs In this section, based on the choice of U(1) current (3.36), we compute the Ramond-Ramond partition function with the corresponding U(1) charge inserted, which can be expected to return elliptic genera for N = 2 superconformal theories. Furthermore, we show that, under the modular transformation and the spectral flow, the partition functions behave in the same manner as weak Jacobi forms. – 16 – JHEP01(2024)130 Xn/3 = { χ ∈ 2S(Λ(C)) | χ · χ = n/3 } . Consider a fermionic CFT constructed from a self-dual code C ⊂ Fnp (singly-even self-dual code for p = 2) and pick a characteristic vector χ ∈ Xn/3 . Using the U(1) current given by (3.36), we define the U(1)-graded partition function n 1 h i ZEG (τ, z ; Λ(C), χ) = TrHR e2πi (z+ 2 ) J0 q L0 − 24 , (3.40) where Jn (n ∈ Z) is the mode expansion of the U(1) current J(z) and q = e2πiτ . The conformal weight and U(1) charge of each state in the Ramond sector are given by (3.39). Then, the partition function can be written as 1 η(τ )n X χ 1 χ 2 1 e2πi (z+ 2 ) χ·(λ+ 2 ) q 2 (λ+ 2 ) , (3.41) λ ∈ Λ(C) where η(τ ) is the Dedekind eta function. For an N = 2 superconformal theory, one can expect that the graded partition function gives the elliptic genus and becomes a weak Jacobi form of weight 0 and index n/6. The following proposition guarantees that the partition function exhibits the expected modular transformation for n ∈ 12Z. Proposition 3.3 Let Λ(C) be an odd self-dual lattice of rank n ∈ 12Z and χ ∈ Xn/3 . Then, the partition function ZEG (τ, z ; Λ(C), χ) satisfies  ZEG cz 2 aτ + b z , ; Λ(C), χ = e2πim cτ +d ZEG (τ, z ; Λ(C), χ) , cτ + d cτ + d  ZEG (τ, z + lτ + l′ ; Λ(C), χ) = e−2πim(l where a b c d
2 τ +2lz) (3.42) ZEG (τ, z ; Λ(C), χ) , (3.43) ∈ SL(2, Z) and l, l′ ∈ Z, and m = n/6. Proof. The modular transformation by SL(2, Z) is generated by the modular T transformation τ → τ + 1 and S transformation τ → −1/τ . First, we consider the T transformation ZEG (τ + 1, z ; Λ(C), χ) = 1 η(τ + 1)n χ 2 X χ 1 1 χ 2 eπi(λ+ 2 ) e2πi (z+ 2 ) χ·(λ+ 2 ) q 2 (λ+ 2 ) , λ ∈ Λ(C) (3.44) where we used q → e2πi q under the T transformation. By definition of a characteristic vector χ 2 χ, the phase factor eπi(λ+ 2 ) becomes χ 2 πi πin eπi(λ+ 2 ) = eπi(λ·λ+λ·χ) · e 4 χ·χ = e 12 . (3.45) This exactly cancels an overall phase coming from the T transformation of η(τ ). Then, we conclude the invariance of ZEG (τ, z ; Λ(C), χ) under τ → τ + 1, which agrees with (3.42). Second, we consider the modular S transformation by fixing the parameters a = d = 0, b = −1, c = 1. Then it implements τ → −1/τ and z → z/τ : ZEG (−1/τ, z/τ ; Λ(C), χ) = 1 η(−1/τ )n X λ ∈ Λ(C) – 17 – z 1 χ πi χ 2 e2πi ( τ + 2 ) χ·(λ+ 2 ) e− τ (λ+ 2 ) . (3.46) JHEP01(2024)130 ZEG (τ, z ; Λ(C), χ) = Since the Fourier transform is given by fˆ(ω) = Z z Rn χ 1 χ 2 πi dn λ e−2πiλ·ω e2πi ( τ + 2 ) χ·(λ+ 2 ) e− τ (λ+ 2 ) , z2 n χ 2 1 (3.47) χ 1 = (−iτ ) 2 e2πim τ q 2 (ω− 2 ) e2πi (z+ 2 ) χ·(−ω+ 2 ) , the Poisson summation formula tells us ZEG (−1/τ, z/τ ; Λ(C), χ) = 1 η(−1/τ )n 2 2πim zτ ω ∈ Λ(C)∗ X q 1 (ω+ χ )2 2 2 (3.48) e 2πi (z+ 12 ) χ·(ω+ χ ) 2 , ω ∈ Λ(C) √ where we used χ · χ = n/3 ∈ 4Z, the modular S transformation η(−1/τ ) = −iτ η(τ ) and self-duality Λ(C)∗ = Λ(C), and we changed the variable ω → −ω. Hence, we conclude the modular S transformation (3.42). Finally, we consider the transformation (3.43) under the spectral flow: e2πim(l 2 τ +2lz) ZEG (τ, z + lτ + l′ ) = 1 η(τ )n 1 = η(τ )n X χ 1 χ 2 1 e2πi (z+ 2 ) χ·{(λ+lχ)+ 2 } q 2 {(λ+lχ)+ 2 } , λ ∈ Λ(C) X e 2πi(z+ 12 )χ·(λ+ χ ) 2 q 1 (λ+ χ )2 2 2 (3.49) , λ ∈ Λ(C) where we used χ · χ = n/3 ∈ 2Z and changed the variable λ → λ − lχ ∈ Λ(C) (l ∈ Z). (The above transformation suggests that the shift z → z + lτ can be understood as the shift of Λ(C) by lχ in terms of a lattice.) Since the above proposition only ensures the modular transformation of ZEG (τ, z ; Λ(C), χ), we need to require an additional condition to ensure that it is a weak Jacobi form. In the presence of supersymmetry, any operator in the Ramond sector must satisfy h ≥ n/24, which is a unitarity bound from the superconformal algebra. Then, from (3.40), the U(1)-graded partition function can be expanded as ZEG (τ, z ; Λ(C), χ) = X c(n, l) q n y l . (3.50) n≥0,l∈Z In this case, by combining the transformation law, we can conclude that ZEG (τ, z ; Λ(C), χ) is a weak Jacobi form. For fermionic code CFTs, the unitarity bound h ≥ n/24 in the Ramond sector can be rephrased as in the following proposition. Proposition 3.4 Let Λ(C) be an odd self-dual lattice. Then, the Ramond sector satisfies h ≥ n/24 if and only if any characteristic vector χ ∈ 2S(Λ(C)) satisfies χ · χ ≥ n/3. Proof. Let us rewrite the bound h ≥ n/24 in the Ramond sector. We choose an element χ ∈ 2S(Λ(C)). Then, from (3.39), the unitarity bound becomes 1 χ λ+ 2 2  2 – 18 – ≥ n . 24 (3.51) JHEP01(2024)130 e = η(τ )n fˆ(ω) , X Using a characteristic vector χ ∈ 2S(Λ(C)), any characteristic vector can be represented by χ′ = χ + 2λ (λ ∈ Λ(C)). Therefore, the above inequality can be written as 1 2  ′ 2 χ 2 ≥ n 24 ⇔ χ′ 2 ≥ n . 3 (3.52) Therefore, the unitarity bound in the R sector is equivalent to the bound χ · χ ≥ n/3 for any characteristic vector. Theorem 3.1 Let Λ(C) be an odd self-dual lattice of rank n ∈ 12Z and the minimum norm of characteristic vectors in Λ(C) is n/3. For an element χ ∈ Xn/3 , ZEG (τ, z ; Λ(C), χ) is a weak Jacobi form of weight 0 and index m = n/6. Under the assumption in Theorem 3.1, the partition function ZEG (τ, z ; Λ(C), χ) becomes a weak Jacobi form of weight 0 and index n/6. This is the characterization of the elliptic genus, so it is expected to return the elliptic genus. In section 4, we exploit the method described above to reproduce extremal elliptic genera, which gives further evidence of the validity of our computation of elliptic genera. Note that the U(1)-graded partition function depends on the choice of a characteristic vector χ ∈ Xn/3 . Generally, there can be several inequivalent choices for χ ∈ Xn/3 , which give different weak Jacobi forms ZEG (τ, z ; Λ(C), χ). This can reflect that there can exist several ways of realizing the N = 2 superconformal symmetry depending on the choice of the U(1) current. In what follows, we separately discuss how to choose characteristic vectors in fermionic code CFTs for an odd prime p and the binary case (p = 2). Characteristic vector for odd prime p. For an odd prime p, the Construction A lattice is obtained from a self-dual code C ⊂ Fnp . Then, a characteristic vector of Λ(C) is given by the following proposition, which is an improved statement of Proposition 3.7 in [10]. Proposition 3.5 Let p be an odd prime and C ⊂ Fnp a self-dual code. Then χ ∈ Rn is a characteristic vector √ of the Construction A lattice Λ(C) if and only if χ = m/ p ∈ Λ(C) where each component mi is an odd integer for i ∈ {1, 2, · · · , n}. Proof. We explicitly prove the necessary condition (“only if”). From the construction of Λ(C), λ ∈ Λ(C) can be written as λ = √1p s, s ∈ Zn . Since Λ(C) is self-dual and p is odd, λ · λ is an integer and λ · λ ≡ pλ · λ = s · s ≡ n X si mod 2 . (3.53) i=1 On the other hand, if χ is a characteristic vector, χ · λ must be an integer, which means that √ χ ∈ Λ(C). Then χ can be written as χ = m/ p , m ∈ Zn and χ · λ ≡ pχ · λ = n X mi s i i=1 – 19 – mod 2 . (3.54) JHEP01(2024)130 Therefore, if one assumes that the minimum norm of characteristic vectors is n/3, then the U(1)-graded partition function ZEG (τ, z ; Λ(C), χ) has a Fourier expansion (3.50). Combining Proposition 3.3, we arrive at the following theorem. Since any Λ(C) contains vectors with one non-zero element such as λ = definition of characteristic vector: ∀λ ∈ Λ(C) , λ · λ = χ · λ √1 (p, 0, · · · p mod 2 , 0), the (3.55) is satisfied if and only if all mi are odd. ZEG (τ, z; Λ(C), χ) = 1 η(τ )n χ 1 X 1 χ 2 e2πi(z+ 2 )χ·(λ+ 2 ) q 2 (λ+ 2 ) , λ ∈ Λ(C) (3.56) 1 = WC ({fa (τ, z)}) , η(τ )n Here, the complete weight enumerator of C ⊂ Fn3 is defined by WC ({xa }) = X Y (3.57) a (c) xwt , a c ∈ C a ∈ F3 where wta (c) = |{i | ci = a}|, and fa (τ, z) = X 1 a 1 3 a 1 2 e2πi(z+ 2 )(m+ 3 + 6 ) q 2 (m+ 3 + 6 ) , a ∈ F3 . (3.58) m∈Z Characteristic vector for p = 2. For a binary singly-even self-dual code C ⊂ Fn2 , one can define the shadow of the code, from which we can obtain the shadow of the lattice Λ(C). We divide C into two subsets C0 and C2 consisting of doubly-even and singly-even codewords of C, respectively: C0 = {c ∈ C | c · c ∈ 4Z} , C2 = {c ∈ C | c · c ∈ 4Z + 2} , (3.59) where · is the Euclidean inner product: c · c′ = i ci c′i ∈ R. Note that C0 is a doubly-even self-orthogonal code and C0 ⊂ C ⊂ C0⊥ . The shadow S(C) of the code C is defined by ([67]) P S(C) = C0⊥ \ C . (3.60) We can relate the shadow of the code to characteristic vectors of the lattice by the following proposition. Proposition 3.6 ([61]) Let C ⊂ Fn2 be a singly-even self-dual code. Then χ ∈ Rn is a characteristic vector of Λ(C) if √ and only if χ = 2 (s + 2m) where s ∈ S(C) and m ∈ Zn . – 20 – JHEP01(2024)130 Table 1 shows characteristic vectors χ = √1p (c + pm) with χ · χ = n/3. For p > 3 (and p = 2 as we will see shortly), the number of types increases as n becomes larger. For p = 3, there is only one type of vector at any n. In particular, if C contains the codeword 1n ∈ C, √ we can take the characterstic vector χ = 1n / 3. Then, the U(1)-graded partition function can be written in terms of the complete weight enumerator of C as (a) p = 3 χ ∈ Xn/3 n 12 √1 ([±1 3 1 √ ([±1 3 1 √ ([±1 3 24 36 c∈C × 12]) ([(1 or 2) × 12]) × 24]) ([(1 or 2) × 24]) × 36]) ([(1 or 2) × 36]) χ ∈ Xn/3 n √1 ([±1 5 √1 ([±1 5 √1 ([±1 5 √1 ([±1 5 12 24 36 c∈C × 11, ±3 × 1]) ([(1 or 4) × 11, (2 or 3) × 1]) × 22, ±3 × 2]) ([(1 or 4) × 22, (2 or 3) × 2]) × 33, ±3 × 3]) ([(1 or 4) × 33, (2 or 3) × 3]) × 35, ±5 × 1]) ([0 × 1, (1 or 4) × 35]) Table 1. The characteristic vectors χ ∈ 2S(Λ(C)) ⊂ Rn with χ · χ = n/3 and the corresponding vectors in Fnp for odd prime p = 3, 5. The meaning of “corresponding” is that there exists m ∈ Zn such that χ = √1p (c + pm). For example, ([±1 × 2, 0 × 10]) refers to a vector such that two components are 1 or −1 and ten components are 0. χ ∈ Xn/3 n √ 12 24 36 2([±1 × 2, 0 × 10]) ([1 × 2, 0 × 10]) 2([±1 × 4, 0 × 20]) ([1 × 4, 0 × 20]) 2([±2 × 1, ±1 × 2, 0 × 33]) √ 2([±1 × 6, 0 × 30]) ([1 × 2, 0 × 34]) √ √ s ∈ S(C) ([1 × 6, 0 × 30]) Table 2. The characteristic vectors χ ∈ 2S(Λ(C)) ⊂ Rn with χ · χ = n/3 and the corresponding √ vectors in Fn2 . The meaning of “corresponding” is that there exists m ∈ Zn such that χ = 2(s + 2m). √ Table 2 shows characteristic vectors χ = 2(s + 2m) with χ · χ = n/3. Note that √ ([1 × 2, 0 × 34]) ∈ C at n = 36 also corresponds to the characteristic vector χ′ = 2([±1 × 2, 0 × 34]), which does not satisfy the unitarity bound from Proposition 3.4. In the rest of this subsection, we show a practical way to find shadows for specific codes. Here, we use the inclusion map ι : F2 → {0, 1} ⊂ Z to avoid some confusion. Proposition 3.7 Let C ⊂ Fn2 be a singly-even self-dual code that has an the left side is the identity matrix In/2 . Then,  n 2 × n generator matrix G such that  s = P (G1 ), . . . , P (Gn/2 ), 0, . . . , 0 ∈ Fn2 – 21 – (3.61) JHEP01(2024)130 (b) p = 5 is an element of the shadow S(C) where Gi ∈ C : the i-th row of G , ( P (c) = (3.62) 0 (c · c ∈ 4Z) , 1 (c · c ∈ 4Z + 2) . (3.63) n/2 Proof. For any codeword c = x G ∈ C, x ∈ F2 , there exists m ∈ Zn such that ι(c) = ι(x)ι(G) + 2m. Then, via mod 4 computation, we obtain ≡ (ι(x)ι(G))2 = X ι(xi )2 ι(Gi ) · ι(Gi ) + 2 i ≡2 X ι(xi )ι(xj ) ι(Gi ) · ι(Gj ) (3.64) i̸=j X ι(xi P (Gi )) . i In the last line, we used c · c ≡ 2P (c) and c · c′ ∈ 2Z for c, c′ ∈ C from the self-duality of C. On the other hand, since the left part of G is the identity matrix In/2 ,   c · s = x G · P (G1 ), . . . , P (Gn/2 ), 0, . . . , 0 = X ι(xi P (Gi )) , mod 2 . (3.65) i Thus, the equivalences c · s = 0 mod 2 ⇔ c ∈ C : doubly-even c · s = 1 mod 2 ⇔ c ∈ C : singly-even hold, which means that s ∈ S(C). From the proposition, we can read off an element of the shadow S(C) from the generator matrix G. Since the shadow of C is a coset of C in Fn2 : s + C = S(C), the whole set of S(C) can be easily obtained once we have an element s ∈ S(C). 3.4 Spectral flow and chiral ring We have shown that the U(1)-graded partition function becomes a weak Jacobi form under some assumptions. This strongly supports the existence of N = 2 supersymmetry in the corresponding fermionic code CFT. In this subsection, we consider the N = 2 structure and, more concretely, aim to identify the spectral flow and chiral rings in fermionic code CFTs. Let us consider a fermionic CFT constructed from an odd self-dual lattice Λ(C) where the minimum norm of characteristic vectors is n/3 and specify the U(1) current by (3.36) where χ ∈ Xn/3 . For the fermionic code CFT, a state with charge Q = χ · κ can be represented by ( Vκ (z) = : e iκ·X(z) :, κ∈ – 22 – Λ(C) (NS sector) , S(Λ(C)) (R sector) , (3.66) JHEP01(2024)130 c · c = (ι(x)ι(G) + 2m)2 up to neutral operators. The spectral flow by θ can be obtained by shifting the momentum κ by θχ, i.e. κ → κ − θχ. In fact, under the flow, a state Vκ (z) with h = κ2 /2 and Q = κ · χ transforms into Vκ−θχ (z) = : exp[i (κ − θχ) · X(z)] : with 1 n h′ = (κ − θχ)2 = h − θQ + θ2 , 2 6 n ′ Q = χ · (κ − θχ) = Q − θ , 3 (3.67) where we used χ · χ = n/3. This reproduces the transformation rule (2.7) of quantum numbers under the spectral flow. Therefore, we identify the spectral flow operator as (3.68) This is also consistent with the observation in the proof of Proposition 3.3: the spectral flow can be understood as the shift of Λ(C) by χ in terms of a lattice. From Proposition 3.4, the assumption that the minimum norm of characteristic vectors is n/3 implies that any operator in the R sector satisfies h ≥ n/24. In this case, we may identify the Ramond ground states as Vχ′ /2 (z) = : exp(i χ′ · X(z)/2) : , (3.69) where χ′ ∈ Xn/3 . The vertex operators have the expected conformal weight h = n/24. Furthermore, there is another unitarity bound (2.16) in the NS sector. Interestingly, the fermionic code CFT also satisfies the unitarity bound h ≥ Q/2 in the NS sector. To see this, suppose a characteristic vector χ ∈ 2S(Λ(C)) with the minimum norm. Then, any characteristic vector χ′ ∈ 2S(Λ(C)) is written in the form χ′ = χ − 2λ (λ ∈ Λ(C)). Since χ ∈ 2S(Λ(C)) is minimum: χ′ · χ′ ≥ χ · χ, we obtain λ · λ ≥ λ · χ, (3.70) which implies h ≥ Q/2 for a vertex operator Vλ (z). The bosonic oscillators increase the L0 eigenvalue and do not change the U(1) charge, so any operator in the NS sector satisfies the bound. As explained in section 2.1, the spectral flow by θ = −1/2 flows the Ramond ground states into chiral primary fields. Now we obtain V(χ+χ′ )/2 (z) = : exp[i (χ + χ′ ) · X(z)/2] : . (3.71) The conformal weight h and U(1) charge Q of the states are 1 χ + χ′ 2 h= = χ · (χ + χ′ )/4 , 2 2 Q = χ · (χ + χ′ )/2 ,   (3.72) which implies h = Q/2, reproducing the known relation between quantum numbers for chiral primary fields. Also, the conformal weight h satisfies h = χ · (χ + χ′ )/4 ≤ χ · (χ + χ)/4 = n/6 . – 23 – (3.73) JHEP01(2024)130 Uθ = : exp (−i θ χ · X(z)) : . This also agrees with the property of chiral primary fields as shown in (2.18). Let us consider the operator product expansion of two chiral primary fields χ+χ′ χ+χ′′ · 2 2 V(χ+χ′ )/2 (z) V(χ+χ′′ )/2 (w) ∼ (z − w) Vχ+(χ′ +χ′′ )/2 (w) . (3.74) Using the fact that any operator satisfies h ≥ Q/2 and chiral primary fields saturate it, in this case, we obtain χ + χ′ χ + χ′′ · ≥ 0. 2 2 (3.75)  R = ra a = χ + χ′ , χ′ ∈ Xn/3 2  . (3.76) Here, the product is ra · rb = ra+b subject to ra · rb = 0 if a · b > 0 where a = (χ + χ′ )/2 and b = (χ + χ′′ )/2. In the ternary case. For a ternary code (p = 3), the above discussion can be further simplified. In the rest of this section, we move on to the convenient frame F3 = {−1, 0, 1} using the isomorphism φ : {0, 1, 2} → {−1, 0, 1} where φ : 0 7→ 0 , 1 7→ 1 , 2 7→ −1 . (3.77) Then, a characteristic vector χ ∈ Xn/3 takes the form χ = √13 ([(±1) × n]), and the corresponding codeword is c = ([(±1) × n]) ∈ C. This type of codeword is called maximal and we denote the set of maximal codewords by Cmax . Note that a self-dual ternary code contains maximal codewords only when n ∈ 12Z. Then, a characteristic vector χ ∈ Xn/3 can be written as √ χ = c/ 3 , (3.78) c ∈ Cmax . √ Choosing a characteristic vector by χ = c/ 3 (c ∈ Cmax ), the spectral flow operator is √ Uθ = : exp(−i θ c · X(z)/ 3) : , (3.79) where θ is a twist parameter. The Ramond ground states can be identified as Vχ′ /2 (z) = : e 2 i √ 3 c′ ·X(z) :, c′ ∈ Cmax , (3.80) √ where we used χ′ = c′ / 3. Correspondingly, the chiral primary fields in the NS sector are V(χ+χ′ )/2 (z) = : e 2 i √ 3 (c+c′ )·X(z) :, (3.81) which gives the chiral ring n o R = r(c+c′ )/2 c′ ∈ Cmax . – 24 – (3.82) JHEP01(2024)130 Therefore, if one takes the limit z → w, the operator product does not vanish if and only if (χ + χ′ ) · (χ + χ′′ ) = 0. Hence the chiral ring can be written as The U(1) character valued degeneracy of the Ramond ground states is h TrR tJ0 i G± 0 =0 = ′ tχ·χ /2 = X χ′ ∈X c′ ∈C n/3 ′ X (3.83) tc·c /6 , max √ where we used χ = c/ 3. By the spectral flow, we obtain the counterpart in the NS sector called the Poincaré polynomial [36] h i P (t) := TrNS tJ0 |R = X ′ tc·(c+c )/6 = tn/6 c′ ∈Cmax X ′ tc·c /6 , (3.84) c′ ∈Cmax Extremal N = 2 elliptic genera 4 In this section, we construct extremal N = 2 superconformal theories with the central charge n = 12 and 24. Originally, bosonic and N = 1 extremal CFTs are introduced to understand the holographic duality of the pure AdS3 gravity [37].4 For N = 2 theories, extremal CFTs are defined using extremal elliptic genera [40]. The extremal N = 2 SCFTs with the central charge n = 12 and 24 have been explicitly constructed in [6, 41]. We will give an alternative systematic construction of the extremal elliptic genera from linear codes. In this and the following sections, we use the graded character defined in appendix A. Since we only consider (R) the partition function in the R sector, we abbreviate Chl;h,Q as Chl;h,Q . 4.1 Central charge 12 In this subsection, we employ our method to obtain the extremal elliptic genus with the central charge n = 12 (correspondingly, m = 2) [41]. As in (2.45), the q 0 term in the extremal elliptic genus at m = 2 is y 2 + 22 + y −2 . (4.1) From the discussion around (3.69), the corresponding lattice Λ(C) must have 24 orthogonal characteristic vectors in Xn/3 , i.e., |Xn/3 | = 24 and χ · χ′ = 0 for χ, χ′ ∈ Xn/3 , χ ̸= ±χ′ . In what follows, we search for classical codes that give the extremal elliptic genus by imposing constraints from the orthogonality of characteristic vectors. Here, we consider classical codes over Fp where p = 2, 5. When p = 2, from table 2, the shadow S(C) of the code C ⊂ F12 2 must have 6 vectors with two 1 and ten 0. In addition, from the orthogonality, all ‘1’s must be in different positions such as (1, 1, 010 ) , (02 , 1, 1, 08 ) , (04 , 1, 1, 06 ) , (06 , 1, 1, 04 ) , (08 , 1, 1, 02 ) , (010 , 1, 1) . (4.2) There have been numerous works on the relation between extremal CFTs and pure gravity. See, e.g., [68–79] for the developments. 4 – 25 – JHEP01(2024)130 where we used c · c = n. These polynomials are completely determined by the set of maximal codewords. The Poincaré polynomial is known to be closely related to the cohomology of the target manifold [36]. In this case, the code C must include the codewords as (1, 1, 1, 1, 08 ) since s − s′ ∈ C for any s, s′ ∈ S(C) and t ∈ C2 ⊂ C must be in the form like (1, 0, 1, 0, 1, 0, 1, 0, 1, 0, 1, 0) since t · s = 1 for any s ∈ S(C). In summary, the code must be generated by 1 1  1   1  1 1  1 1 1 1 1 0 1 0 0 0 0 1 1 0 0 0 0 0 0 1 0 0 0 1 0 1 0 0 0 0 0 0 1 0 0 1 0 0 1 0 0 0 0 0 0 1 0 1 0 0 0 1 0 0 0 0 0 0 1 1 0 0  0  , 0  1 0  (4.3) 3 1  1   1  1 1  1 3 4 4 4 4 1 4 3 4 4 4 1 4 4 3 4 4 1 4 4 4 3 4 1 4 4 4 4 3 1 4 1 4 4 4 1 4 4 1 4 4 1 4 4 4 1 4 1 4 4 4 4 1 1 1 1 1 1 1 1 1  4  . 4  4 4  (4.4) The Construction A lattice from either code is D+ 12 (see [29, 81] for the names of lattices). We can explicitly check that for any characteristic vector χ ∈ Λ(C) with norm n/3 = 4, the elliptic genus is ZEG (τ, z; Λ(C), χ) = y 2 + 22 + y −2 + O(q) 1 5 = ϕ20,1 + ϕ2−2,1 E4 6 6 = 23 Ch 3 ; 1 ,0 + Ch 3 ; 1 ,2 2 2 + ∞  X (4.5) 2 2 At,1 (Ch 3 ; 1 +t,1 + Ch 3 ; 1 +t,−1 ) + At,2 Ch 3 ; 1 +t,2 2 2 t=1 2 2  2 2 where At,1 = {770, 13915, . . . } and At,2 = {231, 5796, . . . } for t = {1, 2, . . . } [7]. This means that both codes with p = 2, 5 correspond to the CFT with the extremal elliptic genus. In the ternary case, we can perform further analysis using the complete weight enumerator. We consider the ternary Golay code C ⊂ F12 3 generated by 1 0  0   0  0 0  0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 2 2 2 2 2 1 0 1 2 2 1 1 1 0 1 2 2 1 2 1 0 1 2 1 2 2 1 0 1 1 1  2  , 2  1 0  (4.6) whose corresponding lattice Λ(C) is D+ 12 . The complete weight enumerator is 12 12 6 6 6 6 6 6 3 3 6 3 6 3 6 3 3 WC (x0 , x1 , x2 ) = x12 0 + x1 + x2 + 22(x0 x1 + x0 x2 + x1 x2 ) + 220(x0 x1 x2 + x0 x1 x2 + x0 x1 x2 ) . (4.7) – 26 – JHEP01(2024)130 up to permutations of the columns. When p = 5, from table 1, the code C ⊂ F12 5 must have 24 codewords with eleven 1 or 4 and one 2 or 3. All self-dual codes over F5 with length 12 are known [80] and there are 3 such codes up to permutations, all satisfying the orthogonality. One of them is generated by The code contains the codeword 1 = (1, . . . , 1) ∈ C and thus we can take the characteristic √ vector χ = 1/ 3. From (3.56), it can be verified that the q 0 term of the elliptic genus 6 6 12 ZEG (τ, z; Λ(C), χ) is y 2 + 22 + y −2 , which comes from x12 0 + 22x0 x2 + x2 . 4.2 Central charge 24 In this subsection, we obtain the extremal elliptic genus with the central charge n = 24 (correspondingly, m = 4) [6]. As in (2.46), the q 0 term in the extremal elliptic genus at m = 4 is y 4 + 46 + y −4 . (4.8) (m=4) SF1/2 Zext  1 τ, z − 2  1 = q −1 + 24 + q 2 (2048y + 2048y −1 ) + O(q) . (4.9) i |0⟩, thus the The terms q −1 and 24 come from the vacuum |0⟩ and its descendants α−1 1 minimum norm of the lattice Λ(C) must be 3, which corresponds to q 2 . There is only one such odd self-dual lattice: the odd Leech lattice [82]. In the ternary case, the odd Leech lattice can be constructed from the code with the minimum Hamming weight 9. There are two such self-dual codes and one is generated by 1  0  0  0  0  0   0  0  0  0   0 0  0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 1 2 0 1 2 2 0 2 2 2 1 0 1 2 0 0 2 0 2 1 2 0 1 2 0 2 1 0 2 2 1 0 2 1 1 0 0 0 2 1 1 2 1 0 1 1 1 2 2 1 2 1 0 1 2 2 1 1 2 1 2 0 2 2 2 1 1 1 1 2 2 1 0 0 0 2 1 2 0 2 1 0 0 1 1 1 1 2 2 1 1 0 0 2 1 1 1 1 2 0 2 2 1 2 1 1 0 0 1 1 0 1 1 1 2 0 2 0 2 0 2 0 2 1 1 1 0 2 2 1 0   2  0  1  2  , 0  1  2  0   2 2  which is modified from the top matrix for F3 , n = 24, d = 9 in [80]. – 27 – (4.10) JHEP01(2024)130 From the discussion around (3.69), the corresponding lattice Λ(C) must have 48 orthogonal characteristic vectors in Xn/3 . As in the previous case, we would like to find classical codes that return the extremal elliptic genus. Below we consider classical codes over Fp where p = 2, 3. When p = 2, from table 2, we have to choose the U(1) current such as J(z) = χ · √ L(z), χ = 2 (1, 1, 1, 1, 020 ). However, in this case, s = √12 (−1, 1, 1, 1, 020 ) is an element of S(Λ(C)) and χ · s = 2, thus Vs (z) corresponds to q 0 y 2 in the elliptic genus. The existence of this term is inconsistent with y 4 + 46 + y −4 , which means that the extremal elliptic genus cannot be constructed from classical codes over F2 . When p = 3, from table 1, the code C ⊂ F24 3 must have 48 codewords consisting of only 1 and 2. In addition, from the orthogonality, if we choose χ ≡ √13 c, c ∈ C, then 46 codewords except for ±c must have 12 components with the same value as c (both 1 or both 2) and 12 components with different values (one is 1 and the other is 2). In this case, it is also useful to consider the NS sector. The corresponding partition function to the extremal elliptic genus is √ For any characteristic vector χ ∈ Λ(C) with norm n/3 = 8 such as χ = 1/ 3, the elliptic genus is ZEG (τ, z; Λ(C), χ) = y 4 + 46 + y −4 + O(q) 1 4 1 11 67 4 = ϕ + ϕ2 ϕ2 E4 + ϕ0,1 ϕ3−2,1 E6 + ϕ E2 432 0,1 8 0,1 −2,1 27 144 −2,1 4 = 47 Ch 7 ;1,0 + Ch 7 ;1,4 2 + (4.11) 2 ∞ X 3 X t=1 k=1 ! At,k (Ch 7 ;1+t,k + Ch 7 ;1+t,−k ) + At,4 Ch 7 ;1+t,4 2 2 2 5 Other examples In the previous section, we constructed the extremal elliptic genera. In this section, we give other examples of the elliptic genera, especially near-extremal elliptic genera, which are slightly modified in the polar regions from extremal ones as in (2.47). By Theorem 3.1, for the partition function ZEG (τ, z ; Λ(C), χ) to be a weak Jacobi form, the minimum norm of characteristic vectors in Λ(C) ⊂ Rn must be n/3. From the fact that χ · χ = n mod 8 for any characteristic vector χ, if n = 12, 24, it is sufficient to verify the existence of a characteristic vector with norm n/3, i.e., Xn/3 ̸= ∅. In terms of codes, this is equivalent to the existence of the codewords listed in table 1 and 2. If n = 36 or greater, there may be characteristic vectors with a norm less than n/3. Therefore, we should choose a code C whose corresponding lattice Λ(C) does not contain such vectors. In the ternary case, the minimum norm of the characteristic vectors is always greater than or equal to n/3 from Proposition 3.5, thus at any n ∈ 12Z the conditions are summarized as the existence of the maximal codewords (consisting of only 1 and 2), i.e., Cmax ̸= ∅. 5.1 n = 12 , p = 2 4 12 In dimension n = 12, there are only 3 odd self-dual lattices: D+ 12 , E8 ⊕ Z , and Z . As + we have seen in section 4.1, D12 corresponds to the CFT with the extremal elliptic genus. Since Z12 is trivial, we consider the case E8 ⊕ Z4 . For a code C ⊂ F12 2 generated by 1 0  0   0  0 0  1 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 1 – 28 – 0 0 0 1 1 1 0 0 1 0 1 1 0 0 1 1 0 1 0 0  1  , 1  1 0  (5.1) JHEP01(2024)130 where At,1 = {32890, 2969208, . . . }, At,2 = {14168, 1659174, . . . }, At,3 = {2024, 485001, . . . }, and At,4 = {23, 61984, . . . } for t = {1, 2, . . . }. Therefore, the CFT constructed from this code certainly has the extremal elliptic genus. √ For χ = 1/ 3, from (3.56), the terms y 4 + 46 + y −4 in the elliptic genus come from 12 12 24 x24 0 + 46 x0 x2 + x2 in the complete weight enumerator. the Construction A lattice Λ(C) is E8 ⊕ Z4 . We can easily verify that the first four columns correspond to Z4 and the rest to E8 . For any characteristic vector χ ∈ Λ(C) with norm n/3 = 4 such as χ= √ 2 (1, 0, 1, 0, 0, 0, 0, 0, 0, 0, 0, 0) , (5.2) the elliptic genus is = ϕ2−2,1 E4 (5.3) = 7 Ch 3 ; 1 ,0 + 4(Ch 3 ; 1 ,1 + Ch 3 ; 1 ,−1 ) + Ch 3 ; 1 ,2 2 2 + ∞  X 2 2 2 2 2 2 At,1 (Ch 3 ; 1 +t,1 + Ch 3 ; 1 +t,−1 ) + At,2 Ch 3 ; 1 +t,2 2 2 t=1 2 2  2 2 where At,1 = {750, 13875, . . . } and At,2 = {255, 5868, . . . } for t = {1, 2, . . . }. This is not extremal but a β-extremal elliptic genus with β > 1 since it includes the term q 0 y. 5.2 n = 24 , p = 3 We consider a code C ⊂ F24 3 generated by 1  0  0  0  0  0   0  0  0  0   0 0  0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 2 2 0 2 1 1 1 2 2 1 1 1 1 2 2 0 2 2 2 1 2 1 1 0 0 1 1 1 0 2 2 0 2 1 2 0 0 0 0 2 2 2 1 1 2 2 1 1 2 2 2 2 1 2 0 2 1 0 0 2 0 2 0 0 2 0 0 1 2 2 0 2 0 1 1 1 2 0 2 2 0 0 2 2 1 1 2 0 1 0 2 1 0 0 1 0 2 2 0 1 0 0 0 0 1 1 0 1 1 0 1 1 2 2 1 0 0 2 2 1 2 0 2 0 0 2 1 1 0 2 1   1  1  0  2  , 1  0  1  0   0 2  (5.4) which is the matrix for F3 , n = 24, d = 6 in [80]. In this case, the elliptic genus ZEG depends on the choice of U(1) current, i.e., characteristic vector χ ∈ Xn/3 . There are 72 characteristic vectors in Xn/3 , which are divided into two types. For 24 characteristic vectors, e.g., 1 χ1 = √ (1, 1, 1, 1, 1, −1, 1, 1, −1, 1, 1, −1, 1, −1, 1, −1, −1, −1, −1, −1, −1, 1, −1, 1) , 3 – 29 – (5.5) JHEP01(2024)130 ZEG (τ, z; Λ(C), χ) = y 2 − 4y + 6 − 4y −1 + y −2 + O(q) the elliptic genus is ZEG (τ, z; Λ(C), χ1 ) = y 4 − 24y + 22 − 24y −1 + y −4 + O(q) =− 1 4 7 41 137 4 ϕ0,1 + ϕ20,1 ϕ2−2,1 E4 + ϕ0,1 ϕ3−2,1 E6 + ϕ E2 864 48 108 288 −2,1 4 = 23 Ch 7 ;1,0 + 24(Ch 7 ;1,1 + Ch 7 ;1,−1 ) + Ch 7 ;1,4 2 + 2 ∞ X 3 X t=1 k=1 2 (5.6) 2 ! At,k (Ch 7 ;1+t,k + Ch 7 ;1+t,−k ) + At,4 Ch 7 ;1+t,4 2 2 2 1 χ2 = √ (1, 1, 1, 1, −1, 1, 1, 1, 1, 1, −1, −1, 1, 1, −1, 1, −1, −1, 1, −1, 1, 1, −1, 1) , 3 (5.7) the elliptic genus is ZEG (τ, z; Λ(C), χ2 ) = y 4 + y 2 − 12y + 44 − 12y −1 + y −2 + y −4 + O(q) = 1 4 5 83 137 4 ϕ0,1 + ϕ20,1 ϕ2−2,1 E4 + ϕ0,1 ϕ3−2,1 E6 + ϕ E2 864 36 216 288 −2,1 4 = 45 Ch 7 ;1,0 + 12(Ch 7 ;1,1 + Ch 7 ;1,−1 ) + (Ch 7 ;1,2 + Ch 7 ;1,−2 ) + Ch 7 ;1,4 2 + 2 ∞ X 3 X t=1 k=1 2 2 2 2 ! At,k (Ch 7 ;1+t,k + Ch 7 ;1+t,−k ) + At,4 Ch 7 ;1+t,4 2 2 2 (5.8) where At,1 = {32922, 2969505, . . . }, At,2 = {14125, 1658880, . . . }, At,3 = {1989, 484920, . . . }, and At,4 = {45, 62120, . . . } for t = {1, 2, . . . }. The difference with the extremal one in the polar region is the terms q 0 y 2 and q 0 y, thus this is a β-extremal elliptic genus with β > 4. This means that the earlier case is nearer to the extremal elliptic genus. From the generator expansion, it can be seen that the absolute values of the coefficients 4 of ϕ0,1 are equal in both cases. It can be understood as follows. Let ϕ(τ, z) be a weak Jacobi form of weight w = 0 and index m ∈ Z. Since ϕ0,1 (τ, 0) = 12 and ϕ−2,1 (τ, 0) = 0, ϕ(τ, 0) is a constant determined by the coefficient of ϕm 0,1 . On the other hand, ZEG (τ, 0 ; Λ(C), χ) = ZR e (τ ; Λ(C), χ) and the dependence of ZR e on χ is only the overall m sign. Thus, the absolute value of the coefficient of ϕ0,1 in ZEG (τ, z ; Λ(C), χ) does not depend on the choice of characteristic vector χ. We can also observe that the coefficients of ϕ4−2,1 E42 are equal in both cases. This is not n/6 n/12 a coincidence. In what follows, we show that the coefficient of ϕ−2,1 E4 in the U(1)-graded partition function (3.41) does not depend on the choice of characteristic vectors χ ∈ Xn/3 . Let ϕ(τ, z) be a weak Jacobi form of weight w and index m ∈ Z. Substituting a = d = 0, b = −1, c = 1 and τ = i in (2.22), we get 2 ϕ(i, −iz) = iw e2πmz ϕ(i, z) . – 30 – (5.9) JHEP01(2024)130 where At,1 = {32866, 2969208, . . . }, At,2 = {14192, 1659246, . . . }, At,3 = {1976, 484929, . . . }, and At,4 = {47, 62056, . . . } for t = {1, 2, . . . }. The difference with the extremal one in the polar region is the term q 0 y, thus this is a β-extremal elliptic genus with β > 1. For 48 characteristic vectors, e.g., When z = 1 2 (1 − i), from −iz = z − 1 and (2.23), 1 1 ϕ i, (1 − i) = iw e−iπm ϕ i, (1 − i) 2 2     (5.10) . 1 1 ZEG (i, (1 − i) ; Λ(C), χ) = 2 η(i)n 1 = η(i)n X χ χ 2 eπχ·(λ+ 2 ) e−π(λ+ 2 ) λ ∈ Λ(C) X (5.11) 2 e −π(λ2 − χ4 ) . λ ∈ Λ(C) If we fix χ2 = n/3, this does not depend on the choice of χ. Combining them, we can conclude n/12 n/6 that the coefficient of E4 ϕ−2,1 in ZEG (τ, z ; Λ(C), χ) does not depend on χ. 5.3 n = 36 , p = 3 For the central charge 36, the polar terms of the extremal elliptic genus are y 6 + 0(y 5 + y 4 + y 3 + y 2 + y) + q(y 6 − y 5 ) . (5.12) However, as shown in section 2.3, the extremal elliptic genus cannot be realized as a weak Jacobi form. Moreover, it is not possible to construct the elliptic genus such that the coefficient of qy 6 is 1 from codes over any Fp since if we choose the U(1) current as J(z) = χ · L(z), χ ∈ Xn/3 , then the state |χ/2⟩ in the R sector corresponds to q 0 y 6 and its descendants i |χ/2⟩ , i = 1, . . . , 36 to 36qy 6 in the elliptic genus. α−1 From the discussion at n = 24, it is natural to expect that a code with a large minimum Hamming weight gives a near-extremal elliptic genus. In the ternary case, the largest minimum Hamming weight that a self-dual code with length n = 36 can have is known to be 12. Thus, – 31 – JHEP01(2024)130 Thus, ϕ(i, 12 (1 − i)) can be nonzero only if w + 2m ∈ 4Z. For the four generators, it follows that E6 (i) = 0 and ϕ0,1 (i, 12 (1 − i)) = 0. Then, when a weak Jacobi form ϕ(τ, z) such that w + 2m ∈ 4Z is decomposed by generators, only the term consisting of E4 (τ ) and ϕ−2,1 (τ, z), i.e. (E4 )(w+2m)/4 (ϕ−2,1 )m , can have nonzero contribution at τ = i, z = 12 (1 − i). In other words, the coefficient of such a term is determined solely by the value ϕ(i, 12 (1 − i)). On the other hand, from (3.41), the elliptic genus at that values is we consider a code C ⊂ F36 3 generated by ([n, k, d] = [36, 18, 12] code in [83]) 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 2 2 1 2 1 1 1 2 2 1 1 1 2 1 2 2 1 2 0 2 2 1 2 1 1 1 2 2 1 1 1 2 1 2 1 2 2 0 2 2 1 2 1 1 1 2 2 1 1 1 2 1 1 1 2 2 0 2 2 1 2 1 1 1 2 2 1 1 1 2 1 2 1 2 2 0 2 2 1 2 1 1 1 2 2 1 1 1 1 1 2 1 2 2 0 2 2 1 2 1 1 1 2 2 1 1 1 1 1 2 1 2 2 0 2 2 1 2 1 1 1 2 2 1 1 1 1 1 2 1 2 2 0 2 2 1 2 1 1 1 2 2 1 2 1 1 1 2 1 2 2 0 2 2 1 2 1 1 1 2 1 2 2 1 1 1 2 1 2 2 0 2 2 1 2 1 1 1 1 1 2 2 1 1 1 2 1 2 2 0 2 2 1 2 1 1 1 1 1 2 2 1 1 1 2 1 2 2 0 2 2 1 2 1 1 1 1 1 2 2 1 1 1 2 1 2 2 0 2 2 1 2 1 2 1 1 1 2 2 1 1 1 2 1 2 2 0 2 2 1 1 1 2 1 1 1 2 2 1 1 1 2 1 2 2 0 2 2 1 2 1 2 1 1 1 2 2 1 1 1 2 1 2 2 0 2 1 2 2 1 2 1 1 1 2 2 1 1 1 2 1 2 2 0 1 1 1  1   1  1  1  1   1  1 . 1  1   1  1  1  1   1  1 0  (5.13) As in the previous example, the elliptic genus ZEG depends on the choice of U(1) current, i.e., characteristic vector χ ∈ Xn/3 . There are 888 characteristic vectors in Xn/3 , which are divided into two types. For 72 characteristic vectors, the elliptic genus is ZEG (τ, z; Λ(C), χ) = y 6 − 408y + 70 − 408y −1 + y −6 + (36y 6 − 36y 5 + . . . )q + O(q 2 ) =− + 31 365 4 2 269 3 3 3071 2 4 ϕ60,1 + ϕ0,1 ϕ−2,1 E4 + ϕ0,1 ϕ−2,1 E6 + ϕ ϕ E2 124416 41472 7776 13824 0,1 −2,1 4 271 3281 6 1231 6 ϕ0,1 ϕ5−2,1 E4 E6 + ϕ−2,1 E43 + ϕ E2 648 13824 15552 −2,1 6 (5.14) = 71 Ch 11 ; 3 ,0 + 408(Ch 11 ; 3 ,1 + Ch 11 ; 3 ,−1 ) + Ch 11 ; 3 ,6 2 + 2 ∞ X 5 X t=1 k=1 2 2 2 2 2 2 ! At,k (Ch 11 ; 3 +t,k + Ch 11 ; 3 +t,−k ) + At,6 Ch 11 ; 3 +t,6 2 2 2 2 2 2 where At,1 = { 580550 , 167398422 , . . . } , At,2 = { 298452 , 104775760 , . . . } , At,3 = { 71400 , 39702684 , . . . }, At,4 = {7140, 8410920, . . . }, At,5 = {0, 843115, . . . }, and At,6 = {35, 29154, . . . } for t = {1, 2, . . . }. – 32 – JHEP01(2024)130 1 0   0  0  0  0   0  0  0  0  0   0  0  0  0   0  0 0  For 816 characteristic vectors, the elliptic genus is ZEG (τ, z; Λ(C), χ) = y 6 + 93y 2 − 36y + 628 − 36y −1 + 93y −2 + y −6 + (36y 6 − 36y 5 + . . . )q + O(q 2 ) = 241 4 2 569 3 3 3133 2 4 31 ϕ60,1 + ϕ0,1 ϕ−2,1 E4 + ϕ0,1 ϕ−2,1 E6 + ϕ ϕ E2 124416 41472 15552 13824 0,1 −2,1 4 + 2137 3281 6 631 6 ϕ0,1 ϕ5−2,1 E4 E6 + ϕ−2,1 E43 + ϕ E2 5184 13824 7776 −2,1 6 (5.15) 2 + 2 ∞ X 5 X t=1 k=1 2 2 2 2 2 2 2 2 2 2 ! At,k (Ch 11 ; 3 +t,k + Ch 11 ; 3 +t,−k ) + At,6 Ch 11 ; 3 +t,6 2 2 2 2 2 2 where At,1 = { 581480 , 167402049 , . . . } , At,2 = { 296499 , 104768320 , . . . } , At,3 = { 72795 , 39708264, . . . }, At,4 = {6768, 8409525, . . . }, At,5 = {0, 842185, . . . } and At,6 = {35, 29712, . . . }, for t = {1, 2, . . . }. They are both β-extremal elliptic genera with β > 12 since they include the term 36qy 6 . In addition, the coefficients of ϕ6−2,1 E43 and the absolute values of the coefficients of ϕ60,1 are equal respectively, as shown in the previous example. 6 Discussion In this paper, we have considered fermionic CFTs constructed from classical code over Fp [10] and given a systematic way of computing the U(1)-graded partition function that returns elliptic genera for N = 2 theories. Taking the U(1) current to be consistent with the N = 2 algebra, we have shown that the U(1)-graded partition function exhibits modular invariance and spectral flow invariance. Using some examples of classical codes, we demonstrated the unified construction of extremal N = 2 elliptic genera for central charges n = 12, 24. Also, we discussed other examples that give near-extremal elliptic genera. Except for the ternary case, the existence of N = 2 supersymmetry is not manifest in fermionic code CFTs. However, Theorem 3.1 dictates that, under some assumptions, the U(1)-graded partition function becomes a weak Jacobi form, which is invariant under the modular transformation and spectral flow. Additionally, we can consistently identify the spectral flow operator and chiral primary fields. These observations naturally lead us to the following conjecture. Conjecture 6.1 If Λ is an odd self-dual lattice of rank n ∈ 12Z and the minimum of characteristic vectors in Λ is n/3, then the fermionic CFT whose set of vertex operators is specified by Λ admits N = 2 supersymmetry. If an odd self-dual lattice can be constructed from a ternary self-dual code up to rotation, then the resulting CFT admits N = 2 supersymmetry [9]. However, as of now, we are not certain whether any lattice satisfying the assumption in Conjecture 6.1 accepts the construction from ternary codes. It would be interesting to pursue the validity of the conjecture. – 33 – JHEP01(2024)130 = 629 Ch 11 ; 3 ,0 + 36(Ch 11 ; 3 ,1 + Ch 11 ; 3 ,−1 ) + 93(Ch 11 ; 3 ,2 + Ch 11 ; 3 ,−2 ) + Ch 11 ; 3 ,6 Whereas this paper focuses on N = 2 supersymmetry in fermionic code CFTs, there is a possibility that N = 4 supersymmetry emerges in fermionic code CFTs. In this case, the R-symmetry becomes SU(2) symmetry rather than U(1). Note that, in the binary case, any fermionic code CFT contains affine SU(2) symmetry as discussed in the bosonic case [3]. Hence, the binary construction might be helpful to provide N = 4 superconformal theories. In section 4, we constructed extremal N = 2 elliptic genera for central charges n = 12 and n = 24. To construct extremal CFTs with larger central charges, we have to care about the descendants of the bosonic oscillators. Those descendants always appear in CFTs based on lattices and are obstacles to constructing extremal CFTs. One possible candidate to resolve the problem is to take the orbifold by an appropriate symmetry as in the construction of the Monster CFT [1], which is an extremal bosonic CFT. As discussed in the ternary case [9], it would be interesting to study the fermionic orbifold of code CFTs. In section 4 and 5, we computed the elliptic genera and decompose them into N = 2 superconformal characters. The Mathieu moonshine was discovered by noting the relationship between the coefficients of the characters in the elliptic genus and the dimensions of the irreducible representations [84] (see, e.g., [85] and references therein). Our construction of elliptic genera uses classical error-correcting codes, and their automorphisms (symmetries) have been studied extensively [29]. There may be an intriguing relationship between automorphisms of classical codes and coefficients of characters in elliptic genera. It would help acquire a better understanding of the moonshine phenomena. In the present paper, we computed examples of the U(1)-graded partition function with the Fourier expansion (3.50). In other words, the unitarity bound (h ≥ n/24) in the R sector holds. However, one can consider the U(1)-graded partition function whose Fourier expansion has singular terms. For example, if we take the last example in appendix B of [10], the (supersymmetric) unitarity bound (h ≥ n/24) in the R sector does not hold. However, the – 34 – JHEP01(2024)130 Although this paper deals with the construction of extremal N = 2 CFTs from classical codes, it would be interesting if one could construct extremal N = 4 CFTs, some of which have been realized in actual CFTs at small central charges [7, 40, 41]. For the central charge n = 12, it is known that extremal N = 2 and N = 4 elliptic genera are the same functions, so the fermionic code CFT constructed in section 4.1 realizes both the extremal N = 2 and N = 4 theories. For the central charge n = 24, the extremal N = 2 elliptic genus is different from the N = 4 one. To construct N = 4 extremal CFT, we attempted to find binary codes (p = 2) that return the Ramond-Ramond partition function consistent with the N = 4 extremal elliptic genus [7]. However, we could not find such binary codes from the database [80]. Also, in the ternary case (p = 3), it is straightforward to deduce from Theorem 1.3 in [9] that any ternary code cannot yield the N = 4 extremal elliptic genus. If one considers classical codes over p ≥ 5, there would be some chance to obtain the N = 4 extremal CFT. Additionally, there would be some possibilities that N = 4 extremal CFTs arise if one can generalize the construction of fermionic code CFTs to the finite rings Zk for an integer k. Recently, the construction of Narain CFTs from quantum codes over the rings has been given [13]. Analogously, we may construct fermionic CFTs from classical codes over rings. We can expect that such an extension widely broadens the scope of fermionic code CFTs and yields theories inaccessible in the present paper. Construction A lattice contains a characteristic vector with norm n/3 = 12. Therefore, we can construct the U(1)-graded partition function that shows modular invariance and spectral flow invariance while it does not admit the Fourier expansion (3.50). It would be interesting to figure out its property with the help of number theory [51, 86, 87]. Acknowledgments A N = 2 superconformal characters We list the graded characters of the representations of the N = 2 superconformal algebra. The following notation is consistent with [41] up to overall signs. For more details, see e.g. [88–91]. The elliptic genus of an N = 2 SCFT is invariant under integer spectral flows. Therefore, it should be decomposed into graded characters Ch(R) (τ, z) in the R sector defined by Ch(R) (τ, z) = X  SFθ ch(NS) τ, z + θ∈ 12 +Z 1 2  (A.1) where SFθ is defined in (2.41) and ch(NS) (τ, z) is an irreducible character in the NS sector. The characters are classified into three types: massive, massless, and graviton. Let the central charge n ∈ 6Z, l = n6 − 12 , q = e2πiτ , y = e2πiz , with h and Q representing the conformal weight and the U(1) charge, i.e., the eigenvalues of L0 and J0 . Massive. For the NS sector, the irreducible character of the massive representation with the highest weight h, Q is 1 (NS) chl;h,Q (τ, z) =q n h− 24 Q y 1 ∞ Y (1 + yq k− 2 )(1 + y −1 q k− 2 ) (1 − q k )2 k=1 . (A.2) The contribution (1 − q k )−1 = 1 + q k + q 2k + . . . comes from L−k and J−k , 1 + yq r from − −1 r G+ −r , and 1 + y q from G−r . For the R sector, the graded character of the massive representation with the highest weight h′ , Q′ is given by (R) Chl;h′ ,Q′ (τ, z) = X (NS) SFθ chl;h,Q θ∈ 12 +Z where Q n h′ = h + + , 2 24 Q′ =  1 τ, z + 2  Q + n Q + – 35 – 6 n 6  (Q′ > 0) −1 (Q′ < 0) (A.3) . (A.4) JHEP01(2024)130 We are grateful to Hee-Cheol Kim, Minsung Kim, and Yutaka Matsuo for their valuable discussions. The work of K. K. and S. Y. was supported by FoPM, WINGS Program, the University of Tokyo. The work of K. K. was supported by JSPS KAKENHI Grant-in-Aid for JSPS fellows Grant No. 23KJ0436. From (2.6), we obtain a more explicit form as (R) Chl;h′ ,Q′ (τ, z) 1 = X q n h− 24 +Qθ+ n θ2 6 (−y) Q+ n θ 3 θ∈ 12 +Z = X (1 − q k )2 k=1 n n 1 1 ∞ Y (1 − yq k− 2 +θ )(1 − y −1 q k− 2 −θ ) 1 2 1 − 2 m(m+1) q h− 24 +Q(m+ 2 )+ 6 (m+ 2 ) n 1 (−y)Q+ 3 (m+ 2 )−m m∈Z ∞ Y (1 − yq k )(1 − y −1 q k−1 ) (A.5) (1 − q k )2 k=1 ′ ′ n ′ ∞ Y (1 − yq k )(1 − y −1 q k−1 ) X (1 − q k )2 k=1 (−1)m q lm 2 +(Q′ +δ− 1 )m 2 y 2lm m∈Z where δ is 1 if Q′ < 0 and 0 if Q′ > 0. Massless. For the NS sector, the irreducible character of the massless representation with the highest weight h = − Q2 , Q is (NS) ch (τ, z) l;h=− Q ,Q 2 =q n h− 24 Q y 1 1 ∞ Y (1 + yq k− 2 )(1 + y −1 q k− 2 ) 1 (A.6) (1 − q k )2 1 1 + y −1 q 2 k=1 1 Q where 1/(1 + y −1 q 2 ) comes from G− −1/2 |h = − 2 , Q⟩ = 0. For the R sector, the graded character of the massless representation with the highest n weight h′ = 24 , Q′ is given by (R) Chl;h′ = n ,Q′ (τ, z) 24 = X θ∈ 12 +Z (NS) SFθ ch l;h=− Q ,Q 2  1 τ, z + 2  (A.7) where Q′ = Q + n6 . From a similar discussion as the massive representation, ′ (R) Chl;h′ = n ,Q′ (τ, z) = (−1)Q y Q 24 ′ ∞ Y (1 − yq k )(1 − y −1 q k−1 ) (1 − q k )2 k=1 × 1 X m∈Z 1 − y −1 q (−1)m q lm −m 2 +(Q′ − 1 )m 2 (A.8) y 2lm . Graviton. According to the equation (7.5) in [41], the graded character of the graviton n representation with the highest weight h′ = 24 , Q′ = n6 in the R sector is given by n −1 6  (R) (R) Chl;h′ = n ,Q′ = n (τ, z) = q −s Chl; n +s, n + 24 6 24 n 6 6 X    (R) (R) (−1)k Chl; n +s, n −k + Chl; n +s,k− n  24 k=1 6 24 6 (A.9) (R) + (−1) Chl; n ,0 24 where s is an arbitrary positive integer. Using these graded characters, we can decompose the elliptic genus of an N = 2 SCFT as ZEG (τ, z) = (R) A0,0 Chl; n ,0 24 + ∞ X t=0 n −1 6  X (R)  At,k Ch n k=1 l; 24 +t,k  + (R) Chl; n +t,−k 24  + At, n6 (R) Chl; n +t, n  24 6 (A.10) where At,k ∈ Z. – 36 – JHEP01(2024)130 = (−1)Q +δ q h − 24 y Q +δ Open Access. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. References [1] I.B. Frenkel, J. Lepowsky and A. Meurman, A natural representation of the Fischer-Griess monster with the modular function j as character, Proc. Nat. Acad. Sci. 81 (1984) 3256. [3] L. Dolan, P. Goddard and P. Montague, Conformal field theories, representations and lattice constructions, Commun. Math. Phys. 179 (1996) 61 [hep-th/9410029] [INSPIRE]. [4] J.H. Conway and S.P. Norton, Monstrous moonshine, Bull. London Math. 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PLOS ONE RESEARCH ARTICLE Genetic risk factors associated with gestational diabetes in a multi-ethnic population Paula Benny1, Hyeong Jun Ahn2, Janet Burlingame1, Men-Jean Lee ID1, Corrie Miller1, John Chen2, Johann Urschitz ID3* 1 Department of Obstetrics, Gynecology, and Women’s Health, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI, United States of America, 2 Department of Quantitative Health Sciences, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI, United States of America, 3 Department of Anatomy, Biochemistry and Physiology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * johann@hawaii.edu Abstract Aims OPEN ACCESS Citation: Benny P, Ahn HJ, Burlingame J, Lee M-J, Miller C, Chen J, et al. (2021) Genetic risk factors associated with gestational diabetes in a multiethnic population. PLoS ONE 16(12): e0261137. https://doi.org/10.1371/journal.pone.0261137 Editor: Carla Pegoraro, PLOS, UNITED KINGDOM Received: May 18, 2021 Accepted: November 24, 2021 Genome-wide association studies have shown an increased risk of type-2-diabetes (T2DM) in patients who carry single nucleotide polymorphisms in several genes. We investigated whether the same gene loci confer a risk for gestational diabetes mellitus (GDM) in women from Hawaii, and in particular, Pacific Islander and Filipino populations. Methods Blood was collected from 291 women with GDM and 734 matched non-diabetic controls (Pacific Islanders: 71 GDM, 197 non-diabetic controls; Filipinos: 162 GDM, 395 controls; Japanese: 58 GDM, 142 controls). Maternal DNA was used to genotype and show allele frequencies of 25 different SNPs mapped to 18 different loci. Published: December 20, 2021 Copyright: © 2021 Benny et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All raw data files have been made freely accessible and have been uploaded to a public data repository; doi:https:// data.world/pbenny/gdm-snp-analysis. Funding: Funding for this project was partially supported by NIH Awards U54MD00760131, U54GM104944, 1R21HD092716 and P30GM131944. Competing interests: The authors have declared that no competing interests exist. Results After adjusting for age, BMI, parity and gravidity by multivariable logistic regression, several SNPs showed significant associations with GDM and were ethnicity specific. In particular, SNPs rs1113132 (EXT2), rs1111875 (HHEX), rs2237892 (KCNQ1), rs2237895 (KCNQ1), rs10830963 (MTNR1B) and rs13266634 (SLC30A8) showed significant associations with GDM in Filipinos. For Japanese, SNPs rs4402960 (IGFBP2) and rs2237892 (KCNQ1) were significantly associated with GDM. For Pacific Islanders, SNPs rs10830963 (MTNR1B) and rs13266634 (SLC30A8) showed significant associations with GDM. Individually, none of the SNPs showed a consistent association with GDM across all three investigated ethnicities. Conclusion Several SNPs associated with T2DM are found to confer increased risk for GDM in a multiethnic cohort in Hawaii. PLOS ONE | https://doi.org/10.1371/journal.pone.0261137 December 20, 2021 1 / 15 PLOS ONE Genetic risk factors for type II diabetes and gestational diabetes Introduction Normal pregnancy is associated with a period of relative insulin resistance that progressively leads to maternal hyperglycemia and transplacental passage of glucose to promote fetal growth. This is similar to the type of insulin resistance found in patients with type 2 diabetes (T2DM) [1–5]. Maternal hyperglycemia usually results in an increase in insulin secretion by maternal pancreatic beta-cells [6]. However, in approximately 1 out of 15 pregnant women in the USA [7], the diagnosis of gestational diabetes (GDM) is made when this compensatory mechanism is impaired. It has been postulated that there may be an underlying baseline level of insulin resistance that predates the pregnancy and that changes of glycemic regulation associated with the secretion of human placental lactogen, also known as human chorionic somatomammotropin, from the placenta, leads to a compromise in glycemic control and the diagnosis of GDM. Maternal age, overweight/obese status and a family history of diabetes are risk factors for GDM [8, 9]. Recent studies have also described combinatorial effects of risk factors in the manifestation of GDM, together with the integration of first trimester fasting glycemia measurements [10, 11]. Fetuses of pregnant women with poorly controlled GDM are at an increased risk for macrosomia, polyhydramnios, birth injury, neonatal hypoglycemia, and other neonatal metabolic disturbances. In addition to these perinatal complications, the likelihood of long-term consequences for the health and wellbeing of both mothers and their offspring is significantly higher compared to normoglycemic pregnancies. GDM is not only associated with a higher incidence of adult obesity and the development of T2DM later in life, but also with a predisposition for cardiovascular diseases [12, 13] and metabolic disorders [14– 16]. While GDM usually resolves after delivery, the increased probability of future deterioration in insulin resistance and beta-cell insufficiency puts women who have had GDM at a greater risk of developing T2DM than those who had a normal pregnancy [17]. Women with a previous history of GDM have a 35% to 60% chance of developing diabetes in the next 10–20 years [18]. GDM prevalence rates have been reported to vary in direct proportion to the prevalence of T2DM in several ethnic populations [8]. Along with high rates of T2DM, GDM is particularly prevalent in certain minority populations including Asian, Native American, Hispanic and African-American women, who are at higher risk for GDM than non-Hispanic Caucasian women [19]. The local prevalence of T2DM in Hawaii is increasing, especially among Asian and Pacific Islander parturients. These groups have demonstrated significantly higher rates of impaired glucose tolerance (15.5%) and T2DM (20.4%) compared to the overall U.S. population [20]. This increase in T2DM in Asian and Pacific Islanders is likely to correlate with increasing number of pregnancies complicated by GDM [20, 21]. A recent report on the prevalence of GDM in Hawaii found that GDM rates were highest in Filipino women, followed by Chinese, Japanese and Native Hawaiian/Pacific Islander women. Caucasians in Hawaii had the lowest GDM rate of all ethnic groups [22]. Such differences in GDM incidences are likely to be multi-factorial and are expected to include genetic factors [23]. Several pioneering genome-wide association studies (GWAS) have identified over 60 genetic loci in genes responsible for insulin secretion, insulin resistance, lipid and glucose metabolism, and other pathways that are associated with type 2 diabetes [24–29]. While most of these studies were from European populations, more recent GWAS have identified novel loci specific to other populations such as South Asians [30], Japanese [31, 32], Mexicans [33], Chinese [34, 35], African Americans [36, 37], and Asian Indians [38]. Since women with GDM are at increased risk of T2DM and both T2DM and GDM are polygenic, multifactorial diseases, it has been suggested that both diseases are manifestations of similar PLOS ONE | https://doi.org/10.1371/journal.pone.0261137 December 20, 2021 2 / 15 PLOS ONE Genetic risk factors for type II diabetes and gestational diabetes pathophysiological processes [39]. Several studies have validated these loci to also infer an increased risk of gestational diabetes [40–43]. While these studies have examined several different ethnic populations, few prior studies have investigated the specific associations of T2DM in the multiethnic Hawaiian populations [44]. One such study to focus on the Hawaiian population is the MEC-PAGE (Multi-Ethnic Cohort Population Architecture using Genomics and Epidemiology). While this large study provided data on risk assessments of chronic diseases such as diabetes [45], colorectal [46] and prostate cancer [47] in Native Hawaiians, this was predominantly in an older population (age 45–75 years old). Another recent study focused on disease occurrence in Native Hawaiians and identified a locus on Chr6 associated with T2DM [48]. However, these loci could not be validated as the replication study size was a small, predominantly Samoan cohort. As Native Hawaiians comprise approximately 10% of the State’s population, a more accurate representation of the different populations of Hawaii is currently lacking. Therefore, in this study, we investigated whether a select panel of 25 significantly reported single nucleotide polymorphisms (SNPs) known to be associated with T2DM conferred a risk for GDM in Pacific Islander, Filipino and Japanese women in a unique cohort of pregnant women living in Hawaii. The results of this cross-sectional study would provide new knowledge on the genetic risks of GDM susceptibility occurring in the distinct populations of Hawaii. Materials and methods Subjects A case–control study was performed comparing genotype frequencies of 25 different SNPs (Table 1) in healthy pregnant women and pregnant women with GDM. Ethical approval for the study was obtained from the Institutional Review Board (IRB) of the University of Hawaii. All participants provided written informed consent. Samples were obtained from the RMATRIX Biorepository of the John A. Burns School of Medicine, University of Hawaii following the study’s inclusion/exclusion criteria. The Hawaiian Biorepository houses over 9,000 biospecimens collected from pregnant women from 2006–2012. All pregnant women had been routinely screened with a 1-hour 50-g oral glucose challenge test (GCT) at 24–28 weeks of gestation. Gestational age was determined by ultrasound examination. Individuals with blood Table 1. Clinical characteristics of participants according to ethnic group. Variable N Mean Std Dev Min Max N Mean Std Dev Min Max Filipino Maternal Age 395 30.5 5.7 18.0 44.0 162 31.7 5.5 19.0 42.0 0.029 BMI 395 24.2 4.4 15.4 43.5 155 25.9 5.2 15.9 44.6 <0.001 Gravida 395 2.8 1.7 1.0 13.0 162 2.8 1.6 1.0 8.0 0.930 Parity 395 1.2 1.1 0.0 6.0 162 1.4 1.3 0.0 5.0 0.031 Japanese Pacific Islander Control p Ethnicity GDM Maternal Age 142 35.0 4.8 19.0 45.0 58 35.8 5.0 27.0 50.0 0.300 BMI 141 22.4 3.6 15.7 43.5 51 24.9 3.8 17.7 34.5 <0.001 Gravida 142 2.5 1.5 1.0 7.0 58 3.2 1.9 1.0 7.0 0.020 Parity 142 0.9 0.9 0.0 4.0 58 0.9 1.1 0.0 4.0 0.950 Maternal Age 197 28.0 5.9 18.0 43.0 71 30.0 6.1 19.0 46.0 0.016 BMI 192 29.4 6.2 16.5 50.0 56 32.7 7.1 18.3 47.8 <0.001 Gravida 197 3.4 2.2 1.0 12.0 71 3.3 2.3 1.0 12.0 0.870 Parity 197 2.0 1.9 0.0 8.0 71 1.8 1.8 0.0 8.0 0.410 https://doi.org/10.1371/journal.pone.0261137.t001 PLOS ONE | https://doi.org/10.1371/journal.pone.0261137 December 20, 2021 3 / 15 PLOS ONE Genetic risk factors for type II diabetes and gestational diabetes glucose levels greater than 130 mg/dL underwent a 3-hour 100-g oral glucose tolerance test (OGTT) in the morning after an overnight hour fast. Pregnant women were diagnosed with GDM when 2 or more glucose values were at or above the specified Coustan-Carpenter thresholds for diagnosing gestational diabetes; fasting 95 mg/dL, 1-hour 180 mg/dL, 2-hour 155 mg/dL, and 3-hour 140 mg/dL [49]. Blood was collected from 291 women who met Coustan-Carpenter criteria for GDM and 734 non-diabetic controls (normal glucose tolerance). Only subjects, where self-reported ethnicity was concordant with that of all four biological grandparents’ ethnicity were included in this study. According to the US Census Bureau, race and ethnicity are defined as a person’s self-identification with one or more social groups [50]. Subjects may report multiple races and ethnicities based on geographical, social, and cultural affiliations associated with human civilization. Selfidentified population stratifications and geographic ancestry have been demonstrated to be highly correlated with self-identified race/ethnicity as well as a determinant of genetic structure [51]. The GDM cohort consisted of 71 Pacific Islander, 162 Filipino, and 58 Japanese women from Hawaii. The control cohort consisted of 197 Pacific Islander, 395 Filipino, and 142 Japanese women from Hawaii with normal glucose tolerance (NGT). Information regarding Height (m), weight (kg), age, BMI, ethnicity (self-reported) and GCT, OGTT results were obtained from prenatal records. Genotyping The 25 single nucleotide polymorphisms (SNPs) were chosen due to their strong association with T2DM in other ethnic groups including Caucasians/United States [25], Europeans [26] and Caucasians/Canada [27]. While these SNPs were associated with T2DM in other ethnic groups, this is the first study to evaluate the association in Native Hawaiian, Pacific Islander, Filipino and Japanese populations. Genomic DNA was extracted from whole blood using the Autopure DNA isolation system (Gentra Systems, Minneapolis, MN) following the manufacturer’s protocol. All SNPs selected for genotyping (Table 2) were candidate or GWAS-identified variants shown to be associated with T2DM or GDM. Genotyping was performed on the TaqMan OpenArray platform (Life Technologies, Foster City, CA) using 125ng of genomic DNA. TaqMan OpenArray Genotyping Mastermix was added to the DNA and this solution was then distributed across the OpenArray plates pre-loaded with TaqMan genotyping assays for 32 SNPs. Polymerase chain reaction (PCR) was performed on a Bio-Rad Slide Chambers Dual-Block Alpha unit using PCR conditions set by the manufacturer. Allelic discrimination was done on a Biotrove OpenArray System using the OpenArray SNP Genotyping Analysis Software Version 1.0.3. All SNPs were analyzed using TaqMan Genotyper Software Version 1.0.1. Quality control criteria included fingerprint blanks, which are used to identify the plate and verify cross-contamination and or sample error and no template controls (NTC), which serve as a background detector and review of any reagent problems. DNA samples that failed in more than 25% of genotyping assays were excluded, assuming poor DNA quality and uncertain measurements. A genotyping error of 0.3% was determined by blind duplicate genotyping. Automated call rates for all 25 SNPs exceeded 97%. Statistical analysis Demographic and clinical variables (age, BMI, gravidity, and parity) were summarized by descriptive statistics; means, standard deviations, minimums and maximums. Differences in patient characteristics between case and control groups for each ethnicity were examined using independent two-sample t-test and differences in patient characteristics among the three race/ethnicity groups were also examined using one-way Analysis of Variance (ANOVA). PLOS ONE | https://doi.org/10.1371/journal.pone.0261137 December 20, 2021 4 / 15 PLOS ONE Genetic risk factors for type II diabetes and gestational diabetes Table 2. Association of 25 SNPs with GDM risk. Filipino Gene SNP ADCY5 Japanese Pacific Islanders Alleles MAF HWE Alleles MAF HWE Alleles MAF HWE rs2877716 C/T 0.7 0.028 C/T 0.3 1.000 C/T 4.3 1.000 ARAP 1 rs11603334 G/A 5.5 1.000 G/A 3.4 1.000 G/A 5.5 0.415 CDKAL1 rs7754840 G/C 49.6 0.258 G/C 39.4 0.718 G/C 45.6 0.877 CDKN2A/2B rs10811661 T/C 30.8 0.285 T/C 42.4 0.300 T/C 36.4 0.754 DGKB rs2191349 T/G 24.3 0.476 T/G 31.9 0.565 T/G 15.1 0.030 � EXT2 rs1113132 C/G 27.1 0.082 G/C 34.3 0.856 C/G 14.2 0.546 FTO rs8050136 C/A 15.5 0.843 C/A 16 1.000 C/A 11.7 0.737 G6PC2 rs3755157 C/T 35.0 0.571 C/T 38.2 0.258 T/C� 48.5 0.748 G6PC2 rs16856187 A/C 37.3 0.330 A/C 33.9 0.573 A/C 43.8 1.000 <0.010 G6PC2 rs560887 C/T 2.0 0.015 C/T 1.8 1.000 C/T 1.2 GCK rs4607517 G/A 13.9 <0.010 G/A 22.7 1.000 G/A 18.9 0.815 GCKR rs780094 C/T 41.7 0.210 T/C� 49.2 0.735 C/T 27.2 0.207 HHEX rs1111875 T/C 17.0 0.032 T/C 30.9 1.000 T/C 35.7 0.02 HHEX rs7923837 A/G 9.5 0.325 A/G 18.7 0.567 A/G 27.1 0.077 IGFBP2 rs4402960 G/T 26.5 0.358 G/T 29.4 0.551 G/T 22.4 0.399 KCNQ1 rs2237892 C/T 38.7 0.462 C/T 40.9 0.601 C/T 28.7 1.000 KCNQ1 rs2237895 A/C 33.1 0.406 A/C 32.3 0.848 A/C 24.1 1.000 MADD rs10501320 G/C 0.5 1.000 G/C 0.3 1.000 G/C 1.2 1.000 MTNR 1B rs10830963 G/C 47.8 0.606 C/G� 43.2 0.286 G/C 48.9 0.281 PKN2 rs6698181 C/T 18.8 0.623 C/T 40.2 0.476 C/T 8.7 1.000 SLC30A8 rs13266634 T/C 49.9 0.749 C/T� 38.8 0.858 C/T� 41.9 0.084 TCF7L2 rs7903146 C/T 1.8 1.000 C/T 5.2 1.000 C/T 11.5 0.041 TCF7L2 rs7901695 T/C 1.7 1.000 T/C 5.2 1.000 T/C 9.7 0.399 TCF7L2 rs12255372 G/T 1.7 1.000 G/T 3.6 1.000 G/T 9.2 0.688 TCF7L2 rs11196205 G/C 4.1 0.134 G/C 6.7 1.000 G/C 11.2 0.720 Alleles: major/minor, MAF = Major Allele Frequency, HWE = Hardy-Weinburg Equilibrium. SNPs not at Hardy–Weinberg equilibrium (p<0.05) are shown in blue. Reversed minor allele compared to Filipino Controls are in red with an asterisk. Risk alleles are bold and underlined. https://doi.org/10.1371/journal.pone.0261137.t002 Genotype minor allele frequencies (MAF) were summarized by percentages. The BreslowDay test [52] was used to assess the homogeneity of MAFs and odds ratios (OR) among the three ethnic groups. We used chi-square tests to determine whether an individual polymorphism was in Hardy–Weinberg equilibrium. For the dominant and recessive model of each polymorphism, unadjusted odds ratios were calculated with p values using chi-square tests within each ethnic group. The dominant/recessive model was chosen over other models such as the additive or multiplicative model as it increased the resolution of assessing disease risk, similar to other studies of diabetes [53, 54]. We evaluated the homogeneity of the unadjusted OR among the three ethnic groups by Breslow-Day test and used Mantel-Haenszel formula [55] to calculate summary unadjusted OR with 95% confidence interval (CI). We then selected SNPs which showed that 1) unadjusted OR were significantly different from 1.0; 2) p values of Breslow-Day test were less than 0.05; 3) the summary of unadjusted OR was significantly different from 1.0 based on Mantel-Haenszel formula. For the selected SNPs, multivariable logistic regression models were used to further adjust and control for confounding factors, such as age, BMI, parity, and gravidity. All data analyses were performed in R-package and SAS 9.3 and a two-tailed p<0.05 was regarded as statistically significant. PLOS ONE | https://doi.org/10.1371/journal.pone.0261137 December 20, 2021 5 / 15 PLOS ONE Genetic risk factors for type II diabetes and gestational diabetes Results We compared the genotype frequencies in 291 women from Hawaii who met Coustan-Carpenter criteria for GDM and 734 matched non-diabetic controls (Pacific Islander: 71 subjects with GDM and 197 non-diabetic controls; Filipinos: 162 GDM patients and 395 controls; Japanese: 58 GDM patients and 142 controls). The clinical baseline characteristics are listed in Table 1. The mean BMI was significantly higher in all three GDM groups (Filipino and Japanese p<0.001, Pacific Islander p = 0.016) when compared to the ethnically matched controls. Additionally, mean maternal age (p = 0.029) and mean parity (p = 0.031) were significantly higher in Filipino women with GDM than in their control (p = 0.001), whereas parity (p = 0.020) and maternal age differed in the Japanese GDM cohort and Pacific Islander women respectively. 25 SNPs reported to be associated with T2DM were genotyped. Table 2 reports the SNP alleles, as well as minor allele frequency (MAF) and the results from the Hardy–Weinberg equilibrium analysis in the control group for all 25 SNPs studied. All SNPs were in Hardy– Weinberg equilibrium at the significance level of p>0.05 except for (i) rs4607517 (GCK) and rs2877716 (ADCY5) in Filipinos, (ii) rs2191349 (DGKB) and rs7903146 (TCF7L2) in Pacific Islanders, (iii) rs1111875 (HHEX) and rs560887 (G6PC2) in both Filipino and Pacific Islanders (Table 2, blue values). For certain SNPs, there was also a reversal of major/minor alleles among the subpopulations (as in an allele that is major in one race becomes minor in another race and vice versa) (Table 2, red alleles). Unadjusted odds ratios (OR) and associated p-values were subsequently calculated for the association of genotypes with GDM in the dominant and recessive model of each polymorphism (Tables 3 and 4 respectively). Interestingly, for each race, several SNPs showed significant associations with GDM (Tables 3 and 4, blue values). However, none of the SNPs appeared to show a consistent association with GDM across all three investigated ethnicities. To evaluate this observation further, we assessed the homogeneity of the unadjusted OR among the three ethnic groups using the Breslow-Day (BD) test as well as the Mantel-Haenszel (MH) formula that calculates a summary of unadjusted OR with 95% confidence interval. In the dominant model, the association of rs2237892 (KCNQ1, Table 3) with GDM differed significantly between our populations (BD p = 0.024, green value, Table 3). Indeed, while highly significant for Filipinos (p<0.001), we detected only a moderate association of rs2237892 (KCNQ1) with GDM in Japanese subjects (p = 0.03) and none in Pacific Islanders (p = 0.49) (Table 3). Similarly, for the recessive model, rs13266634 (SLC30A8, Table 4) showed a significant association with GDM in Pacific Islanders (p = 0.005), but not in Filipinos (p = 0.301) or Japanese (p = 0.230) (BD p = 0.004, green value, Table 4). SNPs with values from MantelHaenszel formula that were significantly different from 1.0 in the dominant and recessive models are highlighted in red in Tables 3 and 4, respectively. Next, we used multivariable logistic regression models to adjust for age, BMI, gravidity, and parity. Such regression analyses were applied only to selected SNPs that originally showed either significant unadjusted OR p values (p<0.05; blue) or significant Breslow-Day test p values (p<0.05; green) or values from Mantel-Haenszel formula significantly different from 1.0 (red) in the dominant and recessive models shown in Tables 3 and 4. The results of the regression analyses for the SNPs that originally showed significant unadjusted OR p values (p<0.05) (blue values in Tables 3 and 4) are shown in Table 5. The results of the regression analyses for the SNPs that originally showed significant Breslow-Day test p values (p<0.05) (green values in Tables 3 and 4) are shown in Table 6. The results of the regression analyses for the SNPs that showed values from Mantel-Haenszel formula significantly different from 1.0 (red values in Tables 3 and 4) are shown in Table 7. PLOS ONE | https://doi.org/10.1371/journal.pone.0261137 December 20, 2021 6 / 15 PLOS ONE Genetic risk factors for type II diabetes and gestational diabetes Table 3. Association of SNPs with GDM risk using a dominant model (unadjusted odds ratios; OR, Breslow-Day test; BD, Mantel-Haenszel formula; MH). Filipino Japanese P OR PI Gene SNP OR P OR BD_FJPI OR.mh Low. B Up. B ADCY5 rs2877716 0.39 ( 0.337 ) 0.61 ( 0.176 ) 0.27 ( 0.046 P ) 0.889 0.34 0.11 1.04 ARAP 1 rs11603334 1.11 ( 0.725 ) 1.08 ( 0.906 ) 0.76 ( 0.601 ) 0.821 1.02 0.64 1.63 CDKAL1 rs7754840 1.5 ( 0.071 ) 0.66 ( 0.195 ) 1.82 ( 0.089 ) 0.057 1.27 0.92 1.75 CDKN2A/2B rs10811661 0.88 ( 0.499 ) 1.17 ( 0.641 ) 0.57 ( 0.054 ) 0.248 0.83 0.63 1.11 DGKB rs2191349 1.13 ( 0.507 ) 0.78 ( 0.433 ) 0.74 ( 0.365 ) 0.406 0.96 0.72 1.29 EXT2 rs1113132 1.52 ( 0.028 ) 4.00 ( 0.032 ) 1.04 ( 0.907 ) 0.218 1.48 1.09 2.02 FTO rs8050136 1.11 ( 0.604 ) 0.9 ( 0.767 ) 0.7 ( 0.335 ) 0.539 0.97 0.71 1.34 G6PC2 rs16856187 0.74 ( 0.12 ) 0.68 ( 0.217 ) 1.17 ( 0.626 ) 0.393 0.80 0.60 1.07 G6PC2 rs3755157 0.83 ( 0.346 ) 0.52 ( 0.047 ) 1.18 ( 0.676 ) 0.252 0.80 0.59 1.07 G6PC2 rs560887 0.44 ( 0.156 ) 0.4 ( 0.35 ) 1.43 ( 0.293 ) 0.745 0.41 0.15 1.12 GCK rs4607517 1.45 ( 0.088 ) 1.17 ( 0.632 ) 0.91 ( 0.753 ) 0.456 1.22 0.90 1.65 GCKR rs780094 0.89 ( 0.56 ) 1.41 ( 0.367 ) 0.67 ( 0.17 ) 0.307 0.89 0.66 1.19 HHEX rs1111875 1.5 ( 0.047 ) 0.61 ( 0.13 ) 1.43 ( 0.229 ) 0.053 1.22 0.91 1.63 HHEX rs7923837 1.51 ( 0.085 ) 1.19 ( 0.605 ) 1.84 ( 0.036 ) 0.607 1.52 1.11 2.08 IGFBP2 rs4402960 0.97 ( 0.888 ) 0.5 ( 0.031 ) 0.83 ( 0.534 ) 0.205 0.82 0.62 1.09 KCNQ1 rs2237892 0.49 ( < .001 ) 0.5 ( 0.033 ) 1.22 ( 0.488 ) 0.024 KCNQ1 rs2237895 1.1 ( 0.624 ) 0.96 ( 0.907 ) 1.79 ( 0.047 ) 0.28 1.20 0.91 1.59 MADD rs10501320 0.59 ( 0.627 ) 0.61 ( 0.176 ) 1.43 ( 0.293 ) 0.556 0.43 0.10 1.95 0.83 MTNR 1B rs10830963 0.63 ( 0.025 ) 0.71 ( 0.368 ) 0.52 ( 0.037 ) 0.799 0.61 0.45 PKN2 rs6698181 0.89 ( 0.551 ) 0.82 ( 0.555 ) 1.02 ( 0.95 ) 0.91 0.89 0.66 1.22 SLC30A8 rs13266634 0.58 ( 0.011 ) 0.74 ( 0.387 ) 1.09 ( 0.781 ) 0.239 0.72 0.53 0.98 TCF7L2 rs11196205 0.95 ( 0.89 ) 0.7 ( 0.465 ) 0.82 ( 0.583 ) 0.878 0.84 0.54 1.31 TCF7L2 rs12255372 0.92 ( 0.876 ) 0.64 ( 0.496 ) 0.72 ( 0.411 ) 0.902 0.76 0.43 1.34 TCF7L2 rs7901695 0.91 ( 0.866 ) 0.8 ( 0.683 ) 0.92 ( 0.817 ) 0.979 0.89 0.52 1.50 TCF7L2 rs7903146 1.2 ( 0.724 ) 0.8 ( 0.683 ) 0.67 ( 0.288 ) 0.655 0.82 0.48 1.38 BD-FJPI = Breslow Day Test for Filipino, Japanese and Pacific Islander. SNPs with significant unadjusted OR p values (p<0.05) are highlighted in blue. SNPs with significant Breslow-Day test p values (p<0.05) are highlighted in green. SNPs with values from Mantel-Haenszel formula significantly different from 1.0 are highlighted in red. https://doi.org/10.1371/journal.pone.0261137.t003 Overall, after adjusting for age, BMI, parity and gravidity, SNPs rs1113132 (EXT2), rs1111875 (HHEX), rs2237892 (KCNQ1), rs10830963 (MTNR1B) and rs13266634 (SLC30A8) showed significant associations with GDM in the dominant model for Filipinos, while rs2237892 (KCNQ1), rs2237895 (KCNQ1) and rs10830963 (MTNR1B) were significantly associated with GDM in the recessive model (Table 8). For Japanese women, the SNPs rs4402960 (IGFBP2) and rs2237892 (KCNQ1) showed significant associations with GDM in the dominant model, but no SNP was significantly associated with GDM in the recessive model (Table 8). For Pacific Islander women, the SNP rs10830963 (MTNR1B) showed significant association with GDM in the dominant model, while rs13266634 (SLC30A8) was significantly associated with GDM in the recessive model (Table 8). Discussion GDM and T2DM are both multifactorial diseases and are believed to share similar epidemiologic risk factors such as obesity, hypertension, polycystic ovarian syndrome, racial-ethnic background, and family history. Several studies have demonstrated a shared genetic susceptibility for both T2DM and GDM among various populations. Cho et al. investigated various PLOS ONE | https://doi.org/10.1371/journal.pone.0261137 December 20, 2021 7 / 15 PLOS ONE Genetic risk factors for type II diabetes and gestational diabetes Table 4. Association of SNPs with GDM risk using a recessive model (unadjusted odds ratios; OR, Breslow-Day Test; BD, Mantel-Haenszel formula; MH). Filipino Japanese P OR PI Gene SNP OR P OR BD_FJPI OR. mh Low. B ADCY5 rs2877716 1.13 ( 0.99 ) NA ( NA ) NA ( NA P ) NA 0 0 Up. B 0 ARAP 1 rs11603334 2.39 ( 0.544 ) NA ( NA ) 2.85 ( 0.471 ) 0.931 2.605 0.364 18.643 CDKAL1 rs7754840 0.82 ( 0.375 ) 1.38 ( 0.441 ) 1.33 ( 0.436 ) 0.366 0.997 0.71 1.4 CDKN2A/2B rs10811661 0.63 ( 0.167 ) 0.36 ( 0.031 ) 1.17 ( 0.701 ) 0.18 0.667 0.425 1.047 DGKB rs2191349 1.48 ( 0.327 ) 0.94 ( 0.903 ) 0.31 ( 0.204 ) 0.32 1.057 0.587 1.903 EXT2 rs1113132 0.73 ( 0.392 ) 1.52 ( 0.201 ) 1.14 ( 0.332 ) 0.131 1.03 0.652 1.627 FTO rs8050136 1.06 ( 0.923 ) 2.56 ( 0.267 ) 1.95 ( 0.484 ) 0.662 1.514 0.655 3.499 G6PC2 rs3755157 1.04 ( 0.898 ) 1.49 ( 0.374 ) 1.19 ( 0.621 ) 0.792 1.168 0.788 1.732 G6PC2 rs560887 1.13 ( 0.99 ) NA ( NA ) 1.14 ( 1 ) 0.372 0.518 0.06 4.461 G6PC2 rs16856187 1.04 ( 0.898 ) 1.44 ( 0.406 ) 1.36 ( 0.387 ) 0.745 1.198 0.821 1.749 GCK rs4607517 1.02 ( 0.969 ) 1.06 ( 0.935 ) 0.48 ( 0.46 ) 0.794 0.919 0.438 1.929 GCKR rs780094 0.76 ( 0.268 ) 1.10 ( 0.785 ) 1.35 ( 0.6 ) 0.521 0.9 0.616 1.316 HHEX rs1111875 1.13 ( 0.798 ) 1.1 ( 0.857 ) 1.15 ( 0.729 ) 0.998 1.128 0.679 1.873 HHEX rs7923837 1.81 ( 0.448 ) 1.65 ( 0.595 ) 1.3 ( 0.592 ) 0.923 1.456 0.703 3.013 IGFBP2 rs4402960 0.75 ( 0.435 ) 0.37 ( 0.156 ) 0.84 ( 0.826 ) 0.679 0.665 0.361 1.224 KCNQ1 rs2237892 0.55 ( 0.03 ) 0.78 ( 0.565 ) 0.82 ( 0.736 ) 0.726 0.634 0.412 0.977 KCNQ1 rs2237895 2 ( 0.015 ) 0.69 ( 0.517 ) 1.64 ( 0.404 ) 0.25 1.604 1.025 2.508 MADD rs10501320 NA ( NA ) NA ( NA ) NA ( NA ) NA 0 0 0 MTNR 1B rs10830963 0.39 ( < .001 ) 0.61 ( 0.15 ) 0.7 ( 0.296 ) 0.339 0.506 0.356 0.72 PKN2 rs6698181 0.59 ( 0.325 ) 1.89 ( 0.117 ) 1.14 ( 1 ) 0.185 1.2 0.653 2.205 SLC30A8 rs13266634 0.79 ( 0.301 ) 0.55 ( 0.23 ) 2.77 ( 0.005 ) 0.004 TCF7L2 rs11196205 1.13 ( 0.99 ) NA ( NA ) 1.14 ( 0.57 ) 0.363 0.432 0.052 3.622 TCF7L2 rs12255372 NA ( NA ) NA ( NA ) 1.14 ( 1 ) NA 0 0 0 TCF7L2 rs7903146 1.13 ( 0.296 ) NA ( NA ) 1.14 ( 0.345 ) 0.028 TCF7L2 rs7901695 NA ( NA ) NA ( NA ) 1.14 ( 0.571 ) NA 0 0 0 BD-FJPI = Breslow Day Test for Filipino, Japanese and Pacific Islander. SNPs with significant unadjusted OR p values (p<0.05) are shown in blue. SNPs with significant Breslow-Day test p values (p<0.05) are shown in green. SNPs with values from Mantel-Haenszel formula significantly different from 1.0 are shown in red. https://doi.org/10.1371/journal.pone.0261137.t004 SNPs known to be associated with an increased risk of T2DM in women with and without GDM in a Korean population [40]. Allelic differences in CDKAL1 (Cdk5 regulatory associated protein 1-like 1), CDKN2A-2B (cyclin-dependent kinase inhibitor 2A), HHEX (hematopoietically expressed homeobox), IGFT2BP2 (Insulin Like Growth Factor Binding Protein 2), SLC30A8 (Solute Carrier Family 30 Member 8), and TCF7L2 (Transcription Factor 7 Like 2) conferred an increased risk of GDM [40]. More recently, MTNR1B (rs10830962) was also found to have an excess association with T2DM and GDM in a Korean population [56]. rs2237895 on the KCNQ1 gene has been confirmed to be associated with both GDM and T2DM in Pakistani and Chinese cohorts [57, 58]. Gene variants of HMG20A (rs7178572) and HNF4A (rs4812829) showed significant association with GDM in Asian Indians [59]. The unique multiethnic population of Hawaii is comprised of both immigrants and multigenerational descendants of Japanese, Chinese, and Filipino heritage, in addition to those of Native Hawaiian ancestry. While many patients presenting for care identify as multiethnic, the inclusion criteria for this study population was strictly limited to those self-reporting one ethnicity according to all 4 grandparents. This allowed us to compare across multiple ethnicities at once without disproportionate heterogeneity. Identifying genotypic differences may account for the variation in disease phenotypes that are seen in various populations of pregnant PLOS ONE | https://doi.org/10.1371/journal.pone.0261137 December 20, 2021 8 / 15 PLOS ONE Genetic risk factors for type II diabetes and gestational diabetes Table 5. Multivariable logistic regression analysis (OR). Filipino Dominant Recessive Gene SNP ADCY5 rs2877716 EXT2 rs1113132 G6PC2 rs3755157 HHEX rs1111875 HHEX rs7923837 IGFBP2 rs4402960 KCNQ1 rs2237892 KCNQ1 rs2237895 MTNR 1B OR Japanese P_value OR 1.53 ( 0.034 ) 1.60 ( 0.027 ) 0.5 ( < .001 ) rs10830963 0.62 ( 0.028 ) SLC30A8 rs13266634 0.49 ( 0.002 ) KCNQ1 rs2237892 0.51 ( 0.019 ) KCNQ1 rs2237895 1.84 ( 0.046 ) CDKN2A/2B rs10811661 MTNR 1B rs10830963 0.34 ( < .001 ) SLC30A8 rs13266634 PI P_value OR 7.40 ( 0.063 ) 0.57 ( 0.133 ) 0.32 ( 0.003 ) 0.45 ( 0.035 ) 0.35 ( 0.053 P_value 0.37 ( 0.153 ) 1.55 ( 0.196 ) 1.72 ( 0.108 ) 0.42 ( 0.021 ) 2.43 ( 0.03 ) ) List of SNPs (adjusted for age, BMI, parity, gravidity) which showed significant unadjusted OR p values (p<0.05) in the Dominant (Table 3) and Recessive (Table 4). https://doi.org/10.1371/journal.pone.0261137.t005 women. For example, an East Asian cohort of women living in New York City with GDM were found to have a normal BMI, have a low risk for fetal macrosomia and GDM phenotype that was relatively easy to control with only diet modification [60]. However, a cohort of Arabian women with GDM living in Scandinavia were found to be much more insulin-resistant than Caucasian Scandinavian pregnant women diagnosed with GDM using a homeostasis model assessment [61]. Of note, multiple testing adjustments such as Bonferroni or Benjamini-Hochberg corrections were not included in this study owing to the modest sample size. This was to maintain the capacity to discover any potential associations between the selected SNPs, ethnicity and diabetes in this unique population. Looking at allelic variations in our population demonstrates the importance of identifying phenotypic factors to screen for gestational diabetes as well as investigating drug treatments that target ethnic-specific mechanisms that can be used to develop personalized medicine. For instance, KCNQ1 gene encodes for potassium inwardly rectifying channel subfamily J, member 11 [62], and polymorphisms associated with potassium channels are likely to control insulin secretion in the pancreatic islet cells. The dominant model demonstrated SNPs at the CDKN2A loci, a gene coding for a cyclin-dependent kinase inhibitor 2A, were highly associated with an increased risk of GDM in Japanese participants. The results from our candidate Table 6. Multivariable logistic regression analysis (BD). Filipino Gene Dominant Recessive SNP OR Japanese P_value OR PI P_value OR P_value KCNQ1 rs2237892 0.5 ( < .001 ) 0.45 ( 0.035 ) 1.18 ( 0.622 ) TCF7L2 rs7903146 NA ( 0.99 ) NA ( NA ) NA ( 0.98 ) SLC30A8 rs13266634 0.73 ( 0.17 ) 0.62 ( 0.41 ) 2.43 ( 0.03 ) List of SNPs (adjusted for age, BMI, parity, gravidity) which showed significant Breslow-Day test p values (p<0.05) in the Dominant (Table 3) and Recessive models (Table 4). https://doi.org/10.1371/journal.pone.0261137.t006 PLOS ONE | https://doi.org/10.1371/journal.pone.0261137 December 20, 2021 9 / 15 PLOS ONE Genetic risk factors for type II diabetes and gestational diabetes Table 7. Multivariable logistic regression analysis. Filipino Dominant Recessive OR Japanese P_value OR PI Gene SNP P_value OR EXT2 rs1113132 1.33 ( 0.13 ) 6.05 ( 0.085 ) 1.16 ( P_value 0.69 ) HHEX rs7923837 1.48 ( 0.095 ) 1.01 ( 0.97 ) 1.56 ( 0.17 ) MTNR 1B rs10830963 0.63 ( 0.025 ) 0.81 ( 0.59 ) 0.39 ( 0.006 ) SLC30A8 rs13266634 0.56 ( 0.006 ) 0.73 ( 0.38 ) 1.02 ( 0.96 ) KCNQ1 rs2237895 1.5 ( 0.14 ) 0.61 ( 0.46 ) 1.19 ( 0.8 ) MTNR 1B rs10830963 0.37 ( < .001 ) 0.52 ( 0.092 ) 0.56 ( 0.14 ) List of SNPs (adjusted for age, BMI, parity, gravidity) which showed values from Mantel-Haenszel formula significantly different from 1.0 in the Dominant (Table 3) and Recessive (Table 4) models. https://doi.org/10.1371/journal.pone.0261137.t007 gene approach are also consistent with prior studies showing B-cell dysfunction and impaired insulin secretion in Japanese populations [63]. Upstream of this locus are sequences that encode for p15INK4b and p16 INK4a, which both inhibit CDK4 and decrease pancreatic beta cell replication [64]. MTNR1B polymorphisms also have associations with GDM in Filipino and Pacific Islander populations. rs10830963 has been reported to have a strong correlation in a meta-analysis of GDM GWAS studies [65], and this finding has been seen among Korean, Greek [66], Russian [67] and Chinese women [42]. MTNR1A and MTNR1B encode for melatonin receptors and high-risk allele carriers at rs10830963 also have higher expression of MTNR1B in pancreatic beta cells, which leads to impaired insulin secretion [68]. Interestingly, the presence of this SNP was associated with a lower frequency of GDM in Pacific Islander women in our cohort. We observe that no single T2DM associated SNP is replicated across all Hawaiian populations who develop GDM, and this could be attributed to our modest sample size. However, we have refined the resolution of SNPs associated specifically with Japanese, Filipino and Pacific Islander populations in Hawaii. Future studies could validate these observations in a larger cohort. In addition, further studies could build on our findings and investigate a larger panel of candidate genes such as BACE2 and HKDC1 which are associated with maternal metabolic traits but were not included in this study [69]. Table 8. Summary table illustrating SNPs with significant associations with GDM, according to race. Filipino Gene Dominant Recessive SNP � OR Japanese P_value OR EXT2( ) rs1113132 1.53 ( 0.034 ) HHEX(� ) rs1111875 1.60 ( 0.027 ) 0.5 ( < .001 ) IGFBP2(� ) rs4402960 KCNQ1(� )(�� ) rs2237892 MTNR 1B (� )(��� ) rs10830963 0.62 ( 0.028 ) SLC30A8(� )(��� ) rs13266634 0.56 ( 0.006 ) KCNQ1(� ) rs2237892 0.51 ( 0.019 ) KCNQ1(� ) rs2237895 1.84 ( 0.046 ) rs10830963 0.37 ( < .001 ) � MTNR 1B( )( ��� SLC30A8(� )(�� ) ) rs13266634 PI P_value OR 0.32 ( 0.003 ) 0.45 ( 0.035 ) P_value 0.42 ( 0.021 ) 2.43 ( 0.03 ) The table lists SNPs that showed significant unadjusted OR p values (p<0.05; � ), significant Breslow-Day test p values (p<0.05; �� ) or values from Mantel-Haenszel formula significantly different from 1.0 (��� ) in the Dominant (Table 3) and Recessive (Table 4) models. Significance was determined after multivariable logistic regression analysis was performed to control for confounding factors. https://doi.org/10.1371/journal.pone.0261137.t008 PLOS ONE | https://doi.org/10.1371/journal.pone.0261137 December 20, 2021 10 / 15 PLOS ONE Genetic risk factors for type II diabetes and gestational diabetes Conclusion These findings elucidate the pathophysiology of increased risk of T2DM later in life for women affected by GDM and highlight the importance of lifestyle modifications to decrease this risk. Such associations need to be validated in larger Asian and Pacific Islander cohorts and further characterized into clinically meaningful phenotypes that can be designed to guide personalized medicine. While deleterious genetic mutations may provide increased susceptibility for gestational diabetes and impaired glucose tolerance postnatally, the environmental risk factors for this condition should also be considered for treatment. Author Contributions Conceptualization: Janet Burlingame, Johann Urschitz. Data curation: Hyeong Jun Ahn, John Chen, Johann Urschitz. Formal analysis: Paula Benny, Hyeong Jun Ahn, Men-Jean Lee, Corrie Miller, John Chen, Johann Urschitz. Funding acquisition: Johann Urschitz. Investigation: Johann Urschitz. Methodology: Hyeong Jun Ahn, Janet Burlingame, Men-Jean Lee, Corrie Miller, John Chen, Johann Urschitz. Project administration: Johann Urschitz. Resources: Johann Urschitz. Software: Hyeong Jun Ahn, John Chen. Supervision: Johann Urschitz. Visualization: Johann Urschitz. Writing – original draft: Paula Benny, Men-Jean Lee, Corrie Miller, Johann Urschitz. Writing – review & editing: Paula Benny, Men-Jean Lee, Corrie Miller, Johann Urschitz. References 1. Buchanan TA, Metzger BE, Freinkel N, Bergman RN. Insulin sensitivity and B-cell responsiveness to glucose during late pregnancy in lean and moderately obese women with normal glucose tolerance or mild gestational diabetes. 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Ethnic differences in perinatal outcome of gestational diabetes mellitus. Diabetes care. 2006; 29(9):2058–2063. https://doi.org/10.2337/dc06-0458 PMID: 16936153 23. Tsai PJ, Roberson E, Dye T. Gestational diabetes and macrosomia by race/ethnicity in Hawaii. BMC research notes. 2013; 6:395. https://doi.org/10.1186/1756-0500-6-395 PMID: 24083634 24. Sandhu MS, Weedon MN, Fawcett KA, et al. Common variants in WFS1 confer risk of type 2 diabetes. Nat Genet. 2007; 39(8):951–953. https://doi.org/10.1038/ng2067 PMID: 17603484 25. Saxena R, Voight BF, Lyssenko V, et al. Genome-wide association analysis identifies loci for type 2 diabetes and triglyceride levels. Science. 2007; 316(5829):1331–1336. https://doi.org/10.1126/science. 1142358 PMID: 17463246 26. Scott LJ, Mohlke KL, Bonnycastle LL, et al. A genome-wide association study of type 2 diabetes in Finns detects multiple susceptibility variants. Science. 2007; 316(5829):1341–1345. https://doi.org/10. 1126/science.1142382 PMID: 17463248 27. Sladek R, Rocheleau G, Rung J, et al. A genome-wide association study identifies novel risk loci for type 2 diabetes. Nature. 2007; 445(7130):881–885. https://doi.org/10.1038/nature05616 PMID: 17293876 28. Langenberg C, Lotta LA. Genomic insights into the causes of type 2 diabetes. Lancet. 2018. Jun 16; 391(10138):2463–2474. https://doi.org/10.1016/S0140-6736(18)31132-2 PMID: 29916387 PLOS ONE | https://doi.org/10.1371/journal.pone.0261137 December 20, 2021 12 / 15 PLOS ONE Genetic risk factors for type II diabetes and gestational diabetes 29. Lotta LA, Pietzner M, Stewart ID, Wittemans LBL, Li C, Bonelli R, et al. A cross-platform approach identifies genetic regulators of human metabolism and health. Nat Genet. 2021 Jan; 53(1):54–64. https:// doi.org/10.1038/s41588-020-00751-5 PMID: 33414548 30. Kooner J, Saleheen D, Sim X, et al. Genome-wide association study in individuals of South Asian ancestry identifies six new type 2 diabetes susceptibility loci. Nature Genetics. 2011; 43(10):984–989 https://doi.org/10.1038/ng.921 PMID: 21874001 31. Yamauchi T, Hara K, Maeda S, et al. A genome-wide association study in the Japanese population identifies susceptibility loci for type 2 diabetes at UBE2E2 and C2CD4A-C2CD4B. Nature Genetics. 2010; 42(10):864–868. https://doi.org/10.1038/ng.660 PMID: 20818381 32. Imamura M, Maeda S, Yamauchi T, et al. A single-nucleotide polymorphism in ANK1 is associated with susceptibility to type 2 diabetes in Japanese populations. Human Molecular Genetics. 2012; 21 (13):3042–3049. https://doi.org/10.1093/hmg/dds113 PMID: 22456796 33. Below J, Gamazon E, Morrison J, et al. Genome wide association and meta-analysis in population from Starr County, Texas, and Mexico City identify type 2 diabetes susceptibility loci and enrichment for expression quantitative trait loci in top signals. 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10523_juvet.qxd 28/05/2008 11:37 AM Page 203 REVIEW Rare lung diseases II: Pulmonary alveolar proteinosis Stephen C Juvet MD1, David Hwang MD PhD2, Thomas K Waddell MD PhD3, Gregory P Downey MD1,4 SC Juvet, D Hwang, TK Waddell, GP Downey. Rare lung diseases II: Pulmonary alveolar proteinosis. Can Respir J 2008;15(4):203-210. Les maladies pulmonaires rares II : La protéinose alvéolaire pulmonaire The present article is the second in a series on rare lung diseases. It focuses on pulmonary alveolar proteinosis (PAP), a disorder in which lipoproteinaceous material accumulates in the alveolar space. PAP was first described in 1958, and for many years the nature of the material accumulating in the lungs was unknown. Major insights into PAP have been made in the past decade, and these have led to the notion that PAP is an autoimmume disorder in which autoantibodies interfere with signalling through the granulocyte-macrophage colony-stimulating factor receptor, leading to macrophage and neutrophil dysfunction. This has spurred new therapeutic approaches to this disorder. The discussion of PAP will begin with a case report, then will highlight the classification of PAP and review recent insights into the pathogenesis of PAP. The approach to therapy and the prognosis of PAP will also be discussed. Le présent article est le deuxième d’une série sur les maladies pulmonaires rares. Il porte sur la protéinose alvéolaire pulmonaire (PAP), un trouble qui provoque l’accumulation de substances lipoprotéinacées dans les espaces alvéolaires. La PAP a été décrite pour la première fois en 1958, et pendant de nombreuses années, la nature de la substance s’accumulant dans les poumons est demeurée inconnue. On a fait d’importantes observations sur la maladie depuis dix ans, lesquelles ont permis d’établir que la PAP est une maladie auto-immune qui provoque l’interférence des autoanticorps avec la signalisation par le récepteur des facteurs stimulant les colonies de granulocytes-macrophages, provoquant un dysfonctionnement des macrophages et des neutrophiles. Ce constat a suscité de nouvelles démarches thérapeutiques pour cette maladie. L’exposé sur la PAP commence par une étude de cas, puis porte sur la classification de la PAP et l’analyse des récentes observations sur la pathogénèse de la maladie. La démarche à l’égard du traitement et du pronostic de la PAP sera également abordée. Key Words: Autoimmunity; GM-CSF; Growth factor; Respiratory failure; Restrictive lung disease CASE PRESENTATION A 46-year-old woman presented with a six-month history of progressive exertional dyspnea and fatigue. There was no history of fever, cough, weight loss, chest pain or peripheral edema. Her medical history included iron deficiency anemia that required parenteral iron replacement. She took no medications, except for iron replacements, and was a lifelong nonsmoker. She worked as a cashier, drank alcohol socially, had no known allergies, no pets and no history of recent travel. On physical examination she appeared well, with no respiratory distress. The following parameters were recorded: blood pressure 110/65 mmHg, heart rate 78 beats/min, respiratory rate 20 breaths/min and body temperature 37.2°C. Her arterial O2 saturation on room air was 91%. Her jugular venous pressure was not elevated. Cardiac examination revealed normal first and second heart sounds, with no murmurs or extra sounds. Auscultation of the chest revealed scattered inspiratory crackles bilaterally. Her abdominal examination was unremarkable, without organomegaly, and there was no peripheral cyanosis, clubbing or edema. Laboratory findings revealed a normal complete blood count and differential. Serum electrolytes, liver and renal function tests were all normal. Arterial blood gas analysis on room air revealed pH 7.37, partial pressure (P) of CO2 37 mmHg, PO2 62 mmHg, and bicarbonate 24 mmol/L. A chest radiograph demonstrated bilateral diffuse airspace opacities affecting both lungs (Figure 1). A computed tomography scan of the chest (Figure 2) revealed diffuse, multifocal, patchy ground glass opacities, with superimposed reticulations resulting in a ‘crazy paving’ appearance. The patient underwent bronchoscopy with bronchoalveolar lavage from the right middle lobe. The lavage fluid was sent for routine cytology, cell count and differential, Gram stain and bacterial culture, acid-fast stain and mycobacterial culture, and fungal stain and culture; all returned negative. A video-assisted thoracoscopic lung biopsy revealed the presence of eosinophilic foamy material in the alveolar spaces (Figure 3). A diagnosis of primary alveolar proteinosis (PAP) was made, and she underwent bilateral whole lung lavage (WLL), which resulted in significant improvement in clinical, radiological and pulmonary function parameters. However, her symptoms recurred after six months. She underwent repeat bilateral WLL, with temporary improvement, followed by a recurrence of symptoms and radiographic abnormalities within a few months. A third WLL resulted in only minor improvement. Therapy with inhaled granulocyte-macrophage colony-stimulating factor (GM-CSF) was initiated and continued for 12 weeks. This resulted in improved clinical and radiological parameters, and she has remained stable over the past two years. PAP Clinical features and classification of PAP PAP is a rare lung disease characterized by the accumulation of excess lipoproteinaceous (surfactant-like) material in the of Respirology, Department of Medicine; 2Department of Laboratory Medicine and Pathobiology; 3Department of Surgery, University of Toronto and the Toronto General Hospital of the University Health Network, Toronto, Ontario; 4Departments of Medicine, Pediatrics and Immunology, National Jewish Medical and Research Center and University of Colorado Denver, Denver, Colorado, USA Correspondence: Dr Gregory P Downey, National Jewish Medical and Research Center, K701b, 1400 Jackson Street, Denver, Colorado 80206, USA. Telephone 303-398-1436, fax 303-270-2243, e-mail downeyg@njc.org 1Division Can Respir J Vol 15 No 4 May/June 2008 ©2008 Pulsus Group Inc. All rights reserved 203 10523_juvet.qxd 28/05/2008 11:37 AM Page 204 Juvet et al Figure 1) Posterior-anterior chest x-ray of a 46-year-old woman with alveolar proteinosis, characterized by bilateral perihilar alveolar infiltrates Figure 3) Open lung biopsy stained by hematoxylin and eosin, illustrating the eosinophilic foamy material present within the alveolar spaces. A The scale bar represents 1.0 mm. B The scale bar represents 0.05 mm Figure 2) Chest computed tomography scan of the patient with alveolar proteinosis, illustrating bilateral ground glass alveolar infiltrates and interlobular septal thickening consistent with a ‘crazy paving’ pattern alveolar compartment. It was first recognized as a distinct clinical entity in 1958 (1). There are three clinically recognized categories of PAP (2): idiopathic PAP, with onset occuring in adulthood; secondary PAP, also typically occurring in 204 adulthood and resulting from another disorder or exposure; and congenital PAP, which occurs in the neonatal period, and causes severe respiratory distress. Idiopathic PAP, the most common form of the disorder, is generally diagnosed in young to middle-aged adults, with a median age at diagnosis of 39 years (2). A recently published analysis (2) of 410 cases of PAP revealed that the median delay between symptom onset and diagnosis is seven months. The disorder is two to three times more common in men, and approximately 72% of patients are smokers (2). More than 90% of cases of PAP are classified as idiopathic (1,3,4), with no identifiable familial or secondary etiology. Patients with PAP report progressive exertional dyspnea, often associated with malaise and low-grade fever. However, prominent fevers should prompt an aggressive search for an underlying infection, as discussed in more detail below. Weight loss may be a clinical feature. A nonproductive cough and, occasionally, hemoptysis may occur. On examination, patients with PAP usually do not exhibit any specific findings (2). Digital clubbing is uncommon; auscultation of the chest may reveal fine crackles, but is more frequently unrevealing. With these nonspecific symptoms, it is Can Respir J Vol 15 No 4 May/June 2008 10523_juvet.qxd 28/05/2008 11:37 AM Page 205 Pulmonary alveolar proteinosis not surprising that many patients are misdiagnosed with chronic bronchitis. When chest radiography is performed, the radiographic findings are often strikingly more severe than would be expected based on the patient’s history and physical examination. When evaluating a patient with suspected PAP, care must be taken to consider possible exposure to environmental hazards that have been associated with secondary PAP. It should be noted, however, that many of these putatively causal exposures are only described in single case reports or small case series, and hence, direct evidence of causation is lacking. Examples of occupational exposures that have been associated with PAP include a variety of organic and inorganic dusts (flour, cement, wood) and fumes (chlorine gas, gasoline, plastics). Exposure to a heavy inhalational burden of silica has long been recognized to trigger acute ‘silicoproteinosis’ (5,6), a condition that has many similarities to alveolar proteinosis, but this disorder is now uncommon in the era of workplace safety standards that mandate the use of personal protective equipment. A variety of systemic disorders have been reported to be associated with PAP, and these cases are classified as secondary PAP. Most frequently, secondary PAP is reported in association with hematological disorders such as chronic myelogenous leukemia and myelodysplastic syndromes (7,8). Altered immunity resulting from HIV infection (9), hypogammaglobulinemia (8), thymic alymphoplasia (10) and immunoglobulin (Ig) A deficiency (11) have all been associated with secondary PAP. The rare genetic disorder known as lysinuric protein intolerance, caused by a mutation in the y+L amino acid transporter-1 gene (12,13), leads to impaired membrane transport of dibasic amino acids. Its consequences include hematological abnormalities and secondary PAP (14,15). Congenital PAP, first described in 1981 (16), is a rare cause of neonatal respiratory distress syndrome. Clinically, such infants present with respiratory distress that is not responsive to surfactant or corticosteroid therapy. In such infants, lung transplantation is the only available life-saving therapy. As discussed further below, the pathogenesis of congenital PAP is quite distinct from that of PAP developing in adulthood. Biology and pathogenesis of PAP As described above, PAP of all types is characterized by the accumulation of surfactant-like material in the alveolar spaces. That the accumulated material in PAP may be a functional surfactant was first proposed in 1965 (17). In an elegant series of in vivo and in vitro experiments, Ramirez and Harlan (18) demonstrated in 1968 that the accumulation of phospholipids in the alveolar space in PAP was the result of decreased clearance, rather than of increased synthesis, of this material. They further demonstrated that the nature of the phospholipid in PAP is chemically similar (but not identical) to that of a naturally occurring surfactant. Although the latter assertion was initially challenged on the basis that the material isolated from the lungs of PAP patients lacked surface-active properties, subsequent studies have demonstrated that the material has bona fide surfactant properties (interested readers are referred to reference 2 for a review). Alveolar macrophage dysfunction: The finding that surfactant clearance is impaired in PAP led to the hypothesis that PAP is the result of a defect in alveolar macrophage function. In the 1970s, several studies demonstrated that alveolar macrophages Can Respir J Vol 15 No 4 May/June 2008 obtained from PAP patients by bronchoalveolar lavage (BAL) were phenotypically immature (19) and exhibited reduced ability to phagocytose Candida pseudotropicalis (19) and Staphylococcus aureus (20). These findings also supported the clinical observation that patients with PAP are predisposed to pulmonary infections. An early case series (21) appeared to support this latter hypothesis, describing an increased frequency of opportunistic infections in PAP patients. Many of the patients included in the studies in the present review, however, likely had secondary PAP related to an underlying hematological malignancy; many had also received immunosuppressive therapies, and it is therefore difficult to conclude from this evidence alone that PAP confers a predisposition to infection. Unexpected insights from knockout mice – the role of GMCSF: In the 1970s and 1980s, intense interest in GM-CSF had developed in the field of experimental hematology. GM-CSF is a potent stimulator of myeloid hematopoiesis, and was cloned in 1984 (22). GM-CSF binds to a cell surface receptor that is comprised of a distinct alpha chain and a beta (β) chain. The latter is also a component of the receptors for interleukin (IL)-3 and IL-5. Knockout mice lacking either the gene for GM-CSF itself (23,24), or for the β chain of the receptor (25,26) were generated in the 1990s. To the surprise of the investigators, these mice did not have abnormal hematopoiesis, but instead reliably developed a pulmonary disorder indistinguishable from PAP. Furthermore, they also exhibited defective clearance of radiolabelled surfactant components, rather than increased surfactant synthesis, in keeping with the earlier observations made by Golde et al (19). The GM-CSF receptor is expressed on type II pneumocytes and on alveolar macrophages (27,28); pulmonary epithelial cells are a source of GM-CSF (27). Local intrapulmonary delivery of exogenous GM-CSF (29) or alveolar epithelial overexpression of GM-CSF (29) can correct the pulmonary pathology observed in GM-CSF-deficient mice. Notably, GMCSF-deficient mice also have other abnormalities, including a predisposition to infections (30,31) and impaired macrophage function (32,33). Together, this series of investigations suggested that a GM-CSF deficiency in mice is a model that very closely resembles human PAP. The link between GM-CSF and human idiopathic PAP was revealed when anti-GM-CSF antibodies were detected in the serum and BAL fluid of patients with the disease (34-36). This key finding explained the attenuated hematopoietic response observed in studies of GM-CSF used as a therapeutic agent for idiopathic PAP (37-39). Furthermore, the presence of antibodies that neutralize GM-CSF in patients with idiopathic PAP provides a pathogenetic explanation for the development of the disease in the absence of genetic abnormalities of GM-CSF or its receptor. Idiopathic PAP can therefore now be classified as an autoimmune disorder (2). Why does the lack of GM-CSF, either due to its complete absence (in the knockout mouse) or to its binding by autoantibodies (in idiopathic PAP), lead to surfactant accumulation? The precise mechanism by which this occurs is not yet known, but it has been demonstrated that alveolar macrophages from GM-CSF-deficient mice exhibit decreased expression of PU.1, a transcription factor required for the functional maturation of these cells (40). These cells were unable to metabolize surfactant; however, when provided with GM-CSF in vitro, they expressed PU.1 and cell surface markers characteristic of mature macrophages, and acquired the 205 10523_juvet.qxd 28/05/2008 11:37 AM Page 206 Juvet et al ability to metabolize surfactant. PAP patients treated with GM-CSF had higher levels of PU.1 expression than healthy controls or PAP patients before treatment (41). Additional signalling events downstream of the GM-CSF receptor also appear to be important for surfactant catabolism. Peroxisome proliferator-activated receptor-gamma (PPAR-γ) is activated by GM-CSF stimulation, which in turn activates the transcription of genes required to metabolize lipids and glucose. PPAR-γ messenger RNA transcripts are absent in the alveolar macrophages of PAP patients, but are present in the alveolar macrophages of healthy control subjects (42). Moreover, GM-CSF therapy restores PPAR-γ expression to control levels in patients with PAP (42). Together, these animal and human observations illustrate the critical role of anti-GM-CSF autoantibodies in the pathogenesis of idiopathic PAP. They reveal the importance of GM-CSF in maintaining surfactant homeostasis in the alveolar microenvironment. Furthermore, they demonstrate that GM-CSF acts by driving the functional maturation of alveolar macrophages. Infections associated with PAP: Although micro-organisms can be isolated from the lungs of PAP patients by BAL, many of these potential pathogens are rarely associated with clinical evidence of infection. For example, while the lung lavages of eight of 19 sequential patients treated between 1984 and 1992 grew nontuberculous mycobacteria, none of these patients manifest clinical evidence of disease related to infection from these organisms (43). In contrast, other investigators have observed an increased prevalence of disseminated opportunistic infections in patients with PAP. In particular, infection of the central nervous system by Aspergillus species (44) and Nocardia species (45-47) has been described. In Seymour and Presneill’s 2002 review (2) of 410 cases of PAP, Nocardia species were isolated in 34 cases. They hypothesized that this observed increase in the frequency of disseminated and opportunistic infections may indicate a systemic defect in immunity in patients with PAP, although they did not detect any difference in neutrophil counts, corticosteroid use or cigarette smoking among patients with and without these infections (2). A recent publication by Uchida et al (48) provided additional insight into the increased prevalence of infections observed in patients with PAP. The authors hypothesized that the presence of GM-CSF antibodies may also contribute to neutrophil dysfunction in patients with PAP, and in the GMCSF knockout mouse. Although neutrophil ultrastructure was normal, neutrophil phagocytic capacity was reduced, as were markers of neutrophil activation in response to GM-CSF stimulation. These deficiencies were observed both in mouse neutrophils and in neutrophils from patients with PAP, but not in those from healthy control subjects. Bacterial killing by neutrophils was also moderately diminished in both the mouse model and in patients with PAP, as compared with unaffected controls. Therefore, anti-GM-CSF antibodies appear to mediate functional impairment in both neutrophils and macrophages in PAP, which translates into a global but subtle defect in the innate immune response to bacterial infection. As previously mentioned, secondary PAP has been described in the setting of HIV (9,49-52). However, this association is still restricted to case reports, and a direct pathogenetic relationship between HIV and secondary PAP has not been established. Some authors have suggested that Pneumocystis jirovecii pneumonia is responsible for these cases of PAP (50) and not 206 HIV infection per se. Moreover, the propensity for patients with either PAP (of any type) or HIV to present with opportunistic pulmonary infections such as those due to P jirovecii and Nocardia asteroides could conceivably lead to a diagnostic dilemma. In the event of uncertainty, clinicians should consider ordering HIV serology to exclude this diagnosis. Pathogenesis of congenital PAP: Deficiency of surfactant protein (SP)-B was reported in a patient with congenital PAP in 1993 (53). In fact, an inherited autosomal recessive mutation in the SP-B gene is the cause of this disorder in many cases (54). However, abnormalities in other proteins have been reported, such as the common β chain of the IL-3, IL-5 and GM-CSF receptors (55), and in SP-C (56). It appears that congenital PAP can result from diverse genetic abnormalities, with many cases as yet unexplained (57). Physiological testing On pulmonary function testing, a restrictive ventilatory defect is usually apparent in PAP patients, with reductions in total lung capacity and vital capacity (2,3). The diffusing capacity for carbon monoxide is often reduced out of proportion to the degree of the restrictive defect. Hypoxemia with an elevated alveolar-arterial PO2 gradient is the result. Arterial blood gas analysis often reveals compensated respiratory alkalosis (8). A markedly elevated shunt fraction while breathing 100% O2 has been reported in PAP patients (58). Radiographic abnormalities In PAP, posteroanterior and lateral chest radiographs typically demonstrate bilateral and symmetrical airspace opacities that seem out of proportion to the relatively modest and indolent symptoms and clinical presentation. The opacities are generally most dense in the perihilar regions, and may exhibit a ‘butterfly’ configuration, similar to pulmonary edema or P jirovecii pneumonia (1,59). Other patterns may be seen, including focal infiltrates, reticular markings, nodules and asymmetrical infiltrates. Pleural effusions and lymphadenopathy are absent but, generally, the plain radiographic findings are nonspecific. Computed tomography provides better visualization of the pulmonary parenchyma and illustrates the nature of the infiltrates. Again, however, opacities may be peripheral or central, focal or diffuse (60). Typically, ground glass opacities and increased interstitial markings are seen. Airspace opacities may be sharply demarcated from adjacent normal parenchyma in a nonanatomical fashion, creating a geographic appearance (60). The combination of interlobular septal thickening and ground glass opacification has been termed the ‘crazy paving’ appearance, due to its resemblance to cobblestones laid in an irregular, unpredictable fashion. The crazy paving appearance was once believed to be pathognomonic for PAP, but has since been described in other conditions. Examples include bronchioloalveolar cell carcinoma, P jirovecii pneumonia, alveolar hemorrhage, sarcoidosis, cryptogenic organizing pneumonia and nonspecific interstitial pneumonia (61). Exogenous lipoid pneumonia and drug-induced lung diseases may also produce the same appearance (61). Given this broad differential diagnosis, a further careful diagnostic evaluation is required. Diagnosis of PAP Laboratory studies: Most routine laboratory studies are normal in patients with PAP. Serum lactate dehydrogenase may be Can Respir J Vol 15 No 4 May/June 2008 10523_juvet.qxd 28/05/2008 11:37 AM Page 207 Pulmonary alveolar proteinosis elevated (58,62), but this is a nonspecific finding. Similarly, elevated levels of SP-A, SP-B and SP-D have been reported in PAP (63-66), but again, this finding is present in other disorders as well. Serum and BAL levels of the mucin-like protein KL-6 (67) are markedly elevated in PAP, and appear to correlate with disease activity. Overall, however, these laboratory indicators have not proven useful in making the diagnosis of PAP. Anti-GM-CSF antibodies, as described above, are central to the pathogenesis of idiopathic PAP, and have shown promise as a diagnostic test. In the initial report by Kitamura et al (34), 11 idiopathic PAP patients, two secondary PAP patients, 53 healthy individuals and 14 patients with other lung diseases were examined. PAP was diagnosed by BAL and lung biopsy. BAL was performed in all of the patients and in 30 of the healthy subjects. Blood samples were obtained from 30 of the healthy subjects, five of the idiopathic PAP patients and one of the secondary PAP patients. The authors found that polyclonal anti-GM-CSF IgG antibodies were present in all serum and BAL fluid samples from idiopathic PAP patients, but were not present in the serum or BAL fluid of any of the other subjects, including those with secondary PAP. Furthermore, serum from the three patients with idiopathic PAP in remission (ie, no symptoms, clear chest radiograph) still contained the antibodies (34). Therefore, although this was a small study, it provides powerful evidence that IgG anti-GM-CSF antibodies drive the development of idiopathic PAP and may represent a useful diagnostic tool. Bronchoalveolar lavage and lung biopsy: The diagnosis of PAP can be established definitively with BAL and/or lung biopsy. BAL fluid from patients with PAP is opaque and milky in appearance. When allowed to stand, the fluid spontaneously separates into a pale yellow, almost translucent supernatant and a thick sediment (8). A cytospin preparation demonstrates the presence of amorphous, granular, eosinophilic material. The enlarged alveolar macrophages obtained in this manner are characteristically vacuolated and foamy in appearance. Electron microscopy demonstrated the presence of tubular myelin, lamellar bodies and fused membrane structures within the amorphous extracellular material (3). These electron microscopical characteristics are morphologically the same as those of surfactant. Open lung biopsy, traditionally considered to be the gold standard diagnostic test, is now largely unnecessary in the majority of cases of PAP; the combination of the clinical presentation, imaging findings and BAL results are generally sufficient to make the diagnosis (2). Biopsy specimens reveal multiple nodular areas of consolidation, with periodic acidSchiff stain-positive acellular material filling the alveoli and terminal bronchioles. There may be a mild associated lymphocytic infiltrate in the interstitium (1), but this feature is minor in comparison with the findings in the alveolar compartment. Importantly, the underlying architecture of the lung is usually preserved. In advanced cases, fibrotic changes may develop, which can lead to architectural distortion, although this is uncommon (68-70). As is the case with BAL samples, electron microscopy can be employed to examine the acellular material for the presence of tubular myelin in difficult cases. Therapy Treatment for idiopathic PAP has evolved from the use of a variety of nonspecific and largely ineffective agents, to the Can Respir J Vol 15 No 4 May/June 2008 physical removal of the lipoproteinaceous material from the lungs, to the development of specific therapy targeted at the underlying pathogenesis of the disorder. The first publication to describe PAP (1) also discussed its unsuccessful treatment with postural drainage, antibiotics and corticosteroids. Inhalation of aerosolized trypsin (71) and lavage with heparin and saline (72) have been reported to be helpful in isolated cases, as has the mucolytic agent ambroxol (73,74). Overall, however, these therapies have not shown success in large numbers of patients (2), unlike WLL. WLL: WLL has long been considered the definitive therapy for PAP. The idea that the accumulated material could be physically removed from the lungs of PAP patients was first advanced in the early 1960s, when selective segmental lavage was reported by Ramirez et al (18,75). This was a trying experience for patients and physicians because it involved the transtracheal blind administration of warmed saline with only minimal analgesia, several times daily, for several weeks (76). However, in addition to providing the conceptual basis for the development of WLL, it represented the advent of the use of BAL as a therapeutic tool in modern pulmonary medicine. WLL is now performed under general anesthesia, with a double-lumen endotracheal tube in place. The lungs are ventilated for 20 min with 100% O2 to wash out nitrogen. The lung to be treated is then isolated at end-expiration, and the volume of that lung is estimated from the preoperative functional residual capacity. Warmed 0.9% saline is then instilled at a rate of 100 mL/min, which approximates the rate of absorption of the 100% O2 from the lung. Once the functional residual capacity has been replaced with saline, tidal volume-sized aliquots of saline are instilled under gravitational force and then allowed to drain out. The procedure is repeated, often using 10 L to 12 L of saline per lung, until the effluent, which is typically initially milky and turbid, becomes clear (77). Once this occurs, residual fluid is aspirated and the lung is ventilated with 100% O2 again. A decision can then be made whether to proceed to WLL of the contralateral lung (77). Chest percussion is commonly included in the procedure to promote clearance of the lipoproteinaceous material (78). The WLL procedure takes 2 h to 4 h and should be performed in a centre with anesthesiologists skilled in the placement and management of double-lumen endotracheal tubes. Care must be taken to avoid barotrauma. Saline thorax is another potential complication; insertion of a chest tube is occasionally required. Diuresis is forced during the procedure and for several days afterward to ensure resorption of all excess extravascular lung water. In severely hypoxic patients with PAP, WLL has been performed with extracorporeal membrane oxygenation and in hyperbaric chambers (79,80). Because no prospective studies of WLL are available, it is difficult to determine the effect of this procedure on prognosis. However, the vast majority of cases of idiopathic PAP have been reported to improve on clinical, radiographic and physiological grounds following WLL (2). Improvement in PO2, forced vital capacity, total lung capacity and diffusing capacity for carbon monoxide is expected for several days following the procedure (81). The goal of therapy is a return to normal functioning, with freedom from symptoms such as exertional dyspnea (77). The median duration of freedom from recurrent symptoms appears to be approximately 15 months, but is highly variable, with many patients needing only a single lavage (2). A small number of patients do not respond to WLL, and 207 10523_juvet.qxd 28/05/2008 11:37 AM Page 208 Juvet et al younger age appears to be the major predictor of a poor response. Nonresponders had a similar long-term prognosis to responders, save for slightly decreased survival (87%±9% versus 97%±2%) in these patients in the first six months after WLL (2). It should be noted that congenital PAP appears to be particularly unlikely to respond to WLL (2). GM-CSF therapy for idiopathic PAP: The discovery that GM-CSF-deficient mice develop a disorder resembling PAP, which resolves when GM-CSF is replaced, led to the hypothesis that GM-CSF therapy could be effective in humans with the disorder. The first prospective study of GM-CSF therapy in PAP patients began in 1995. Fourteen patients with idiopathic PAP were treated with 5 μg/kg/day of GM-CSF given subcutaneously. Five patients (36%) responded, as judged by improvements in the alveolar-arterial gradient for O2; a sixth patient responded following an escalation of the dose to 20 μg/kg/day (38). The remaining eight patients did not respond to GMCSF. All patients had anti-GM-CSF antibodies in serum obtained before the initiation of therapy. A second study, using a dosage of 5 μg/kg/day to 9 μg/kg/day, reported significant clinical, radiographic and physiological improvements in five of seven patients, including improvements in the alveolar-arterial gradient (82,83). Benefits are generally not seen for eight to 12 weeks (83). Interestingly, the hematopoietic response to GM-CSF is blunted, which appears to result from the presence of anti-GM-CSF antibodies (38,83). In summary, although GM-CSF is generally well tolerated, the overall response rate to this therapy in these two small, nonrandomized studies appears to be less than 50%, which is in stark contrast to the ability of WLL to produce rapid improvements in symptoms and pulmonary function in the majority of patients. There are reports of the successful use of inhaled GM-CSF for the treatment of PAP. Further studies of GM-CSF therapy in idiopathic PAP are clearly warranted to better define its role. Treatment of secondary PAP: If secondary PAP is diagnosed, the underlying disorder must be treated, or the patient should be removed from exposure to the suspected environmental agent. Additional treatment with WLL may still be necessary, and has been performed successfully, in secondary PAP (84-86). Because secondary PAP is not associated with anti-GM-CSF antibodies, there is no rationale for GM-CSF in its treatment. Treatment for congenital PAP: As with secondary PAP, GM-CSF therapy should not be used for congenital PAP, which is the result of SP-B deficiency or a mutation in the β chain of the GM-CSF receptor (and perhaps other unrecognized mutations). Unfortunately, congenital PAP does not respond well to WLL (2). At the present time, lung transplantation appears to be the only therapeutic option in cases of congenital PAP. Prognosis The prognosis in idiopathic PAP is considerably better than might be expected, based on the severity of physiological derangements that can arise in this condition. Actuarial fiveand 10-year overall survival rates are approximately 75% and 68%, respectively (2). Disease-specific survival is higher; approximately 88% at five years (2). The only specific adverse prognostic factor identified is age, with patients younger than five years (many of whom likely had congenital PAP) having an actuarial survival of only 14% at five years after diagnosis. Patients older than the median age at diagnosis (39 years) were 208 at higher risk of death than younger patients, but not for diseasespecific mortality (2). Although smokers are at increased risk for the development of PAP, continued smoking did not appear to influence survival (2). Overall, the risk of death from respiratory failure appears to be approximately 10% to 15% (2). Spontaneous improvement, or rarely, complete resolution, occurs in a minority of idiopathic PAP patients. In their review of all reported cases of PAP to 2002 (2), Seymour and Presneill reported that 7.9% exhibited some spontaneous improvement. That such improvement may occur has been recognized since PAP was first described, with five of the original 26 patients reported by Rosen and colleagues (1) demonstrating “definite improvement”. It should be noted that radiographic or physiological improvements are often incomplete (2). Pulmonary fibrosis has occurred rarely in PAP, typically late in the course of the disease (69,70). Its clinical and prognostic significance is unclear, but its presence would be expected to result in incompletely reversible physiological abnormalities. Whether the pulmonary fibrosis resulting from PAP is progressive is unclear. CONCLUSIONS Like lymphangioleiomyomatosis, PAP is a disease in which significant conceptual and therapeutic advances have been made as a result of investigations into the fundamental biological aberrations that characterize the disorder. The serendipitous discovery of PAP-like disease in the GM-CSF-deficient mouse, and the subsequent finding of anti-GM-CSF antibodies in idiopathic PAP patients, has recast it as an autoimmune disorder. Despite these advances, numerous challenges remain. Much about the biology of idiopathic PAP is still incompletely understood. For example, what is the role of cigarette smoking in the pathogenesis of PAP? What is the significance of the interstitial lymphocytic infiltrate that accompanies PAP? Although the evidence strongly implicates anti-GMCSF antibodies in the pathogenesis of the disease, what triggers their synthesis in individual patients and what factors are responsible for the waxing and waning course in some patients? Therapy for PAP is also an area that merits further investigation. What is the effect of treatment on the natural history of the disease? WLL effectively treats PAP in most instances, but it is cumbersome and associated with both morbidity and mortality. A treatment that can be administered by the patient is conceptually much more appealing. However, although GMCSF can be used, its 50% success rate and expense makes it suboptimal. Clearly, more research needs to be done to understand why some patients respond and others do not. Alternative therapeutic approaches are also worth considering. Given that idiopathic PAP results from the presence of an autoantibody, B cell-specific treatments may be useful. Rituximab, a monoclonal antibody directed at the CD20 molecule expressed on the surface of mature B cells, was originally employed in the treatment of non-Hodgkin B cell lymphomas, but has shown efficacy in a variety of autoimmune diseases (87). Conceivably, this agent, or a similar one, may be useful in the treatment of PAP. Similarly, it may be possible to deliver high concentrations of GM-CSF to the alveolar space to overwhelm anti-GM-CSF antibodies at their site of action. Aerosolized GM-CSF has been used successfully in PAP, and it may be possible to induce alveolar epithelial overexpression of GM-CSF using gene therapy, as Can Respir J Vol 15 No 4 May/June 2008 10523_juvet.qxd 28/05/2008 11:37 AM Page 209 Pulmonary alveolar proteinosis has been achieved in animal models (29,88). Congenital PAP, while rare, is a devastating illness that has a completely different pathogenesis. Deficiency of SP-B or the β chain of the GM-CSF receptor could also theoretically be replaced using genetic or cellular therapies directed at the alveolar compartment. With our improved understanding of the various forms of PAP, it is hoped that further advances in therapy are forthcoming. Increased understanding of PAP with the use of careful and well-designed in vitro, animal and human studies will hopefully also stimulate further research into the mechanisms of other unusual and more common lung diseases. REFERENCES 1. Rosen SH, Castleman B, Liebow AA. Pulmonary alveolar proteinosis. N Engl J Med 1958;258:1123-42. 2. Seymour JF, Presneill JJ. Pulmonary alveolar proteinosis: Progress in the first 44 years. Am J Respir Crit Care Med 2002;166:215-35. 3. Prakash UB, Barham SS, Carpenter HA, Dines DE, Marsh HM. 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Pulmonary alveolar proteinosis causing severe hypoxemic respiratory failure treated with sequential whole-lung lavage utilizing venovenous extracorporeal membrane oxygenation: A case report and review. Chest 2001;120:1024-6. Rogers RM, Levin DC, Gray BA, Moseley LW Jr. Physiologic effects of bronchopulmonary lavage in alveolar proteinosis. Am Rev Respir Dis 1978;118:255-64. Mazzone PJ, et al. Granulocyte-macrophage colony-stimulating factor therapy for pulmonary alveolar proteinosis. Am J Respir Crit Care Med 2000;161:A888. (Abst) Kavuru MS, Sullivan EJ, Piccin R, Thomassen MJ, Stoller JK. Exogenous granulocyte-macrophage colony-stimulating factor administration for pulmonary alveolar proteinosis. Am J Respir Crit Care Med 2000;161:1143-8. Yokomura K, Chida K, Suda T, et al. [Secondary pulmonary alveolar proteinosis associated with myelodysplastic syndrome.] Nihon Kokyuki Gakkai Zasshi 2002;40:599-604. Wardwell NR Jr, Miller R, Ware LB. Pulmonary alveolar proteinosis associated with a disease-modifying antirheumatoid arthritis drug. Respirology 2006;11:663-5. Kosacka M, Dyla T, Jankowska R. [Alveolar proteinosis after professional exposure to cotton and linen dust, successfully treated with whole lung lavage – a case report] Pneumonol Alergol Pol 2004;72:217-20. Kazkaz H, Isenberg D. Anti B cell therapy (rituximab) in the treatment of autoimmune diseases. Curr Opin Pharmacol 2004;4:398-402. Huffman JA, Hull WM, Dranoff G, Mulligan RC, Whitsett JA. Pulmonary epithelial cell expression of GM-CSF corrects the alveolar proteinosis in GM-CSF-deficient mice. J Clin Invest 1996;97:649-55. 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Respawn: A Distributed Multi-Resolution Time-Series Datastore Maxim Buevich † Anne Wright ‡ Randy Sargent ‡ Anthony Rowe † Dept. of Electrical & Computer Engineering ‡ Robotics Institute Carnegie Mellon University, U.S.A. {mbuevich,arwright,rsargent,agr}@andrew.cmu.edu † Abstract—As sensor networks gain traction and begin to scale, we will be increasingly faced with challenges associated with managing large-scale time-series data. In this paper, we present a cloud-to-edge partitioned architecture called Respawn that is capable of serving large amounts of time-series data from a continuously updating datastore with access latencies low enough to support interactive real-time visualization. Respawn targets sensing systems where resource-constrained edge node devices may only have limited or intermittent network connections linking them to a cloud-backend. The cloud-backend provides aggregate storage and transparent dispatching of data queries to edge node devices. Data is downsampled as it enters the system creating a multi-resolution representation capable of lowlatency range-base queries. Lower-resolution aggregate data is automatically migrated from edge nodes to the cloud-backend both for improved consistency and caching. In order to further mask latency from users, edge nodes automatically identify and migrate blocks of data that contain statistically interesting features. We show through simulation and micro-benchmarking that Respawn is able to run on ARM-based edge node devices connected to a cloud-backend with the ability to serve thousands of clients and terabytes of data with sub-second latencies. I. I NTRODUCTION Technological advances in low-power processors, communication and sensors are rapidly accelerating our ability to record information about the physical world. This data has the potential to revolutionize application domains including critical infrastructure monitoring, health care, transportation, defense systems, manufacturing, smart buildings and city-wide energy optimization. However, for this data to be actionable, we need scalable and efficient solutions designed to handle the increasingly enormous amount of data being generated. Current large-scale sensing systems are typically composed of a tiered architecture where resource constrained sensor nodes transmit data to a server-backend. Limited networking capabilities (for example in wireless networks) often force designers to significantly filter data at the source. In many cases, this data filtering removes information that could be used to support new functionality beyond the original design scope. Given the current trend in disk and flash memory technologies, it is now possible to store large amounts of data locally on an embedded gateway device. Unlike most of the approaches used in current cloud computing systems, embedded gateways are CPU limited, memory constrained and exist as field devices on networks with high latencies. Many distributed sensing systems also suffer network outages which without coordinated local storage lead to data loss. To support interactive visualization and querying of data, these systems need to intelligently organize, pre-process and pre-fetch data to provide timely access to data. Ideally, a sensor networking datastore should also have provisions to support streaming communication that might be required by control applications. The availability of fresh (recent) data often conflicts with the throughput gains achievable by batching data. In this paper, we present Respawn, a distributed time-series datastore designed to manage hundreds of thousands of sensor feeds while providing range-based queries at sub-second latencies from resource-constrained devices. To achieve this, Respawn leverages two concepts: cloud-to-edge partitioning and multi-resolution storage. In Respawn’s distributed architecture, communication is load balanced between a few serverclass machines and many inexpensive, low-end embedded edge devices. This is achieved by partitioning the data between the cloud and the edge, storing the low-resolution aggregate data in cloud nodes and the high-resolution data at fielddeployed edge nodes. A dispatcher front-end is responsible for directing queries between the cloud and the edge and, in effect, maintaining low request latencies. The dispatcher is designed to operate predominantly out of memory to support tens of thousands of concurrent connections. A bloom filter-based caching layer is used to determine the location of requested data and to avoid costly network accesses. For multi-resolution storage, Respawn leverages the opensource Bodytrack Datastore (BTDS) [1], a light-weight multiresolution datastore for time-series data. On edge node gateway devices, BTDS is responsible for down-sampling incoming data and storing hierarchically-organized copies of the data at differing resolutions. This pre-processing upon ingest of the data significantly improves range-based queries where a subset of data is requested from an arbitrary timespan. In the evaluation section, we show that BTDS can perform rangebased queries running on an embedded platform at nearly the same speed as range-based requests for standard relational databases running on server-class hardware. We also extend BTDS to support lossless compression, where different levels of compression are applied to different levels in the hierarchy, allowing for more commonly accessed data to be served faster. Since edge nodes typically have latencies of almost an order of magnitude greater than the cloud, selective data migration can be used to further improve latency. A Qualityof-Service (QoS) parameter at each gateway node is used to determine how much bandwidth is available for data and hence how aggressively tiles can be migrated. Top-level aggregate tiles are migrated first since these are typically used as starting points for range-based queries. Low-level (higher resolution) tiles are migrated based on both client access patterns and based on data metrics like standard deviation. Standard deviation is one of many metrics that can be used to pinpoint tiles that would be of more interest to clients. We evaluate these different migration schemes through a small user study in which participants are required to search through data. In order to evaluate the performance of Respawn, we benchmark ingest and query times on a single server machine comparing against MySQL, SQLite and OpenTSDB. This includes an evaluation of the impact of using LZ77 compression. We also benchmark BTDS on memory-contrained 450Mhz ARM9 embedded hardware which cannot support MySQL or OpenTSDB. Finally, we evaluate our latency masking techniques using user traces from a network consisting of an embedded gateway board, dispatcher, and cloud datastore. Using trace-driven simulation, we evaluate how the system scales across large networks with thousands of edge nodes. In summary, this paper makes the following three main contributions. First, it provides the design of a highly-scalable time-series datastore that leverages local multi-resolution storage, along with compression, and is able to operate on embedded hardware. Second, we present a cloud-to-edge handoff mechanism with intelligent caching to reduce latencies for interactive queries and visualization. Finally, we evaluate the system in terms of single-server performance and horizontal scalability through the addition of edge nodes. II. R ELATED W ORK It is becoming increasingly apparent that no single approach can solve all data storage problems [2]. For sensor networking applications, it is important to be able to store and process many streams of data simultaneously. There have been several efforts to build upon databases like MySQL to support stream processing functionality like pattern correlation queries [3]. Other similar work has tried to incorporate stream processing engines and historical storage [4]. These efforts illustrate many of the challenges involved with large-scale time series data, even in the context of highly-connected and resource abundant datacenters. In this section, we highlight a few of the key distinctions between traditional databases and those designed for sensing applications. A. Time Series Datastores There have been many examples of time series databases for infrastructure monitoring (for example in data centers) and financial analysis, such as DataGarage[5], RRDtool [6], tsdb [7], TSDS [8], OpenTSDB [9], Dremel [10], Vertica [11] and DataSeries [12]. Some of these databases already support multi-resolution queries [5], [6], [9], [11]. However, none of them natively support sub-second time stamping. RRDtool is one of the few options that can operate on an embedded target, but it is designed to run locally and does not provide any distributed management features. Many of these databases do not natively support access control [5], [6], [7], [9], [10], [12]. Even the ones that do have access control [11] lack perstream permissions and do not operate in a distributed manner. Respawn in contrast embraces a cloud-to-edge distributed operation with per-sensor stream security through use of the a Publish-Subscribe access control model. The OceanStore [13] project has very similar goals and features to Respawn. OceanStore is a global persistent data store designed to be highly scalable. One of its primary goals is to provide consistent and highly-available service to an infrastructure running on top of untrusted devices. It also strives to reduce latency by capitalizing on data locality whenever possible. However, OceanStore is not primarily a time series database and hence does not provide multi-resolution access. Because the data could be significantly more general, it utilizes access-based caching schemes rather than proactive data migration. It is also not optimized to run on embedded targets. B. Streaming and Aggregation Another approach to managing continuous data is to perform on-the-fly aggregation using stream processing techniques [14] [15]. This approach works well for networks that have enough provisioning to transfer data in real-time, but suffer in that they potentially lose information that could be processed later. In Respawn, we leverage an XMPP communication layer to deliver real-time transducer information and then provide a componentized storage facility that operates in the background. Decoupling the design of the stream-processing functionality from the historical data allows the system to more flexibly batch entries to increase ingest throughput. C. Sensor Network Databases Many databases have been proposed that operate within sensor networks, such as Diffusion[16], Tag [17], TinyDB [18], Cougar [19] and DIMENSIONS [20]. Diffusion provides a routing and in-networking processing approach that propagates queries using flooded messages. Cougar uses a similar approach only it uses a static configuration of nodes that is selected using a proxy front-end. This proxy front-end is still responsible for dispatching requests out to the leaf-nodes of the sensor network for data collection. There is no notion of storage or caching at the proxy. TinyDB focuses on running the database within resource constrained sensor nodes on microcontroller hardware. Queries are processed directly on endpoint sensor devices. Respawn is more geared towards data management that runs on sensor gateway collector devices rather than on the leaf nodes themselves. In many cases, these gateway collectors could be connected to proprietary networks where it would be difficult to run custom software. For example, one could connect a Respawn edge node to a buildings BACnet data feed and it would act as a consolidator of potentially hundreds of sensor streams. Dimension further improves upon the idea of in-network storage by using wavelet transforms to deal with data aging and multi-resolution. D. Middleware Certain sensor networking middleware frameworks also provide storage capabilities. sMAP [21], for example, focuses on with data delivery, but also provides querying and retrieval of sensor data. sMAP is compatible with MySQL for storage, but can also use a custom binary time-series datastore called readingDB to help cope with the performance challenges. readingDBs performance is significantly improved over MySQL, latency/bandwidth  barrier   edge  data   request   WSN   client   edge  nodes   address   request   mobile   device   dispatcher   cloud  data     request   cloud   node   migra;on   Fig. 1: Respawn High-Level Architecture but it is still primarily a centralized datastore without builtin load balancing. readingDB does however provide delta encoding and zlib compression and can be manually distributed based on how sMAP is configured. In contrast, Respawns design is centered around the notion of edge-to-cloud partitioning and is highly distributed by default. Its primary goal is latency masking which it achieves through intelligent data migration. E. Why Respawn? Data centers have become increasingly effective at providing low-latency access to vast amounts of data. Services like Google Maps allows people to interactively scroll through databases that are hundreds of Terabytes in size with hundreds of Gigabytes of indexing information. Sensor data and the way in which it is collected differ significantly from the type of information that current data center practices are so efficient at handling. Typical operations performed on time series data include plotting, zooming, correlation, clustering, prediction, pattern matching queries and summarization. Time series processing systems often have both a historical and real-time component. In order to optimize ingest throughput, databases often batch large segments of data. Unfortunately, this introduces significant delay. Most traditional databases deal with read and write transactions that are for the most part independent. In time series databases, there is a constant stream of appends with intermittent queries. Specific to sensing systems, data sources are often distributed and must be collected on field devices. Unlike data centers with high-speed network interconnects, field devices are frequently connected with expensive links like cellular or satellite where customers are charged per kilobyte. Few open-source time series databases provide the timing granularity required for capturing physical events. The majority of these systems were designed for data center monitoring or financial applications that require integer second timing granularity. Sensing systems should be able to distinguish sub-second events. As sensor networks are finding their way into a wider range of large-scale complex applications, there is a growing need for a scalable storage platform customized for these particular needs. III. D ESIGN AND I MPLEMENTATION The Respawn architecture enables interactive exploration and responsive querying of time-series sensor data, despite the data originating from embedded nodes behind a latencybandwidth barrier. A high-level representation of the Respawn system architecture is displayed in Figure 1. Sensor readings are generated at the edge, in most cases from mobile devices and wireless sensor network deployments. The sensor data streams are transmitted through a series of adapters to Respawn edge nodes, low-cost embedded gateways outfitted with large amounts of flash storage. Edge nodes process and store the data locally and periodically notify a Respawn cloud node of new data received. Cloud nodes are server-class machines which handle much of the load of and serve as low-latency data caches for the Respawn system. The cloud nodes also facilitate a continuous migration of a subset of the total data being stored by the edge nodes. When data is migrated from the edge, the cloud node notifies the Respawn dispatcher. The dispatcher serves as the system entry point for almost all data requests initiated by clients. It is designed to handle large volumes of small requests and to maintain thousands of open connections at once. The dispatcher’s primary function is to redirect requests from clients to end-nodes. Redirections are decided based on client-perceived latency, as well as load on individual edge nodes and load on the entire network. In Section IV, we show that despite the centralized nature of the dispatcher, it scales to handle thousands of simulataneous users and does not represent a significant performance bottleneck in the architecture given our target network sizes. A. Multi-Resolution Datastore For the purpose of local data storage, Respawn leverages Bodytrack Datastore (BTDS), an open-source time-series datastore which targets multi-resolution queries and interactive visualization. BTDS stores raw data streams in a lossless format and additionally generates lossy aggregate versions of the streams for query acceleration. In the current implementation, aggregates are calculated by finding a mean average of datapoint values over a set window, however, it is possible to incorporate other aggregation functions. BTDS supports a command line interface for local client requests and an HTTP/JSON interface for web requests. 1) Data Model: A single instance of the datastore is a collection of data streams, with each stream separated into a single raw version and many aggregate version (Figure 2). An aggregate is a representation of the raw data down-sampled by a power of two, and therefore corresponds to a distinct level of resolution. A single aggregate is composed of many tiles, tiles in turn are composed of one-point buckets. Formally, a tile represents a contiguous time-range and a collection of m equally-sized buckets. A bucket is a ”smaller” contiguous time-range containing either a single datapoint or no datapoint. In a single instance of the datastore, all tiles contain exactly m buckets (regardless of resolution level) and thus contain a maximum of m points. Buckets at level L are half the size temporally of buckets at level L+1, thus tiles at level L represent a time-range half the size of tiles at L+1 (please refer to Figure 2). This also means that, for any arbitrarily-chosen range in time, the level L view of the data contains twice the buckets and is up to twice the resolution of the level L+1 view of the data. By convention, the bucket ”width” (time duration represented by a bucket) at an arbitrary level is equal to 2level seconds; thus at level 0 a bucket has a ”width” of 1 second. A tile is uniquely addressed by its resolution level and offset. A tile’s offset at level L corresponds to its distance in time from the Unix epoch as measured in tiles of level L. …   (le   2   offset   1   0   resolu&on  level   bucket   1   1   0   2   3   0   0   2   1   3   4   6   5   each time-series datapoint will contain a timestamp, a mean value, a standard deviation, and a count. Floating point values are stored with 64-bit precision. Count and standard deviation are meaningful for low-resolution aggregate tiles because they represent the number of raw datapoints used for calculating the mean value and the standard deviation of the datapoints. The format for a JSON tile response is given below: 7   -­‐1   (response example:) { …   "fields": [ "time", "mean", "stddev", "count" ], "data": [ [1367968303.677585, 486.696, 12.053, [1367969280.41341, 494.2760, 4.347, [1367970305.185479, 498.36, 3.0824, ... [1368391167.9347, 476.883, 5.690, ], "level": 10, "offset": 2609, 0   4   8   Unix  &me  (seconds)   12   raw  data     stream   Fig. 2: BTDS Multi-Resolution Storage Figure 2 shows an example of how aggregate data tiles are organized within BTDS. The raw data stream and three of the aggregate versions are displayed (other aggregates are not shown). Each aggregate is represented by tiles of m=4 buckets (a small m value was chosen for presentation purposes). The stream in this example begins at the Unix epoch, thus the leftmost tiles have an offset of 0. At each level of resolution, tile offsets are incremented as time progresses. BTDS stores data streams as uncompressed binary files on disk (In Section IV we explore the impact of adding compression to BTDS). Sets of contiguous tiles at a single resolution are organized into files in much the same way that buckets are organized into tiles. Files are named according to their corresponding range of offsets and are organized into two directory levels. Top-level directories separate individual data streams while bottom-level directories separate resolution levels within data streams. The structure of BTDS maps well to the organization of many common filesystems and enables performance benefits associated with filesystem-level caching and optimized lookup. 2) Making Requests: Respawn queries are implemented on top of independent BTDS tile requests. In Respawn, BTDS tile requests are made via HTTP and return JSON (Javascript Object Notation) representations of tiles. A tile request must specify a data stream, a tile resolution level, and a tile offset. Data stream names are split into two parts: a device name and a channel name. Channels which are grouped into devices can share timestamp values, resulting in accelerated ingest and saved disk space. The level and offset specified in a request maps directly to a tile position in the data stream. Because all tiles are precomputed at the time of ingest, requests are made in constant time, regardless of the resolution level. The format for an HTTP tile request is given below: (request format:) GET /tiles/DEVICE.CHANNEL/LEVEL.OFFSET.json (example:) GET /tiles/sensor.temperature/10.2609.json Each tile request yields JSON data for a single tile. A response contains an array of datapoints, an array of fields describing each datapoint, and the tile level and offset. Typically, 1125], 1507], 1520], 1531] } B. Distribution Layer Components Time-series sensor data destined for storage enters through the edge nodes. It is first buffered in memory for a predefined amount of time so as not to incur the overhead of constant writes to the BTDS datastore. Periodically, the buffered datapoints are written to the datastore and committed to disk. The BTDS datastore parses the incoming points and, based on timestamp, collects them into tiles. Raw timestamp and value information is stored in the highest-resolution raw tiles; all other tiles are aggregate tiles generated from raw tiles. In addition, edge nodes run security key generators that publish keys to XMPP, which maintains network-wide access control. A primary function of the cloud node is to serve data summaries collected from the edge nodes in the network. To improve latency, the cloud node maintains a collection of persistent and non-persistent data structures. The first of these is the Summary datastore, an instance of the BTDS datastore containing migrated edge tiles which are likely to be requested in the future. The migrated tiles represent an approximation of the total data in the distributed datastore. The storageconstrained nature of the edge nodes necessitates data aging, but this is not required of the cloud node. Additional modules are maintained to optimize accesses to the Summary datastore and to enforce access control policies; these are discussed further in the section on data migration. The dispatcher acts as the first point of entry for almost all client-initiated data requests. It redirects requests for data tiles of particular channels to the appropriate end-nodes, either edge or cloud. The dispatcher does not serve time-series data; requests and response payloads are kept small (i.e. smaller than 100 bytes). This, coupled with the fact that most dispatcher data structures are kept in memory, enables the dispatcher to serve a steady-state stream of over ten thousand requests per second (as described in Section IV). The dispatcher’s data structures are primarily copies of the structures generated by the cloud node. For most requests, the dispatcher decides where clients are redirected to and, as a result, how load in the system is distributed. (B)  Cloud  Hit   (A)  Unmigrated   (C)  Cloud  Miss   client   client   l/bw  barrier   edge     node   A2   B1   l/bw  barrier   A1   data   store   DS   dispatcher   edge     node   B2   data   store   DS   dispatcher   cloud  node   cloud  node   A1=AddrRequest    A2=EdgeRequest   client   B1=AddrRequest    B2=CloudRequest   C4   l/bw  barrier   edge     node   C3   DS   dispatcher   C1   C2   data   store   cloud  node   C1=AddrRequest    C2=CloudRequest    C3=EdgeAddrRequest    C4=EdgeRequest   Fig. 3: Request Transaction Types An XMPP back-end hosts a publish-subscribe network and is used by the three primary components for the sharing of event notifications and other lightweight metadata. XMPP also provides a delivery system for streaming data, as well as access control and security. D. Data Migration C. Request Redirection 1) Predictive Caching: As an alternative to the classic caching scheme we have developed methods for Predictive Caching in order to minimize the effect of the latencybandwidth barrier. Predictive caching leverages the structure of time-series sensor data by analyzing and isolating certain characteristics of the data. The data characteristics are used in the construction of a prediction model which decides which portions of data are likely to be requested and should be preemptively cached. We have defined and implemented two predictive caching schemes in Respawn, Periodic Migration and Proactive migration. In this section, we discuss the details of the Respawn dispatcher and the movement of a client’s data fetch through the Respawn system as shown in Figure 3. All of the requests described in this section are standard BTDS-format HTTP requests extended with a security key. A data retrieval is initiated by an AddrRequest arriving at the dispatcher. The dispatcher uses an access control list to map the requested channel to the security key currently in use by the channel’s edge node. A key mismatch causes an error response to be sent. Upon a key match, a list of base resolutions and a summary bloom filter are checked. If either match, a redirection to the cloud node is returned. The Bloom filters are sized to keep the probability of false-positives low, implying a reasonable assurance that in the case of a match the requested tile exists at the cloud node. If the previous check resulted in false outputs, the requested tile has not been migrated and a redirection to the edge is returned, causing the client to make a subsequent EdgeRequest. The probabilistic nature of the CTR Bloom filters allows for false-positive cloud redirections to occur. This results in three main types of request traces in the Respawn system: ”Unmigrated”, ”Cloud Hits,” and ”Cloud Misses.” The ”Unmigrated” traces are the simplest of the three. When a request processed by the dispatcher fails to positively match, the tile has not been migrated and the client is redirected to edge. When a match on the bloom filters occurs, it is only likely that the tile has been migrated. Hence, a small percentage (i.e. 1%) of CTR Bloom filter matches result in ”Cloud Miss” traces. In such cases, the client is redirected to the cloud node, causing it to send a CloudRequest. The request is checked against the full Cloud Tile Registry (CTR), which returns a negative result, signifying that the tile has not been migrated. The negative result is returned to the client, causing the client to issue an EdgeAddrRequest. An EdgeAddrRequest is functionally identical to an AddrRequest, with the exception that it is guaranteed to redirect the client to the tile’s edge node. Once the client receives a response to the EdgeAddrRequest, it uses the response to generate an EdgeRequest, which it sends to the appropriate edge node. Upon receiving the request, the edge node verifies the client’s access, retrieves the tile from the local datastore, and serves the client’s request. For distributed storage systems which leverage storage at the edge, the quality of network links to the edge devices can be a substantially limiting factor for performance, especially in terms of latency. In this section we will describe the mechanics of data migration used to reduce access latencies. Periodic Migration: When browsing, clients will frequently request tiles with the lowest resolution first, in order to see a ”big-picture” view of a dataset. From there they will ”drill down” to explore particular sections of the data at a higher resolution. The effect of this phenomenon is that lowresolution tiles are on average requested more often than highresolution tiles. Thus, it is often practical to bias tile migration toward lower-resolutions. The Respawn cloud node periodically migrates all tiles at or below a predefined resolution, which can be assigned on a per-edge-node or per-device basis. Periodic Migration is most effective for periodic data feeds from time-triggered sensing systems. Proactive Migration: Whereas Periodic Migration leverages resolution information about data tiles for migration decisions, Proactive Migration involves extracting characteristics from the data itself for the same purpose. In Respawn, each tile generated at the edge is assigned a value corresponding to the standard deviation of the sensor values contained within the tile. For many datasets, standard deviation was found to be a good scalar approximation of the statistical importance of a time-series data-range. Furthermore, standard deviation over tiles is simple enough that it can be computed upon initial ingest of the data, even on a low-end embedded platform. We found Proactive Migration based on standard deviation to be especially effective at accelerating access times for sparse data feeds containing isolated events of interest. This suggests that Proactive Migration is especially suited for handling data feeds from event-triggered sensing systems. 2) Mechanics of Migration: Respawn maintains and migrates a collection of data structures to facilitate request redirection. The first of these is the Summary datastore, a subset edge  node   *   cloud  node   Edge   Node   dispatcher   IP  address,  security  key   Edge  Node  List   Edge  Node  List   XMPP   Cloud   Node   Dispatcher   IP/Key   Event   Base  Resolu7ons   Summary  Datastore   Summary  Tiles   *   BTDS  Mul7-­‐Res  Data   *   latency/bandwidth  barrier   *   Summary  Bloom  Filters   Base  Resolu7ons   fetch   Cloud  Tile  Registry  (CTR)   HTTP   Summary  Bloom  Filters   New  Tile   Event   XMPP   Summary   Update   Event   fetch   *locally  generated  data  structure   Fig. 4: Data Structure Migration within Respawn of the tiles in the system chosen for migration (Figure 4). Two additional modules are maintained to optimize accesses to the Summary datastore: The Base Resolutions and the Cloud Tile Registry (CTR). The Base Resolutions are a mapping of time-series data channels to their base migration resolution, the resolution R for which the cloud node is guaranteed to migrate all tiles belonging to that channel whose resolution is R or lower. The CTR is a registry containing the addresses of all migrated tiles not represented by the Base Resolutions. Like the rest of the data structures maintained by the cloud Node, the CTR is segregated by channel and is thus implemented as a map from channel to list of unique tile addresses. The cloud node makes migration decisions on a per-datachannel basis and thus a set of migrated tiles for one channel is independent from the set of another channel. Access to CTR addresses are further optimized by the addition of Summary Bloom filters. This enables faster, no-false-negative lookups on the existence of addresses. The last high-level structure maintained by the cloud node is the Edge Node List, which maps channels to their associated IPs, security keys, and other access control metadata. This listing is checked on every request in order to verify that a client requesting cached data originating at an edge node is authorized to access the edge node. 3) HTTP vs. XMPP: Respawn separates two common modes of communication, event notifications and bulk transfers, and implements them with two distinct layer-7 protocols. This choice has the benefit of increasing responsiveness to client requests, horizontal scaling potential, and reliability in overload cases. Event notifications, the first mode, are always sent as the direct or indirect result of an asynchronous sensor event entering the system through an edge node. These notifications are lightweight data transfers whose destination is a single node or a subset of all nodes in the system; global broadcasts are very rare. In many cases, event notifications cannot be predicted and thus it is difficult for a receiver to control data flow without severely hindering communication. Respawn uses the XMPP publish-subscribe protocol for these types of transactions. XMPP provides an event node abstraction for which each event node has one or more publishers and one or more subscribers, in addition to tunable quality-of-service parameters to place limits on data flow. Each event node is also associated with an access control list. Respawn uses these access control features as a basic building block in its security model. XMPP was explicitly designed for lightweight, asynchronous massaging and is thus a good fit for Respawn event messages. Data fetches represent bulk transfers of structured data facilitated by client-server interactions. They can occur both periodically and as a result of an asynchronous event being received. Respawn uses HTTP for transferring time-series tiles and large metadata structures. HTTP serves these purposes well as a result of its simplicity, its bulk transfer efficiency, and its request-driven model. One common limitation of HTTPbased systems is the need to periodically ”poll” for data, which can result in wasted bandwidth in cases where data is not yet available or does not exist. This effect is mitigated in Respawn because most fetches are triggered by the receipt of a lightweight XMPP event. The use of HTTP’s requestdriven model is also key in preventing overload for the most important components of Respawn. In terms of bandwidth, most back-end data transferred by the dispatcher and cloud node are initiated by the dispatcher and cloud node. Thus, it would be difficult to overload the dispatcher and cloud node through the addition of edge nodes. 4) Bloom Filter: A bloom filter is a probabilistic data structure used for quickly and space-efficiently testing an element’s membership in a set. Bloom filters are most commonly implemented as bit arrays in which each element is mapped to multiple bit positions via multiple unique hash functions. A set is queried for an element by hashing the element with the same set of hash functions and checking whether all of the bit positions are set to 1. Querying a bloom filter does not return an exact result; false positives are possible while false negatives are not. Respawn maintains a bloom filter per data stream in the Summary Bloom Filters data structure. As tiles are added to a Bloom filter, the filter becomes less accurate and produces a larger percentage of false positives. A Bloom filter which produces a false positive ratio of 1% contains 9.57 bits of space per element in the set. When the number of elements in a Bloom filter becomes too great to maintain a false positive ratio of 1%, the cloud node will ”resize” the bloom filter by generating an empty one of double size, rehashing the values, and reinserting them. 1K-point range query time (ms) 100000 Fig. 5: ARM9 Embedded Gateway IV. E VALUATION In this section we evaluate the primary components of the Respawn system. By micro-benchmarking BTDS against MySQL, SQLite, and OpenTSDB, we profile the ingest and query performance of a multi-resolution architecture against standard relational and time-series architectures. We also show initial compression ratio and compute the overhead for performing gzip compression on a large environmental sensor dataset. We then perform a design-based simulation of various dispatcher architectures and then benchmark the actual throughput of our implementation. Finally, we use traces collected from users browsing a typical time-series dataset to evaluate various Respawn migration schemes. A. Multi-Resolution Storage Each database was profiled on an 8-core E5320 Intel Xeon CPU clocked at 1.86GHz with 4GB of RAM. Since BTDS fits within a small enough footprint to operate on edge-node routers, we also benchmark it on a 450MHz ARM9 processor with 32MB of RAM operating from a 64GB flash micro-SD card (shown in Figure 5). Unless otherwise specified, each point on the following graphs represents the average of at least one thousand runs. Table I shows the average request time required to fetch a 1024-point (1K) block of data from each database at a random location within a one million point dataset. We see that SQlite performs best and BTDS runs slightly faster than MySQL and significantly faster than OpenTSDB. Enabling compression adds a factor of six slowdown in BTDS performance. The embedded platform runs approximately 13 times slower on the embedded target. Given that each embedded edge node will receive a small fraction of the requests as compared to the main server, the overall response time of 310 ms is promising. Database BTDS BTDS Compressed BTDS Embedded SQlite MySQL OpenTSDB 1K-Point Random Query Time (ms) 23.86 159.51 310.98 11.98 25.49 1206.38 TABLE I: Full resolution query performance BT datastore SQLite MySQL OpenTSDB 10000 1000 100 10 100000 1x106 1x107 Dataset Size (# of datapoints) Fig. 6: Range-based query performance (log-log scale) Next we evaluate the performance of range-based queries that are defined by a starting timestamp, an end timestamp and a resolution. On standard relational databases like MySQL, this requires fetching raw values and then computing aggregates. BTDS performs its processing on ingest. Figure 6 shows the difference in performance of each database with respect to 1Kpoint range-based query. The x-axis shows the performance as the size of the database increases. For standard databases, as the size of the dataset increases this incurs a larger penalty when computing aggregates. Note the log-scale on both the x and y axis. On small datasets, SQLite outperforms BTDS. However as the dataset grows, BTDS’s ability to efficiently traverse pre-computed aggregates allows it to provide a constant query response time independent of the dataset size. Both SQLite, MySQL and OpenTSDB show drastically increasing delay as the data scales. Next, we evaluate the ingest penalty required for fast range-based queries. Table II shows the total time required to ingest a 1M-point dataset. BTDS is approximately 1.7 times slower than SQLite and over 9 times slower than MySQL. The embedded BTDS target is more than 70 times slower than its server counter-part due to poor disk I/O performance. Since the embedded nodes are closest to the streaming data, it would be rare for the system to need to ingest a large (1 year) batch of data. The compression overhead on write ends up being approximately a 400% decrease in performance. As the database increases in size, BTDS must aggregate data across increasingly larger datasets. Figure 7 shows that the BTDS ingest performance scales as the dataset increases but rapidly levels off. MySQL, SQLite and OpenTSDB remain nearly constant since they are typically just seeking to the end of a structure and appending data. With a sensor input rate of 1Hz, the average ingest performance stabilizes at about half a year worth of data and still remains competitive with MySQL. Database BTDS BTDS Compressed BTDS Embedded SQlite MySQL OpenTSDB 1M-Point Ingest Time (seconds) 373.03 1503.32 26404.00 217.18 41.12 6072.56 TABLE II: Ingest performance with 1K batches ingest time for 1K pts per stream (s) 1K-point Ingest Time (seconds) BT Datastore (raw) BT Datastore (smoothed) mySQL (smoothed) Sqlite (smoothed) OpenTSDB (smoothed) 10 1 0.1 0 5x106 1x107 1.5x107 2x107 2.5x107 400 200 0 0 2000 Stored Data Size (number of raw datapoints) 4000 6000 8000 10000 Number of streams Fig. 9: Ingest Performance vs Number of Streams BT datastore time SQLite time MySQL time OpenTSDB 10000 600 500 1000 # of occurrences 1M-point ingest time (seconds) 600 3x107 Fig. 7: Ingest Performance vs Datastore Size (log scale) 100 10 1 G=1, P=1 G=10, P=1 G=100, P=1 G=1, P=10 G=10, P=10 G=100, P=10 400 300 200 100 0 0 5000 10000 15000 batch size (number of raw datapoints) 20000 0 0.5 1 1.5 2 Compression Ratio Fig. 8: Ingest Performance vs Data Batch Size (log scale) Fig. 10: Compression Ratio for Example Dataset The distributed nature of Respawn also horizontally scales to alleviate central ingest bottlenecks. motion, pressure, humidity and audio as well as a wide variety of HVAC sensors that monitor blower speeds and water intake temperatures. We see that in general, the data compresses to 25% of its original size, however the runtime performance overheads are non-trivial. As future work, we intend to investigate utilizing the tile structure of our database to compress only infrequently access tiles or ones with low-standard-deviation numbers that would achieve higher compression ratios. One mechanism used to optimize reads and writes in streaming storage is batching of data. Figure 8 shows how each database scales in terms of ingest time as the batches of points increase in size. They all follow a similar trend and level off with approximately 1K data points. 1K data points on a 1Hz sensor feed corresponds to 15 minutes of data. There is a trade-off with how much a sensor should be willing to batch in memory and how soon data becomes available to incoming queries. Approaches like [22] have proposed making queries on both disk and in-memory to reduce those overheads. Another technique for improving ingest performance is to group sensors that have identical timestamps. In BTDS, grouping can be used to save on the bookkeeping overhead normally required to setup a unique timestamp entry. Figure 9 shows the time required to ingest 1K-points per stream as the number of total streams is increased. Each line represents a different grouping size G and a different number of processor cores P used by the server. We see that grouping sensor improves efficiency up to a point, but saturates at about 10 inputs. We also see that BTDS becomes CPU limited since increasing the number of cores is able to double performance. Figure 10 shows a histogram of the compression ratios achieved when running gzip compression on a 21GB repository containing 6 months of mixed time series sensor data. The data consisted of environmental sensors like light, temperature, B. Dispatcher Next, we compare against the architectures shown in Figure 11. The first is a centralized architecture where all nodes stream data to the cloud for storage and there is no database running on the edge nodes. The second architecture, proxy, is one where storage exists on the edge nodes and requests are routed through the cloud. The final redirection architecture is the most distributed option, where a a lightweight dispatcher redirects requests either to the cloud or, in this case, only to edge nodes that are running a local copy of the database. Figure 12 shows the limit on number of requests per second in each architecture. These lines were generated using a simulation based on hardware profiles. Networking overhead was determined by running requests from a separate machine against the database or dispatcher until its performance saturated. Real requests were generated and rerouted to closely mimic the real system. Since we do not have access to thousands of edge nodes, we assume that these requests can Centralized   Proxy   Client   Redirec2on   Fig. 11: Dispatcher Architectures Request limit (requests/second) 18K Client   Client   Central Storage Classic Cache Redirection/Hand-off 16K 14K 12K 10K 8K 6K 4K 2K 0 0 be serviced outside of the local network independently (i.e. requests are directly handed off to edge nodes do not interfere with each other). We also assume that the requests for each edge node arrive in an equally independent fashion. The centralized storage architecture is limited by the number of simultaneous requests that can be made to BTDS (in this case 80 per second). As the number of nodes in the system scales, the performance gradually worsens as additional edge nodes consume the server’s bandwidth. This approach, which is most common in simple sensor collection systems, scales very poorly. The proxy architecture shows improved scalability due to its ability to offload the majority of the database workload to the edge. The system becomes network bandwidth constrained at about 2000 requests per second. For many applications, this approach is simple and able to handle medium workloads. The Respawn components are flexible enough that they can be easily configured to support this type of access pattern if so desired. The redirection and hand-off approach which best represents the standard Respawn configuration scales quite well supporting up to 16,000 requests per second. Eventually, the limiting bottleneck is the performance of the dispatcher. These operating points can be used as general rules of thumb to determine if a particular scheme meets the expected application demands. C. Migration In this section, we evaluate three different tile migration schemes. The first is a simple periodic migration technique where top-level aggregate tiles are periodically migrated based on available bandwidth of the edge node. Since each Respawn edge node is configured with a QoS limit by the user or is limited by the network connection itself, tiles are transmitted at a fixed rate so as to keep average throughput below the configured setting. The second scheme is a statistical pre-fetch mechanism that prioritizes which tiles get migrated based on standard deviation of the data. The intuition being that clients are more likely to investigate areas where the data is exhibiting significant changes. Again, these tiles are migrated within the specified QoS limits. The final scheme is a combination of the first two approaches. In this case, tiles are selected in a round-robin fashion from the periodic and the stddev-based transmit queues. The cloud caches data periodically at a lower granularity and also maintains a few regions of highly variable data ready to serve. In order to evaluate these migration approaches, we performed a small user study where participants were asked to browse around a web-based plot of a week worth of time-series 2000 4000 6000 8000 10000 Number of edge nodes Fig. 12: Architecture Request Limit vs Number of Edge Nodes data from environmental sensors placed in our lab. Users were asked to determine approximately how many hours in the last week a lab was occupied based on sensor data. This forced users to navigate and zoom into each region to determine the length of each section and if it was continuous. Each click and zoom selected on the plotting tool logged the corresponding range-based query to the database. These traces could then be played back against a version of the system executing each of the migration schemes. Figure 13 shows the latency per time of a characteristic trace performed on the data. In (a), there was no migration. Each request shown by a small circle was redirected to an embedded cloud node that was running on the campus wireless network. These requests took on average 340 ms. The grey background shading shows a sliding window average of the latency to help visualize the impact of clusters of high and low latency requests. In trace (b), the system utilized periodic tile migration. Accesses that went to the cloud instead of the edge are shown with much lower latencies that correspond with connecting to a wired server machine on campus. In trace (c), the variance-based prefetching approach is used to migrate data with higher levels of variation, a scheme which has a slight performance increase over periodic migration. Finally, trace (d) shows both periodic and variance-based prefetching, which achieves the best performance at an average latency of 85 ms. In all of these cases, the migration bandwidth was kept constant, and 5% of edge tiles were migrated. We see that combining both approaches is most effective in our tests. This can be attributed to users making a reasonable number of lowresolution tile accesses to get an overview and subsequently ”drilling down” into regions containing interesting features. V. C ONCLUSIONS AND F UTURE W ORK In conclusion, in this paper we present a highly-scalable distributed time series datastore called Respawn. Respawn is able to run a small multi-resolution datastore on embedded leaf nodes that pre-processes incoming data in order to accelerate range-based queries. A high-speed dispatcher server is responsible for routing client requests to either a cloud storage backend or directly to the edge node. We propose and evaluate various data migration techniques that define how edge nodes should push tiles to the cloud backend to help mask latency for client request. This architecture is ideal for supporting interactive visualization tasks, allowing scientists to Latency (secs) Latency (secs) 0.8 0.6 0.4 0.2 0 0 20 40 60 80 100 0.8 0.6 0.4 0.2 0 120 0 20 40 Trace Time (secs) 0.6 0.4 0.2 0 20 40 60 80 100 120 80 100 120 (b) periodic Latency (secs) Latency (secs) (a) no migration 0.8 0 60 Trace Time (secs) 80 100 0.8 0.6 0.4 0.2 0 120 0 Trace Time (secs) 20 40 60 Trace Time (secs) (c) prefetch (d) periodic and prefetch Fig. 13: Trace latency given different tile migration schemes easily browse large repositories of sensor data. Respawn allows large-scale sensing systems to horizontally scale in a manner that still provides responsive access to raw or aggregate data. Through micro-benchmarking, we show that Respawn is able to support range-based queries on large datasets with nearly constant access time while other commonly used databases scale exponentially. We also show that the data ingest penalty can be kept at levels similar to that of MySQL databases by batching and grouping data. We show that in terms of network scalability, that Respawn can support thousands of edge nodes handling tens of thousands of requests. Finally, we demonstrate how intelligent data migration can be used in cloud-to-edge systems to automatically mask latency. As future work, we intend to investigate different aggregation functions as well as more sophisticated data compression and aging techniques. VI. [9] [10] [11] [12] [13] ACKNOWLEDGMENTS This research was funded in part by the Intel Science and Technology Center on Embedded Computing, the Bosch Research and Technology Center in Pittsburgh and TerraSwarm, one of six centers of STARnet, a Semiconductor Research Corporation program sponsored by MARCO and DARPA. 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Dimensions: why do we need a new data handling architecture for sensor networks? SIGCOMM Comput. Commun. Rev., 33(1):143–148, January 2003. Stephen Dawson-Haggerty, Xiaofan Jiang, Gilman Tolle, Jorge Ortiz, and David Culler. smap: a simple measurement and actuation profile for physical information. In Proceedings of the 8th ACM Conference on Embedded Networked Sensor Systems, SenSys ’10, pages 197–210, New York, NY, USA, 2010. ACM. James Cipar, Greg Ganger, Kimberly Keeton, Charles B. Morrey, III, Craig A.N. Soules, and Alistair Veitch. Lazybase: trading freshness for performance in a scalable database. In Proceedings of the 7th ACM european conference on Computer Systems, EuroSys ’12, pages 169– 182, New York, NY, USA, 2012. ACM. 

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Qeios, CC-BY 4.0 · Review, December 8, 2021 Review of: "Everybody Nose: Molecular and Clinical Characteristics of Nasal Colonization During Active Methicillin-Resistant Staphylococcus Aureus Bloodstream Infection" Wolfdgang Witte Potential competing interests: The author(s) declared that no potential competing interests exist. The manuscript reports a study on nasal MRSA colonization in patients with MRSA blood stream infection (BSI). It was performed from July 2018 until May 2019 at the Mount Sinai Hospital, NYC. 63 patients with BSI were enrolled, finally data on demographic and clinical characteristics as well as results from bacteriology were available for 53 of them. Primary cultures were carefully examined by colony morphology for probably heterogeneous colonization. Representative isolates were subjected to molecular typing by means of spa-typing. Background of this study is the widely accepted concept that nasal colonization with Staphylococcus aureus is a substantial reservoir for infections with this pathogens in these carriers. This had also been demonstrated for methicillin resistant S.aureus (MRSA) by studies performed nearly 20 years ago. The study is aimed to analyse the diversity of colonizing MRSA within hosts and of the demographic and clinical features associated with concomitant nasal MRSA colonization in patients suffering from MRSA BSI. The reported data on heterogeneous nasal colonization, on the frequency of concomitant nasal MRSA colonization (70%) and the congruence of results from typing nasal and blood stream isolates (95%) largely confirm results from studies in Europe where at that time MRSA epidemiology was different from that in the USA at present. Furthermore, these previous studies did not include such a broad analysis of clinical patient characteristics. From my side there are following comments/suggestions: 1. Abstract; conclusions a little bit more concrete explanations on the consequences of the findings for prevention/treatment would be desirable. 2. Introduction: For those who are not active in the field of MRSA infections a short description of the changing MRSA epidemiology including the discrimination of HA-MRSA, CA-MRSA and discrimination of widely disseminated epidemic strains of both categories would be helpful. 3. Methods: 1. Either here or at the beginning of the results section more details on the study setting with respect to prevalence of MRSA infections, and, if performed, on results from MRSA screening at admission would be of interest. Qeios ID: AAHFZA · https://doi.org/10.32388/AAHFZA 1/2 Qeios, CC-BY 4.0 · Review, December 8, 2021 2. 3.2.The study is based on 53 patients (37 colonized and 16 noncolonized), for which at least different 12 clinical variables had been recorded. I am concerned about the statistical power. Was a power calculation carried out when designing the study ? 4. Results. Starting point is the widely accepted concept that nasal colonization with Staphylococcus aureus is a substantial reservoir for infections with this pathogens in these carriers. This had also been demonstrated for methicillin resistant S.aureus (MRSA) by studies performed nearly 20 years ago. We should, however, keep in mind, that nosocomial infections (e.g. surgical wound infections) can result from direct transmission to the site of infection and must not necessarily start from nasal colonization. With respect to the comparatively low prevalence of MRSA BSI it would have been a tremendous effort to screen each patient before medical procedures which may result in an infection for MRSA colonization. One of the results is the observation that 38% of the colonized and 2 from 14 of the noncolonized patients had a history of previous MRSA colonization (Table 2 ). Were the corresponding isolates typed or are they still available for typing ? 5. The authors assume that spa-type t002 is indicative for MRSA CC5 “USA100” which is widely disseminated as HA-MRSA in the USA and t008 for the “notorious” CA-MRSA “USA300”. There are however, nosocomial MRSA that also exhibit t008. Therefor it would be highly advisable to perform at least PCR for luk-PV, better in addition also for arcA. (We have to keep in mind that high resolution typing based next generation sequencing [core genome MLST represents the state of the art). 6. Discussion. It would not be revealing to discuss the differences observed for MRSA CC8 and CC5 against the background of HA-MRSA and CA-MRSA epidemiology. Qeios ID: AAHFZA · https://doi.org/10.32388/AAHFZA 2/2 

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We are IntechOpen, the world’s leading publisher of Open Access books Built by scientists, for scientists 6,900 186,000 200M Open access books available International authors and editors Downloads Our authors are among the 154 TOP 1% 12.2% Countries delivered to most cited scientists Contributors from top 500 universities Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI) Interested in publishing with us? Contact book.department@intechopen.com Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com 4 Pulsed Laser Welding Hana Chmelíčková and Hana Šebestová Institute of Physics of the Academy of Sciences of the Czech Republic, Joint Laboratory of Optics of Palacký University and Institute of Physics of the Academy of Sciences of the Czech Republic Czech Republic 1. Introduction Metal joining by means of components heating to the melting temperatures was known thousands years ago in old age Greece. Heat sources have been developed from forging furnace to modern methods of plasma arc welding, electric resistance welding, oxy-fuel welding or laser welding. Laser, as a source of intensive light beam, starts to be implemented into industrial welding systems due to its advantages in comparison with classic methods, for example narrow heat affected zone, deep penetration, flexibility and many others. Besides the welding of compatible metals it is also possible to weld plastics. The principle of “light” welding is the same for all suitable laser wavelengths. The absorption of laser radiation in thin work piece surface layer leads to the temperature rise to the melting or vaporization point. Due to the conduction of generated heat to the surrounding material volume sufficient weld pool is melted. However heat conduction also causes essential energy losses. Laser welding important processing parameters are laser beam properties (power, beam quality and diameter, wavelength, focusing lens length), weld conditions (focus position towards the material surface, relative motion of the work piece towards the laser spot, weld type, processing gas) and physical properties of welded material and work piece dimensions. Typical power densities applied in laser welding lie between 105 – 107 W.cm-2. Lasers with active media formed by CO2, semiconductors, Yb:YAG, Nd:YAG crystals or Ytterbium doped fibres can be used in industry for various components welding. High power gaseous continual CO2 laser with wavelength 10 600 nm and wall-plug efficiency about 10 % has excellent beam quality, near Gaussian mode and high depth of focus, thus it is suitable for deep penetration welding. Far infrared radiation of this laser type cannot be transferred by means of optical fibre. Therefore typical CO2 laser welding system is equipped with fixed processing head and work piece positioning mechanism. A mixture of helium and nitrogen is recommended as a processing gas to suppress plasma shielding effect. Solid state pulsed Nd:YAG laser’s wavelength 1 064 nm is suitable for fibre guiding from resonator to the processing head, fixed on a robot arm. This allows welding few meters far from the laser source which is especially suitable for large or in shape complicated components. Laser beam has a multimode profile with quality 20 – 30 mm.mrad, wall – plug efficiency of the flash-lamp pumped Nd:YAG lasers reaches only about 3%. In recent years www.intechopen.com 42 Nd YAG Laser new and much more efficient laser types were developed – diode lasers with beam quality up to 30 mm.mrad, diode pumped disc Yb:YAG lasers and Ytterbium doped fibre lasers with excellent beam quality, both in near infrared region. With more compact design, higher efficiency 30 % (fibre laser) to 50 % (diode laser) and lower running costs these systems are going to replace above mentioned Nd:YAG and CO2 lasers (Němeček & Mužík, 2009). Nevertheless many pulsed laser welders are already installed in small and middle enterprises and various materials are being processed. Processing parameters optimization is a goal of many research projects to improve productivity and decrease occurrence of defects that results in lower production costs. 2. Processing parameters optimization To achieve fully penetrated high quality weld it is necessary to set optimal combination of processing parameters. They involved three different groups: laser beam properties (wavelength, power, diameter, divergence) material properties (density, thermal conductivity, specific heat, latent heat of melting and vaporization, thickness, joint configuration) and very important interaction parameters (welding speed, focusing element length, focus plane position towards the material surface, shielding gas direction and flow, absorptivity of material surface) (Duley, 1998). At room temperature almost all metals have absorptivity about 10 % - 20 %. It increases during material heating and leaps to 80 % - 90% when metal melting point is reached. Heat Q necessary to melt material mass m is given by the well known equation Q  m[c(Tm  T0 )  Lm ] (1) where c is specific heat, Tm melting temperature, T0 initial temperature and Lm latent heat of melting. In the case of continual laser welding this equation can be transformed to the following form Q   Dhv[c(Tm  T0 )  Lm ] t (2) where Q/t represents power required for melting, ρ volume density, D spot diameter, h penetration depth and v welding speed. Then, penetration depth can be expressed as follows Q t . h  Dv[c(Tm  T0 )  Lm ] (3) Including surface absorption and heat conduction losses, penetration depth can be roughly estimated hK P Dv (4) where P is laser power and K constant resulting from material physical characteristics including surface reflectivity and other energy losses. Thus, penetration depth is www.intechopen.com 43 Pulsed Laser Welding proportional to the applied laser power and inversely proportional to the spot diameter and welding speed. P/v ratio defines the heat input to a unit length. Melted area cross section linearly rises with the heat input (Ghaini et al., 2007). In the case of continual laser welding processing parameters like laser power and welding speed are simply entered into the welding device control system pursuant to the material thickness and physical properties. Much more complicated setting of pulsed laser parameters is outlined in paragraph 2.1. Surface power density or laser beam intensity is defined as a portion of laser power and laser spot area on material surface. Three welding modes are used in praxis, heat conduction mode, penetration mode and deep penetration (keyhole) mode. Heat conduction welding is characterized by power density in the interval 104 – 106 W.cm-2 that causes only surface melting up to 1 mm. The weld is wide and shallow with aspects ratio about 2:1. Only laser beam without sharp intensity peak can be used, or the focal plane must be shifted some millimetres above the material surface. In the case of pulsed laser, pulse length 1 ms – 10 ms is used. When power density balances around the critical point 106 W.cm-2, produced welds are deeper than in the case of the conduction welding. Aspect ratio is about 1:1 that indicates penetration mode welding (Lapšanská et al., 2010). Keyhole welding mode starts when energy density exceeds 106 W.cm-2. Laser beam is focused on the material surface and the fusion zone rapidly heats up to the boiling point. Melted material begins to vaporize at the centre of the weld spot and creates a blind hole (keyhole) in the centre of the weld line (Fig. 1). The pressure of hot metal vapour keeps the hole open during the welding. Presence of the keyhole allows the laser energy to reach deeper into the fusion zone and consequently to achieve deeper weld with lower aspect ratio (Kannatey-Asibu Jr., 2009). Keyhole mode welding is a typical application of high power continuous lasers or high energy pulsed lasers. During the deep penetration laser welding plasma can be generated above the keyhole. Ionised metal vapour and shielding gas absorb laser light, change its direction and cause lower process efficiency. Inert gases such as argon, nitrogen, helium and their special mixtures are used for plasma reduction. On the other hand, thanks to the plasma plume presence, welding process can be controlled by means of plasma intensity measurement (Aalderink et al., 2005). a) b) c) d) Fig. 1. Keyhole formation, a) surface irradiation, b) surface melting, c) vaporisation and cavity formation and d) light absorption inside the keyhole. www.intechopen.com 44 Nd YAG Laser 2.1 Pulsed laser welding parameters In the case of pulsed laser, more parameters are involved. Three basic parameters that must be set at laser source control panel are frequency f (Hz), pulse length t (ms) and flash lamp charging voltage U (V). These parameters define actual pulse energy E (J). Peak power Ppeak (kW) is defined as a portion of energy and pulse length Ppeak  E . t (5) Peak power determines interaction intensity of laser beam with material for given spot size. According to the material thickness and welding mode peak power values 0.2 kW to 5 kW are recommended in the operation manuals of laser welders. Laser average power P (W) is given as a product of actual energy and pulse frequency P  Ef (6) and determines the welding speed. There are two possible welding methods using pulsed laser. The first and simpler one is a spot welding which often replaces resistance welding nowadays. One or more pulses land material surface to reach required penetration depth in spot welding. No mutual motion between the processing head and material is applied. Spot welding is also often used for rough fastening of components to be subsequently seam welded using either pulsed or continual laser. This procedure reduces final distortions resulting from high thermal gradients corresponding to high value of applied power densities. To achieve continuous tight welds using a pulsed laser, pulse overlap must be applied. This is realized using suitable combination of processing parameters. Pulse overlap PO is defined as follows PO  1  v Df (7) To achieve hermetic tight joints pulse overlap is recommended to be 80 %. In comparison with conventional welding methods and continual laser welding, higher peak power densities in laser pulse mode causes higher heating and cooling rates which can result in weld defects and inhomogeneous microstructure. Many experimental works have been realised to optimise pulsed laser welding parameters for different kind of metals with goal to eliminate defects. For instance (Ghaini and al., 2006) studied overlap bead on plate welding of low carbon steel, (Tzeng, 1999) made successful welds without gas formed porosity in lap joints of zinc-coated steel. Another important parameter was introduced in these studies which is effective peak power density. Effective peak power density EPPD is defined as a product of peak power density PPD and pulse overlapping index F EPPD  F  PPD www.intechopen.com (8) 45 Pulsed Laser Welding where F 1 . 1  PO (9) This parameter was introduced to better formulate the real power reaching material surface. When seem welding is required more pulses land material surface (Fig. 2) and their contribution adds. Thus the effective peak power density can be used to compare energy requirements of welds accomplished with different pulse overlap. a) b) Fig. 2. Top view and longitudinal weld cross section. 2.2 Experimental work Pulsed Nd:YAG laser system LASAG KLS 246 – 102 with maximal average power 150 W and beam parameter product 22 mm.mrad was used to carry out experiments focused on the study of the effect of processing parameters on weld dimensions and its surface character. Material to be welded was 0.6 mm thick stainless steel AISI 304. So as the results were not affected by an accidental misalignment of components to be butt joined, by possible contamination or presence of surface defects of contact areas, or other unsuitable initial conditions influencing weld properties, it was decided not to join two sheets but to make a deep remelting of one sheet, which is, in fact, bead-on-plate welding. This strategy ensures that only the effect of energy changes will be studied. Laser welding is very demanding on pieces to be welded preparation, especially when narrow laser beam is used in near focal position. Therefore, highly precise prepared edges and minimal gap between the components to be joined are always supposed, which are conditions that need to be fulfilled in every precise butt joint laser welding application to prepare a high-quality weld. Then, the results of bead-on-plate experiments can be applied to the real sheet welding at conditions suitable for a high-quality weld joint preparation. This simplification can be used for the effect of processing parameter changes study in different laser applications. Welding itself was realized 4 mm under the focal plane to ensure the sufficient beam diameter on the specimen which was 0.85 mm. Focussing lens with 100 mm focal length was www.intechopen.com 46 Nd YAG Laser used. Cleaned degreased weld pieces were clamped in a mounting jig. Pure argon gas at coaxial 8 l.min-1 flow rate was used to protect the weld pool against its oxidation. In each set of experiments, only one parameter was changed keeping all the other parameters constant to be able to identify the effect of the one examined parameter. Table 1 presents processing parameters of each set of experiments. series 1 2 3 4 5 E (J) 3.5 3.5 – 6.5 5.0 5.0 1.8 – 6.2 P (W) 45.5 45.5 – 80.6 45.5 15.0 – 35.0 23.4 – 80.6 Ppeak (kW) 1.59 – 0.8 1.03 – 1.82 1.47 1.47 1.2 t (ms) 2.2 – 4.4 3.4 3.4 3.4 1.5 – 5.1 v (mm.s-1) 4.0 4.0 1.5 – 6.0 4.0 4.0 f (Hz) 13.0 13.0 13.0 3.0 – 7.0 13.0 Table 1. An overview of processing parameters of realised experiments 2.2.1 Pulse length effect The first set of experiments was focused on the effect of pulse length which was changed in the interval from 2.2 ms to 4.4 ms. To keep constant beam energy 3.5 J charging voltage had to be decreased when pulse length was increased (Fig. 3). Thus the average power remained constant and peak power decreased (Fig. 4). charging voltage (V) 220 200 180 160 140 120 100 2 2.4 2.8 3.2 3.6 4 4.4 4.8 pulse length (ms) Fig. 3. Charging voltage vs. pulse length keeping constant pulse energy. 1800 peak power power (W) 1500 average power 1200 900 600 300 0 2 2.4 2.8 3.2 3.6 4 4.4 4.8 pulse length (ms) Fig. 4. Average and peak power vs. pulse length keeping constant pulse energy. www.intechopen.com 47 Pulsed Laser Welding Pulse length increase led to the decrease of penetration depth (Fig. 5) that corresponds to the decrease of peak power which seems to be a critical parameter. penetration depth (mm) 0.3 penetration depth 0.25 linear fit 0.2 0.15 0.1 0.05 0 2 2.4 2.8 3.2 3.6 4 4.4 4.8 pulse length (ms) Fig. 5. Penetration depth vs. pulse length. These results also showed that applied parameters were not sufficient for the full penetration of 0.6 mm metal sheet (Fig. 6). However the effect of pulse length is evident. b) a) c) Fig. 6. Perpendicular cross sections of welds at a) 2.8 ms, b) 3.2 ms and c) 3.8 ms. Laser scanning confocal microscope LEXT OLS 3100 was used to image and analyse laser weld surfaces. Spot diameter varied in the interval from 0.704 mm to 0.759 mm and its slightly decreasing tendency with increasing pulse length was detected (Fig. 7). Average value of spot diameter reached 0.73 mm. According to the 0.025 mm deviations measured within the each sample, no definite relationship between spot diameter and pulse length can be determined in the investigated region. spot diameter (mm) 0.88 spot diameter 0.83 linear fit 0.78 0.73 0.68 0.63 0.58 2 2.4 2.8 3.2 3.6 pulse length (ms) Fig. 7. Spot diameter vs. pulse length. www.intechopen.com 4 4.4 4.8 48 Nd YAG Laser Fig. 8 presents an example of surface longitudinal central profile. In technical practise, strict claims on weld surface quality are often posed. Suitable combination of welding parameters can minimise post processing mechanical treatment. Fig. 9 presents surface images of selected samples. Pulse overlap 63 % remained constant during all the experiments of this set. Fig. 8. 3D surface reconstruction and central profile of the weld realised at 3.8 ms. a) b) c) Fig. 9. Weld surface in case of pulse length a) 2.6 ms, b) 3.4 ms and c) 4.4 ms. 2.2.2 Pulse energy effect The second series of experiments studied the effect of pulse energy which was set in the interval from 3.5 J to 6.5 J via charging flash lamp voltage changes (Fig. 10). Increasing energy naturally increases average as well as peak power (Fig. 11). www.intechopen.com 49 Pulsed Laser Welding 7 pulse energy (J) 6 5 4 3 2 1 0 160 170 180 190 200 210 220 charging voltage (V) Fig. 10. Pulse energy vs. charging voltage. 1800 power (W) 1500 1200 peak power 900 average power 600 300 0 3 3.4 3.8 4.2 4.6 5 5.4 5.8 6.2 6.6 pulse energy (J) Fig. 11. Average and peak power vs. pulse energy. Higher laser power leads to the deeper penetration which corresponds to the higher applied power resulting in higher heat input. Nevertheless penetration depth evolution is not linear (Fig. 12). penetration depth (mm) 0.7 0.6 0.5 0.4 0.3 0.2 penetration depth 0.1 polynomic fit 0 3 3.4 3.8 4.2 4.6 5 pulse energy (J) Fig. 12. Penetration depth vs. pulse energy. www.intechopen.com 5.4 5.8 6.2 6.6 50 Nd YAG Laser Fig. 13 presents reached aspect ratios (penetration depth to weld width) as a function of effective peak power density. In this case pulse overlap 63 % was applied. Thus pulse overlapping index was 2.7. It means that power really touching a unit of material surface is 2.7 times higher in comparison with laser output power assuming no energy losses between laser output and material surface. 0.9 aspect ratio 0.75 0.6 0.45 0.3 PPD 0.15 EPPD 0 0 2000 4000 6000 8000 10000 (effective) peak power density (W.mm-2) Fig. 13. Aspect ratio vs. effective peak power density. Penetration depth slightly increases with pulse energy until about one half of the sheet thickness is penetrated. Then, when the formation of the keyhole starts (4.7 J), it increases steeply until the full penetration is reached at 5.9 J (1.7 kW). Keyhole formed at lower peak powers is not stable enough to establish true keyhole welding, and penetration welding mode can be observed (Fig. 14). a) b) c) Fig. 14. Perpendicular cross sections of welds at a) 3.8 J, b) 4.7 J and c) 6.2 J. Aspect ratio increases with increasing effective peak power density until the full penetration is reached at about 8.6 kW.mm-2. At the moment of full penetration spot diameter is maximal, and aspect ratio decreases. Once the full penetration has been achieved, increasing effective peak power density does not lead to another weld width grow. Rather, conversely, the width decreases because the beam can escape from the melt pool because of the full penetration and because less energy is absorbed. That is why the aspect ratio increases again. These data corresponds to the weld cross-section fusion area measurement (Fig. 15). www.intechopen.com 51 cross-section fusion area (mm2) Pulsed Laser Welding 0.35 0.3 0.25 0.2 0.15 0.1 fusion area 0.05 polynomic fit 0 3 3.5 4 4.5 5 5.5 6 6.5 pulse energy (J) Fig. 15. Cross-section fusion area vs. pulse energy. Fig. 16 presents the effect of beam energy on spot diameter. Beam diameter on the specimen was 0.84 mm for all carried out experiments. Spot diameter does not reach this value for almost all spots. This fact corresponds to the relative low energy application, non-uniform heat distribution, resulting from characteristic profile of beam intensity, and is supported by relatively low thermal conductivity of welded material. spot diameter (mm) 0.88 0.83 0.78 0.73 0.68 0.63 0.58 3 3.4 3.8 4.2 4.6 5 5.4 5.8 6.2 6.6 pulse energy (J) Fig. 16. Spot diameter vs. pulse energy. Spot shape is also very demanding on pulse energy. The volume of melted material is higher at higher energies which leads to the higher deformation of the spot shape (Fig. 17). a) b) Fig. 17. Weld surface in case of pulse energy a) 3.5 J, b) 5 J and c) 6.2 J. www.intechopen.com c) 52 Nd YAG Laser 2.2.3 Welding speed effect The third experiment was focused on the effect of welding speed. Welding speed was changed from 1.5 mm.s-1 to 6 mm.s-1. pulse overlap (%) 100 80 60 40 20 0 0 0.001 0.002 0.003 0.004 0.005 0.006 0.007 welding speed (m.s-1) Fig. 18. Pulse overlap vs. welding speed. Speed changes correspond to the pulse overlap change from 86 % to 40 % (Fig. 18, Fig. 19). b) a) c) Fig. 19. Weld surface in case of welding speed a) 1.5 mm.s-1, b) 3.5 mm.s-1 and c) 6 mm.s-1. These experiments did not prove any significant change in penetration depth (Fig. 20). On the other hand, weld width decreased with increasing welding speed (Fig. 21). These give us positive information that slight required changes of pulse overlap via welding speed do not significantly influence penetration depth. penetration depth (mm) 0.7 0.6 0.5 0.4 0.3 0.2 penetration depth 0.1 linear fit 0 0 0.001 0.002 0.003 0.004 welding speed (m.s-1) Fig. 20. Penetration depth vs. welding speed. www.intechopen.com 0.005 0.006 0.007 53 Pulsed Laser Welding spot diameter (mm) 0.88 0.83 0.78 0.73 0.68 spot diameter 0.63 polynomic fit 0.58 0 0.001 0.002 0.003 0.004 0.005 0.006 0.007 welding speed (m.s-1) Fig. 21. Spot diameter vs. welding speed. 2.2.4 Pulse frequency effect Another experiment concerned with pulse frequency effect. Pulse frequency was changed from 9 Hz to 17 Hz. In this case peak power remained constant, while the average power increased (Fig. 22). average power (W) 100 80 60 40 20 0 6 9 12 15 18 pulse frequency (Hz) Fig. 22. Average power vs. pulse frequency. Frequency increase naturally increases pulse overlap (Fig. 23, Fig. 24). Pulse overlap must be high enough so as also bottom side of the sheet is continuously penetrated (Fig. 25). 80 pulse overlap (%) 70 60 50 40 30 20 10 0 6 9 12 pulse frequency (Hz) Fig. 23. Pulse overlap vs. pulse frequency. www.intechopen.com 15 18 54 Nd YAG Laser a) b) c) Fig. 24. Weld surface in case of frequency a) 9 Hz, b) 13 Hz and c) 17 Hz. a) b) c) Fig. 25. Weld bottom side in case of frequency a) 9 Hz, b) 13 Hz and c) 17 Hz. Power increase led to the increase of penetration depth (Fig. 26) as well as the weld width. Frequency 11 Hz was sufficient for the full penetration. Spot diameter slowly increases with increasing frequency (Fig. 27). penetration depth (mm) 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 6 9 12 pulse frequency (Hz) Fig. 26. Penetration depth vs. pulse frequency. www.intechopen.com 15 18 55 Pulsed Laser Welding spot diameter (mm) 1 0.8 0.6 0.4 spot diameter 0.2 polynomic fit 0 6 9 12 15 18 pulse frequency (Hz) Fig. 27. Spot diameter vs. pulse frequency. 2.2.5 Combination of pulse energy and pulse length effect In the last experiment beam energy and pulse length were changed simultaneously to keep constant peak power (Fig. 28). Although the peak power is still the same average power increases (Fig. 29). pulse length (ms) 6 5 4 3 2 1 constant peak power 0 0 1 2 3 4 5 6 7 pulse energy (J) keeping peak power constant pulse energy and pulse overlap Fig. 28. Pulse length vs. pulse energy keeping constant peak power. 7 6 5 4 3 2 pulse energy (J) 1 pulse length (ms) 0 0 20 40 60 80 100 average power (W) Fig. 29. Combination of pulse energy and pulse length (keeping constant peak power) vs. average power. www.intechopen.com 56 Nd YAG Laser The higher energy and pulse length the deeper penetration depth (Fig. 30). This result points out the fact that even peak power can not be used as a definite indicator of penetration depth in pulsed laser welding. Fig. 31 presents a comparison of these results with results of the effect of pulse energy (chapter 2.2.2). It is obvious that pulse energy is a critical parameter since it defines the volume of melted material. penetration depth (mm) 0.6 0.5 0.4 0.3 0.2 0.1 constant peak power 0 0 20 40 60 80 100 average power (W) Fig. 30. Penetration depth vs. average power keeping constant peak power. weld dimensions (mm) 1 0.8 0.6 0.4 h (const. t) d ( const. t) h (const. Ppeak) d (const. Ppeak) 0.2 0 0 1 2 3 4 5 6 7 pulse energy (J) Fig. 31. Weld width d and penetration depth h vs. pulse energy for constant and variable pulse length. 3. Conclusion High power Nd:YAG lasers with millisecond pulses are used in industry for spot or overlap welding. Many research papers reporting on pulsed laser welding parameters optimisation leading to the production of sufficiently deep welds without defects have been published. Usually more parameters were simultaneously changed in such optimisation processes. The aim of our research work was to identify the effect of each parameter separately. www.intechopen.com Pulsed Laser Welding 57 Pulsed overlap bead-on-plate welding of 0.6 mm thick AISI 304 stainless steel was realised in our laboratory. Flash lamp pumped pulsed Nd:YAG laser KLS 246-102 with multimode beam profile was used for five series of welding experiments. The influence of different process parameters – pulse length, pulse energy, welding speed, pulse frequency and combination of pulse energy and pulse length on weld dimensions was examined in five separate experiments. Weld cross sections and laser spots overlap on samples surfaces were observed and measured by means of laser scanning confocal microscope LEXT OLS 3100. The effect of penetration depth and surface spot diameter on applied parameters was outlined. Following from the above mentioned results, each processing parameter more or less influences weld characteristics. It is obvious that the knowledge of only one parameter, for example beam energy or average power, is not sufficient for the prediction of weld dimensions in pulsed laser seam welding. Peak power, pulse length and frequency in combination with processing speed are also very important. Suitable combination of processing parameters must be always found. Experiments with flash lamp pumped solid state laser with very low efficiency will be followed by new studies on modern laser systems – diode and fibre lasers. 4. Acknowledgment The Academy of Sciences of the Czech Republic supports this work under the project no. KAN301370701. 5. References Aaldering, B. J.; Aarts, R. G. K. M.; Jonker, J. B. & Meijer, J. (2005). Weld Plume Emission During Nd:YAG Laser Welding, Proceedings of the third International WLT Conference on Lasers in Manufacturing, Munich, Germany, June 13-16, 2005, Available from http://doc.utwente.nl/52666/2/weld_plume.pdf Duley, W. W. (1998). Laser welding, A Wiley-Interscience publication, ISBN 978-0-471-246794, Hoboken, New Jersey, USA Ghaini, F.M.; Hamedi, M. J.; Torkamany & Sabbaghzadeh, J. (December 2006). Weld metal microstructural characteristics in pulsed Nd:YAG welding, In: Scipta Materiala 56 (2007), 955 – 958, Elsvier Ltd., Available from http://www.sciencedirect.com/science/article/pii/S1359646207001194 Kannatey-Asibu Jr., E. (2009). Principles of Laser Material Processing, A John Wiley & Sons, Inc., Publication, ISBN 978-0-470-17798-3, Hoboken, New Jersey, USA Lapšanská, H.; Chmelíčková H. & Hrabovský, M. (2010). Effect of Beam Energy on Weld Geometric Characteristics in Nd:YAG Laser Overlapping Spot Welding of Thin AISI 304 Stainless Steel Sheets, Metallurgical and Materials Transactions B, Vol. 41, No. 5 (2010), pp. 1108-1115. ISSN 1073-5615 Němeček, S. & Mužík T. (2009). Laser Material Processing – Hardening and Welding, Proceedings of METAL 2009 18th International Conference on Metallurgy and Materials, Hradec nad Moravicí, Czech Republic, May 19-21, 2010, Available from http://www.nanocon.cz/data/metal2009/sbornik/List/Papers/014.pdf www.intechopen.com 58 Nd YAG Laser Tzeng, Y. F.(1999). Pulsed Nd:YAG Laser Seam Welding of Zinc-Coated Steel Welding research supplement, Vol.6, No.4, (July 1999), pp. 238-s -211-s, ISSN 1729-8806 www.intechopen.com Nd YAG Laser Edited by Dr. Dan C. Dumitras ISBN 978-953-51-0105-5 Hard cover, 318 pages Publisher InTech Published online 09, March, 2012 Published in print edition March, 2012 Discovered almost fifty years ago at Bell Labs (1964), the Nd:YAG laser has undergone an enormous evolution in the years, being now widely used in both basic research and technological applications. Nd:YAG Laser covers a wide range of topics, from new systems (diode pumping, short pulse generation) and components (a new semiorganic nonlinear crystal) to applications in material processing (coating, welding, polishing, drilling, processing of metallic thin films), medicine (treatment, drug administration) and other various fields (semiconductor nanotechnology, plasma spectroscopy, laser induced breakdown spectroscopy). How to reference In order to correctly reference this scholarly work, feel free to copy and paste the following: Hana Chmelíčková and Hana Šebestová (2012). Pulsed Laser Welding, Nd YAG Laser, Dr. Dan C. Dumitras (Ed.), ISBN: 978-953-51-0105-5, InTech, Available from: http://www.intechopen.com/books/nd-yaglaser/pulsed-laser-welding InTech Europe InTech China University Campus STeP Ri Slavka Krautzeka 83/A 51000 Rijeka, Croatia Phone: +385 (51) 770 447 Fax: +385 (51) 686 166 www.intechopen.com Unit 405, Office Block, Hotel Equatorial Shanghai No.65, Yan An Road (West), Shanghai, 200040, China Phone: +86-21-62489820 Fax: +86-21-62489821 © 2012 The Author(s). Licensee IntechOpen. This is an open access article distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 

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Vol. 8 No. 1 Language and Semiotic Studies Spring 2022 Exploring Saussure’s Analogy between Linguistic and Monetary Signs Kristian Bankov New Bulgarian University, Bulgaria Abstract Beginning with a few of Saussure’s ideas from Course of General Linguistics1 the author tracks the development of money as signs fulfilling their pecuniary function. The principle of the arbitrariness of linguistic signs, postulated in Course, is the leading concept under which all forms of money are grouped for study. In its evolution from the primitive consumable forms such as salt, leather and grain to the last abstract forms of accountable digital writing, the money sign is increasing its level of arbitrariness between signifier and signified value and thus becomes “better for thinking” (Levy-Strauss) and communicating. The second part of the paper considers the main contributions to the field of semiotics pertaining to money and economic value, and thus highlights the originality of the model, in which the arbitrariness of monetary signs and the scarcity of money tokens in circulation form the model’s foundation. The author’s analysis, applying the dyadic sign, reveals the relationship between the value or meaning of a monetary sign and the temporal dimension of our experience. The article concludes with some hypotheses about the homology between the primordial finitude of human temporality (Heidegger) and the principle of scarcity/finitude of money in circulation as a condition of their value. Keywords: arbitrariness, money sign, fungibility, scarcity, sign production To determine what a five-franc piece is worth one must therefore know: (1) that it can be exchanged for a fixed quantity of a different thing, e.g., bread; and (2) that it can be compared with a similar value of the same system, e.g., a one-franc piece, or with coins of another system (a dollar, etc.). In the same way a word can be exchanged for something dissimilar, an idea; besides, it can be compared with something of the same nature, another word. (Course, 1959, p. 115) 1. An Analogy for the Arbitrariness of Linguistic and Monetary Signs One of the characteristics of Saussure’s Course is its essentialness. Its exact, short and clear formulations have inspired innumerable researchers and form the basis of Structuralism, one of the most popular research paradigms. The short passage, which postulates the necessity of semiology, is a good example of Saussure’s style (and that of his students who published the Course). Several references to the analogy between linguistics and economics (and between linguistic and monetary signs in particular), 114 Kristian Bankov which have great potential for further development, are also set out in the text. It is these that I intend to examine here, and though I am not the first to undertake such a study, I argue that the approach proposed here is the first of its kind. At the beginning of the third chapter of Course, the various sciences are reviewed in order to separate those with a distinct synchronic and diachronic approach from those in which there is no reason for such a division. It is there that Saussure separates economics and linguistics from the rest (Course, 1959, p. 119), and compares political economy to the innovative “synchronic” linguistics he offers. Old-fashioned comparative linguistics is compared to economic history. In a second passage, quoted above (but see also p. 149), he develops the analogy specifically at a sign level. Until now, however, there has been no analysis of the analogy between the two types of sign in terms of the linguistic sign’s fundamental characteristic, arbitrariness. According to many scholars of Saussure, and particularly in De Mauro’s classical reading, the discovery of linguistic signs’ arbitrariness is the most important and mature step in the establishment of Saussure’s research paradigm (De Mauro, 2005, pp. 325-334). However, Saussure captures the language and the market in a so to speak “synchronic parallel” that would function differently in a different moment of the evolution of the money sign. He uses the value of a 5-franc silver coin as an analogy for the semantic value of the linguistic sign but does not compare it with a paper or commodity monetary sign. This is where my contribution comes in. A brief overview of the different historical money forms shows that they follow a semiotic development, going from a very low degree of arbitrariness between the signifier (e.g., the mental image of the currency token) and its value, as in the primitive forms, to a degree of arbitrariness and formal perfection beyond that of the linguistic sign (as in the case of electronic money). However, a small misunderstanding may come from the way the study is set up. For Saussure the study of the origin and development of language “is not even worth asking” (Course, 1959, p. 72), when it comes to the object of linguistics and where the notion of arbitrariness belongs. On the contrary here I am using this notion for a diachronic comparative analysis. For Saussure anything in language that is “prehistory”, e.g., before written records, was not suitable for scientific analysis for obvious reasons. The money sign, however, evolves after the linguistic written records were available and what follows is a series of synchronic comparisons, made with the same logic as Saussure’s comparison with the silver coin. In philosophy Simmel gives quite concrete examples of the evolution of the material substance of money in Chapter II of Philosophy of Money (see Simmel, 1990), but his perspective is different and I would say complementary to the one proposed here. Also, we know not very much on the origin of language, but still there has been a consistent scientific research on the matter since the early nineties of the XX century and it will not be too speculative if we imagine that it evolved in a similar way as the money sign—from contextually dependent forms of social interaction with zero degree of arbitrariness for prehistoric man, to the strongly formalized “arbitrary” languages of the present day.2 What would have changed Saussure’s mind on the study of the prehistoric linguistic practices is the scientific evidence which today is far more available. 2. The Different Forms of Money Signs Bartering is the first and most primitive form of economic exchange. In a barter system, commodities are exchanged according to their utilitarian value. A minimum 115 Exploring Saussure’s Analogy between Linguistic and Monetary Signs degree of conventionality is required to determine what quantity of one commodity corresponds to another. Here, hypothetically, we can assume that any good possessed in excess functions as a sign for a future need that could be satisfied as the result of a future exchange. But the conventions are too numerous, and above all, the whole system is contextually dependent—there must be a coincidence of excess and need in time to motivate the two exchanges. Barter systems easily evolved into the commodity money system. Some of the merchandise typically exchanged under the barter system acquire a privileged function in the commodity money system by combining a higher utilitarian value with a higher durability. This includes grain, livestock, salt, leather, etc.—consumable goods, which become a convertible object as they provide a greater guarantee for future exchange. These are also sought after by those who do not have the need (for salt, leather, wheat, etc.), but are acquired with a view to their future conversion to goods available in excess. In this sense, they have a clearer semiotic function (and a higher degree of arbitrariness), since their possession is independent of both the possessor’s needs and his productive abilities. However, this is still reliant on the persistence of the market as a social convention. Economists often neglect the difference between this kind of commodity money and commodity money made from precious metals, cowries, ritual objects, stones, beads, etc. According to a semiotic analysis, the latter has a higher degree of arbitrariness, since their value comes not from the utilitarian function but from their symbolic value. They are not consumed, but just owned and exchanged. A hybrid option, and an important step to the next phase of the evolution of money, are instruments as monetary signs (mostly in China). The most well-known are spade money and knife money. A real turning point in currency development is the invention of the coin, as evinced by both a semiotic analysis and the historical facts that followed the spread of the use of coins. In the official histories of money,3 however, there is again no explicit emphasis placed on this link. The qualitative difference that the coin introduces into market-based economic relations is expressed by the English term fungibility. Unlike commodity money, which was consumable or bearers of other functions and had to be judged separately for each deal, the coins in their conception were identical and countable. The coin introduces structural discretion into the thinking of economic value, just as phonetically distinct words emerged from the amorphous sound and visual matter of the gesture and shouting phase. Saussure quite accurately compares the word as an acoustic image pattern with a coin that, regardless of the particular token in our hands, always carries the same value. The coin turns money signs into “economic” writing. Much has been written about the changes in our civilization since the discovery of the printing machine, but only few note that coin minting is the first form of printed writing. Accordingly, its effects are remarkable: the coin provides a much more complete tool for political governance and a completely new stage in the organization of military campaigns and, above all, a qualitatively new level of entrepreneurship and market exchange. There are convincing studies about the role of coins in the Hellenic world, the Roman Empire, and ancient China.4 All this, according to the analysis suggested herein, is due to the fact that the monetary sign, before all else, becomes good for thinking with its increased level of arbitrariness. As the hypothesis that thinking is linguistic in nature dominated the 20th century, and as Saussure’s insights reflect the mechanism of this dependence, civilization’s progress as a result of the invention of a monetary sign with the properties of a linguistic sign becomes easier to explain. But money does not stop developing with the invention and refinement of the 116 Kristian Bankov coin, though it has dominated economic exchange for nearly two millennia. Financial institutions began to develop intensively particularly from the fourteenth century onwards, with the beginning of the great geographic discoveries and the Trade revolution. Meanwhile we witness the emergence of bank institutions, credit, shared financial risk in investments, etc. All this multiplies the money turnover and so-called “representative money”, which, unlike the coins, do not have their own material value but only guarantee that they can be exchanged for gold or other valuables by a bank. From the name itself, it is clear that the new monetary form is a next step towards the arbitrariness of the monetary signs. Being much easier and safely portable than gold, they open a new level in trade and financial manipulation. If a coin makes economic value better for thinking, representative money makes it better for communication. The banknote is the most perfect form of representative money. It is no coincidence that the most complex work about the profound social function of money—Simmel’s Philosophy of Money (1900)—is inspired by the historical period dominated by the banknote. Banknotes still exist today, but after the phase of representative money there is another structural change in the monetary system, which brings the symbolic function of money (defined by its level of arbitrariness) to that of the linguistic sign, on the side of the signified. For a long time, national banks have complied with banknote coverage. Banknotes were guaranteed with deposits of gold. Accordingly, the monetary sign had a triadic structure: 1) a banknote - 2) an economic value - 3) a gold equivalent deposited in the bank. But the dynamics of socio-economic processes, expressed in monetary relations, have surpassed the capacity of the “gold standard”. Its crisis began after the World War I; one by one countries started to abandon it; and the USA was the last in 1971. The next money form was the so-called fiat money (or legal tender). The value of this money form does not depend on an external reference, but on state law. This law serves as the social convention in a linguistic sign, defining the relation between signifier and signified. The sign structure of fiat money is identical from many perspectives to Saussure’s linguistic sign. In contrast to natural language, however, the level of arbitrariness also gains an ethical dimension in monetary signs. The value of money depends on the amount of money put into circulation. Some see the lagging gold standard as an emanation of neoliberalism and financial-corporate order, wherein the arbitrariness of a currency is a prerequisite for arbitrary financial manipulations, for example, with which a government can impoverish the savers through a single decision. But this topic exceeds the objectives of the article. The last form of money to date is e-money. Within this category we can distinguish two main types—electronic banking of ordinary national currencies, and new forms of digital currency such as Bitcoin. In the first case, we have a direct continuation along the axis of a currency’s rising arbitrariness. I get my salary by bank transfer, meaning that the sensory indicator of the occurrence of this important event for me is an electronic record that I could reproduce on a screen. For conventional currency, the same “signified”, e.g., the same monetary value, I receive as a bundle of banknotes that, although extremely comfortable for daily purchases, carry a large number of constraints. By contrast, electronic payments make it possible for me to pay for a good or service at any point on the planet, and that does not require the recipient to be present to receive the sum. E-money is perfect from a communication point of view, and this is a qualitatively different shift in monetary sign forms. Regarding the independence of the signified from the signifier, e-money is of a different nature than the classic, arbitrary linguistic signs described by Saussure. 117 Exploring Saussure’s Analogy between Linguistic and Monetary Signs Even more interesting is the case of digital currencies. The main difference from electronic banking of national currencies is that digital currencies are not issued by central banks; there is no legal government protection behind them. As an analogy with language, digital currencies are like jargon, whose importance depends on the number of people who use them, naturally in parallel with the literary language / official currency. In fact, my overall interest in the semiotic study of money was inspired by digital currencies, and from the lack of satisfactory socio-cultural models for their nature. This study is part of the theoretical basis for a larger research project, and so will not discuss digital currencies analysis, which would, however, keep us from linking with Saussure’s fundamental work. Here the main focus was to track the evolution of the sign-value relation’s arbitrariness to the type of currency, in the course of the emerging different forms of money. Accordingly, as a central difference between the two sign systems on which a reliable model of economic value can be built, “scarcity” is a necessary condition, on which the value of a currency in circulation depends. This has long been a central problem for central banks after the penetration of fully “arbitrary” monetary value, independent from any third-party valuables like the Gold standard. In the second part of the article, I will try to position the above-mentioned theoretical hypothesis among the most significant existing contributions to sociosemiotic analyses of money and economic value. 3. Dynamic, Triadic, and Holistic Approaches As with many other topics, as well as that of economic value and money, semiotics is divided into two major paradigms. The two authors who devoted most of their work to this problem belong to different paradigms and although chronologically there is a complete coincidence in their contributions (both debuted in 1968 and during the next 10-12 years published several books), they entirely ignore each other. These are Ferruccio Rossi-Landi and Jean Baudrillard. Both authors are fierce critics of capitalism and bourgeois ideology, and they both rely on the theories of Karl Marx, in particular the theme of alienation (although at the end Baudrillard turns even against Marx), but their hypotheses are totally mutually incompatible. Rossi-Landi’s critique is based on a broadly developed analogy (or homology, as he prefers to call it) between physical and linguistic work (Rossi-Landi, 1975, pp. 1862-1888). In his model of the sign, the third member is always present, and in most cases, it constitutes the embodied work of “linguistic workers”. Not only does he disregard the above-mentioned difference in language and currency, but he insists that it is ideologically condemnable to assume that systems of natural language spontaneously ​​ arise beyond the individual wishes of community members (pp. 1932 ff.). It is precisely such an assumption that conceals the fact that they, as the material artefacts, are the product of human labor that the masses have produced, but that elites control the channels of distribution and accordingly take advantage of something that does not rightfully belong to them. Augusto Ponzio, a student and principal follower of Rossi-Landi, goes even further in this doctrine by developing a direct analogy between the model of the Saussurian sign and the marginalist (liberal) school in economics. His main criticism is that both emphasize the moment of exchange, the market in one case, and the exchange of messages in the other, completely ignoring production and subjectivity, which are the real cause and source of exchange, the latter only constituting the surface of these processes (Ponzio, 1990, pp. 185 ff.). Rossi-Landi’s influence extends beyond the Bari circle. Umberto Eco himself 118 Kristian Bankov is strongly influenced by Rossi-Landi’s doctrine, to such an extent that in Theory of Semiotics the typology of signs is replaced by a typology of the various forms of sign production (Eco, 1975, Chapter 3.6). However, it should be noted that in one of the most important chapters, which clarifies the characteristics of culture as a semiotic phenomenon, Eco pays special attention to prove that economic exchange can be considered as a semiotic interaction (together with the production of tools and the exchange of women). There he uses an entirely Marxist model of economic value, and at the same time he is the only one of the authors cited in this tradition who explicitly formulates the principle of currency’s scarcity: The only difference between a coin (as sign-vehicle) and a word is that the word can be reproduced without economic effort while a coin is an irreproducible item (which shares some of the characters of its commodity-object). This simply means that there are different kinds of signs which must also be differentiated according to the economic value of their expression-matter. (Eco’s emphasis, ibid., pp. 25-26) Eco, however, never returns to this typology of signs according to the economic value of their expressive matter—something that I have tried to do in the first part of this article. Baudrillard, opposite to Rossi-Landi, not only uses the dyadic model of the sign to criticize the capitalist economy, but also directs all his inquiries toward consumption rather than production. That is why both discourses are so distant, although their purpose and disciplinary membership are the same. More a visionary than a researcher, Baudrillard opens a discursive perspective on a semiological reading of the consumer society. His main diagnosis is that in new post-war capitalism, the leading instrument for class exploitation is no longer labor but consumption. Thus, the system deforms its logic. From a material process based on the real needs of people, consumption develops into an autonomous network of signifieds that (typical of the overall logic of postmodernism) loses touch with material reality and begins to refer to its components in a meaningless carousel of signifiers. In such a system media, advertising, and fashion accelerate the course of the carousel, which distracts the individuals rotated by it more and more from their authentic existence, thus increasing their alienation. Baudrillard exclusively uses Saussure’s conceptual apparatus in this analysis, though he does not pay attention to monetary signs’ analysis anywhere. Instead, he makes a direct parallel between the post-modern situation in culture and the transition from the financial logic of the gold standard (currency) to the wholly de-facto (arbitrary) monetary system: [...] this process culminates in the virtual international autonomy of finance capital, in the uncontrollable play of floating capital. Once currencies are extracted from all productive cautions, and even from all reference to the gold standard, general equivalence becomes the strategic place of the manipulation. (Baudrillard, 1975, p. 129 n. 9) He also offers some valuable insights into the function of money and credit in the following direction: “The illusionism is truly remarkable: society appears to extend credit to you in exchange for a formal freedom, but in reality, it is you who are giving credit to society, alienating your future in the process.” (Baudrillard, 1968, p. 160) Thus, he points out the relationship of finance to the temporal dimension of human existence, although his reading is entirely negative. In confirming that the dyadic tradition going from the Course more accurately captures the dynamics of monetary signs, we turn to Derrida’s remarkable 1991 work Given 119 Exploring Saussure’s Analogy between Linguistic and Monetary Signs Time (Derrida, 1992). This is his main treatise on economics, money and gifts. Derrida begins a long deconstruction of the metaphysical idea of giving ​​ or donating time, the link between economic value and temporality being direct. In the chapter entitled “The Madness of the Economic Reason”, he deconstructs the paradoxical situation described in one of Baudelaire’s short stories where one of the characters gives a beggar a fake coin, and at the end of the book expresses the analogy that credit is différance. Of course, Derrida’s aphoristic and blatant way of expression does not allow for identifying any model directly from his work, but the genius of some of his insights into money and finance is attested to by the detailed comment that Michel Tratner makes in his vast article entitled “Derrida’s Debt to Milton Friedman” (Tratner, 2003). It is here that the paradoxes of the “deconstructed” linguistic sign projected on the financial history of the nineteenth and twentieth centuries have quite logically led to the financial relativism of fiat money after 1971. Deconstruction in culture and monetarism in financial politics are similar in their deep structure, their apogee being in the seventies and eighties of XX century, and the socio-economic effect of the latter is probably part of the conditions that contributed to the enormous success of the former (Derrida’s debt, namely, p. 798). Baudrillard and Derrida also direct us to a third type of approach where money and economic value are at the heart of the study, and the semiotic paradigm is pragmatically chosen in terms of its adequacy for their research goals. In these approaches, which I call “holistic”, it is common that, thanks to semiotics, the greater importance of money for economic reality is emphasized above the economic and financial thought accepted in the mainstream. In his article from 1989 “Making Semiotic Sense of Money as a Medium of Exchange” Alan Dyer confronts the deep semiotic understanding of money as a medium of thinking with the major economic theories of money. In his words “By re-presenting objects and experiences as comparable containers of exchange value, money symbolically transforms life and, thus, mediates our understanding of it” (Dyer, 1989, p. 505). This also means that market transactions are not just culturally neutral economic exchanges, but a deeply human interaction of sharing a common understanding and a worldview. Steven Horwitz departs from these premises in his article “Monetary Exchange as an Extra-Linguistic Social Communication Process” (1992) and elaborates on the affinity between the subjectivist theory of the economic value (the famous Austrian school) and the hermeneutic ontology of Gadamer. His most important insights are about the parallel between the two systems where money does not correspond to messages as most of the researchers think, but they correspond to the language itself, within which prices have the function of word or message (p. 208). In his model also the notion of text is projected on the economic reality and it corresponds to the notion of market (pp. 210 ff.) A detailed presentation of these methods as well as his own contribution is proposed by Wennerlind (see Wennerlind, 2001), and in any case the social dimension lies at the center of the hypotheses. So, he divided the institutional, liberal, and Marxist approaches. 4. Instead of a Conclusion: Difference vs Scarcity Saussure’s insight into the analogy between political economy and linguistics (as well as between linguistic and monetary signs) opens a phenomenological research phenotype for the typology of the different monetary signs in terms of how “good” they are for thinking. From the review of existing contributions, those inspired by the dyadic model of the sign go further in that direction, comprising even the temporal 120 Kristian Bankov dimension of the phenomenon and bringing our attention to a radical difference in the basic principle of semiosis between the linguistic and the monetary systems. If in the Saussurean langue system is the difference between the signifiers to determine the value of the sign, in the monetary system, on the contrary, the more identical the signifiers the better for thinking they are. Therefore, in our analysis the invention of the coin is a cornerstone in the evolution of the money sign. But if it is not from the difference then where does the value of the money sign come from? As we have seen, the value of each money sign comes from the principle of scarcity, from its position in a system of quantity rather than in a system of differences. Now, can we identify a phenomenological ground for the perception of the economic value/scarcity, 5 comparable to the “condemnation” to semantic meaning, postulated by Merleau-Ponty – Greimas, as an actualization of the Saussurean heritage? Derrida often includes Heidegger in his arguments, precisely in relation to the paradoxical status of the given time and the metaphysical implications that the German philosopher exposes, interpreting everyday expressions close to giving and receiving time. Derrida, however, does not come to the central question from the point of view of my hypothesis, though his analysis often revolves around it. And this hypothesis is related to what Heidegger calls “the thesis of the primordial finitude of temporality” (Heidegger, 1996 [1927], p. 330). This means that the fundamental characteristic of human existence is its finitude in time, the ontological inevitability of death to which each life project is oriented. According to Heidegger, this situation precedes and is the condition for the everyday conceptualization of time, for the common meaning of temporality (p. 331). This means that despite any cultural differences in the perception of time, religious beliefs and poetic fantasies about the immortality of the human soul, the inevitable finitude is grounding the primordial comprehension of the surrounding world. On a more superficial level, where this topic meets economic issues, we can note that the finitude of human life and its phases from birth to death are at the heart of all public institutions. They would lose sense, just like any normal subject of conversation, at the moment when the finitude of human existence is suspended and life becomes eternal. I believe that the principle of scarcity of the money sign corresponds to the primordial finitude of human temporality, the being of the being there as a thrown project. “Time is money” is a cliché, behind which lies the deep homology between two dimensions, whose meaning and value depend on the limitations of their availability. It is this homology that makes money good for thinking, and gives authors such as Goetzmann the insistence that finance is the foundation of “the ability of humans to imagine and calculate the future” (Goetzmann, 2016, p. 2). Notes 1 2 3 4 5 Saussure, F. de (1959 [1916]). A course in general linguistics (Trans., W. Baskin). New York: Philosophical Library (quoted as Course in the text). […] it is only in the context of inherently meaningful collaborative activities, coordinated by “natural” forms of communication such as pointing and pantomiming, that totally arbitrary linguistic conventions could have come into existence. (Tomasello, 2008, pp. 327-328) Davies, 1997; Ferguson, 2008; Goetzmann, 2016. Seaford, 2004; Goetzmann, 2016. “Scarcity is the fundamental economic problem of having seemingly unlimited human wants in a world of limited resources” (Sexton, 2017, p. 65). 121 Exploring Saussure’s Analogy between Linguistic and Monetary Signs References Baudrillard, J. (1968). Le Système des objectes. Paris: Gallimard. Baudrillard, J. (1975). Mirror of production (Trans., M. Poster). St. Louis: Telos Press. Davies, G. (1997). A history of money from ancient times to the present day. Cardiff: University of Wales Press. Derrida, J. (1992). Given time: I. Counterfeit money (Trans., P. Kamuf). Chicago: University of Chicago Press. Dyer, A. W. (1989). Making semiotic sense of money as a medium of exchange. Journal of Economic Issues, 23(2), 503-510. Eco, U. (1975). A theory of semiotics. Bloomington: Indiana University Press. Ferguson, N. (2008). The ascent of money: A financial history of the world. New York: Penguin. Goetzmann, W. N. (2016). Money changes everything: How finance made civilization possible. Princeton and Woodstock: Princeton University Press. Heidegger, M. (1996 [1927]). Being and time (Trans., J. Stambaugh). Albany: State University of New York Press. Horwitz, S. (1992). Monetary exchange as an extra-linguistic social communication process. Review of Social Economy, 50(2), 196-214. Mauro, T. de (2005). Notizie biografiche e critiche su F. De. Saussure. In Saussure, F. de 2005/1916 Corso di linguistica generale. Bari: Laterza. Ponzio, A. (1990). Man as a sign. Essays on the philosophy of language (Edited and translated from Italian and with an introduction by S. Petrilli). Berlin and New York: Mouton de Gruyter. Rossi-Landi, F. (1975). Linguistics and economics (= Janua Linguarum, Series Maior 81). The Hague: Mouton, 2nd ed. 1977 [first publ. in: Current Trends in Linguistics. Vol. XII: Linguistics and Adjacent Arts and Sciences. The Hague: Mouton 1974, Part 8, 1787-2017] Saussure, F. de (1959 [1916]). A course in general linguistics (Trans., W. Baskin). New York: Philosophical Library. (quoted as Course in the text) Seaford, R. (2004). Money and the early greek mind. Cambridge: Cambridge University Press. Sexton, R. L. (2017). Exploring economics. CTI Reviews, Content Technologies. Simmel, G. (1990). The philosophy of money (Ed., D. Frisby; Trans.,T. Bottomore & D. Frisby). London and New York: Routledge. Tomasello, M. (2008). Origins of human communication. Cambridge, MA: MIT Press. Tratner, M. (2004). Derrida’s Debt to Milton Friedman. New Literary History, 34, 791-806. Wennerlind, C. (2001). Money talks, but what is it saying? Semiotics of money and social control. Journal of Economic Issues, 35, 557-574. About the author Kristian Bankov (kbankov@nbu.bg) is Professor in Semiotics at New Bulgarian University (NBU), visiting professor at Sichuan University and Director of the Southeast European Center for Semiotic Studies at NBU. His interests in semiotics started during the early nineties when he was studying in Bologna, following the courses of Ugo Volli and Umberto Eco. He graduated in 1995 and since then teaches semiotic courses at New Bulgarian University. In 2000 he defended his PhD in Helsinki. In 2006 he became Associate Professor in Semiotics, and he became Professor in 2011. He has written five books and numerous articles in Bulgarian, English, and Italian. Since 2006 he has directed the organization of the international Early-Fall School of Semiotics (EFSS); in 2014 he was elected as the Secretary General of the International Association for Semiotic Studies. 122 

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Jurnal Diversita, 7 (2) Desember (2021) ISSN 2461-1263 (Print) ISSN 2580-6793 (Online) DOI: https://doi.org/10.31289/diversita.v7i2.5038 Jurnal Diversita Available online http://ojs.uma.ac.id/index.php/diversita Studi Meta-Analisis : Efektivitas Cognitive Behavioural Therapy untuk Meningkatkan Self-Esteem Meta-Analysis Study : The Effectiveness Cognitive Behavioural Therapy for Increase Self-Esteem Rizki Sakinah Dewi(1*) & Ananta Yudiarso(2) Fakultas Psikologi, Universitas Surabaya, Indonesia Disubmit: 23 Maret 2021; Diproses: 02 Agustus 2021; Diaccept: 02 September 2021; Dipublish: 02 Desember 2021 *Corresponding author: E-mail: rizkisakinahdewi@gmail.com Abstrak Teknik Cognitive Behavioural Therapy adalah sebuah teknik intervensi yang cukup efektif untuk dapat meningkatkan self-esteem individu. Self-esteem yang dimiliki oleh individu akan memengaruhi tingkat kesejahteraannya dan akan memengaruhi bagaimana ia dapat memandang dirinya sendiri secara positif atau negatif. Oleh sebab itu, penelitian ini dilakukan untuk dapat mengetahui apakah teknik intervensi Cognitive Behavioural Therapy dapat membantu meningkatkan self-esteem pada individu. Metode yang digunakan untuk melihat tingkat efektivitas penggunaan teknik Cognitive Behavioural Therapy tersebut ialah metode meta-analysis, yang dilakukan dengan melihat effect size dari 20 jurnal eksperimen penelitian terdahulu. Effect size didapatkan dengan melihat jumlah partisipan (N), mean (M), dan standard deviasi (SD) pada setiap kelompok eksperimen dan kontrol. Hasil penelitian ini menunjukkan bahwa teknik Cognitive Behavioural Therapy tidak cukup efektif untuk meningkatkan self-esteem pada individu (Hedges’g = 0.34), (95%CI = 0.11 hingga 0.3; dan I2 = 88.0% ), hal ini dikarenakan keberhasilan seorang individu untuk meningkatkan self-esteem juga dapat dipengaruhi oleh dukungan sosial yang diterimanya dari lingkungan, baik keluarga maupun sosial. Kata Kunci: Cognitive Behavioural Therapy; Meta-Analisis; Self-Esteem Abstract Cognitive Behavioral Therapy technique is an intervention technique that is quite effective to increase individual selfesteem. The possessed self-esteem will affects individual level of well-being and how to evaluate theirself positively or negatively. Therefore, this study was conducted to determine whether Cognitive Behavioral Therapy intervention techniques can help increase self-esteem in individuals. Method: The method used to see the level of effectiveness of the Cognitive Behavioral Therapy technique is the meta-analysis method, which was carried out with effect sizes from 20 previous research experimental journals. Effect size was obtained by looking at the number of participants (N), mean (M), and standard deviation (SD) in each experimental and control group. Result: The results of this study indicates that the Cognitive Behavioral Therapy technique is not effective enough to increase self-esteem in individuals (Hedges'g = 0.34), (95%CI = 0.11 to 0.3; and I2 = 88.0%), this is due to the success of an individual to improve self-esteem. Self-esteem can also be influenced by social support received from the environment, both family and social. Keyword: Cognitive Behavioural Therapy; Meta-Analysis; Self-Esteem How to Cite: Dewi, R.S. & Yudiarso, A. (2021). Studi Meta-Analisis : Efektivitas Cognitive Behavioural Therapy untuk Meningkatkan Self-Esteem. Jurnal Diversita, 7 (2): 231-239. 231 Dewi, R.S. & Yudiarso, A. Studi Meta-Analisis : Efektivitas Cognitive Behavioural Therapy PENDAHULUAN Self-esteem menjadi topik yang menarik dalam penelitian ini dikarenakan tingkat self-esteem pada seorang individu akan sangat berpengaruh pada kesejahteraan individu tersebut. Terlebih self-esteem merupakan bagian penting dalam konsep diri seseorang, bagaimana seseorang tersebut akan menanamkan orientasi secara positif maupun negatif terhadap dirinya (Ismi Isnani Kamila & Mukhlis, 2013). Self-esteem pada setiap individu akan menetapkan apakah individu tersebut dapat tampil dengan maksimal dan kompeten pada bidang-bidang yang penting bagi mereka, baik dalam hal akademik, sosial, keterampilan maupun keadaan atau kondisi fisiknya (Santrock, 2012). Self-esteem sendiri merupakan cara yang digunakan seseorang untuk dapat mengevaluasi dirinya sendiri (Santrock, 2012). Menurut Branden (1969) self-esteem diartikan sebagai keyakinan seseorang untuk kompeten dalam menjalani hidupnya dan layak hidup (Waite et al., 2012). Menurut Rosenberg (1965) selfesteem adalah sebuah evaluasi secara global yang dilakukan individu terhadap dirinya sendiri. Self-esteem sendiri merupakan komponen dari skema kognitif yang difokuskan pada dirinya sendiri (Neac, 2013). Menurut skala pengukuran self-esteem milik Rosenberg 1965 (Nurhidayati, 2014) terdapat dua aspek dalam pengukuran self-esteem, diantaranya ialah: 1) Self-worth 2) Selfacceptance. Menurut Harter (dalam Brooks, 1999) terdapat dua faktor yang berhubungan dengan tinggi dan rendahnya self-esteem pada individu, yaitu: 1) Perasaan dan pikiran individu akan kemampuannya serta bahwa dirinya 232 berharga; 2) Individu mendapatkan dukungan sosial, baik dari lingkungan keluarga, sosial, maupun teman sebaya (Nurhidayati, 2014) Individu dengan rendahnya selfesteem ini sebagian besar berasal dari latar belakang keluarga yang bermasalah atau terdapat konflik, individu akan merasa ditolak oleh keluarga dan sosial, pernah mengalami kekerasan, serta tidak mendapatkan dukungan sosial (Santrock, 2012). Individu yang mengalami rendahnya self-esteem cenderung lebih rentan terhadap berbagai macam gangguan klinis, diantaranya ialah kecemasan, kesepian, dan mengalami gangguan makan. Selain itu, ketika berada dalam kondisi yang kurang baik, mereka akan memandang semuanya secara negatif dan hanya mengingat dan memikirkan perilaku-perilaku yang buruk saja (Myers, 2014). Menurut McClure., et. al. (2010) rendahnya self-esteem akan memengaruhi kondisi psikologis, fisik dan sosial seseorang, selain itu bagi perkembangan anak juga akan memengaruhi prestasi akademiknya (Wibowo, 2016). Dalam sebuah studi menyatakan bahwa dari 326.641 individu dengan rentang usia 9 – 90 tahun, self-esteem pada masa remaja cenderung menurun dan mulai meningkat di usia 20 hingga 50 tahun, lalu Kembali menurun diusia 70 hingga 80 tahun. Pada anak remaja perempuan self-esteem biasa menurun pada usia 12 hingga 14 tahun, sedangkan pada remaja laki-laki biasa menurun di usia 16 tahun (Ismi Isnani Kamila & Mukhlis, 2013). Cognitive Behaviour Therapy (CBT) merupakan sebuah teknik intervensi yang dapat meningkatkan self-esteem pada seseorang. Teknik CBT sendiri memiliki 2 Jurnal Diversita, 7 (2) Desember 2021: 231-239. model dasar yang dilakukan, yaitu Cognitive restructuring therapy, dimana pada model ini klien akan diajak untuk merubah distorsi kognitif yang mempertahankan perilaku menyimpang mereka dan merubahnya menjadi kognitif yang lebih adaptif, serta Cognitivebehavioural coping skills therapy mengajarkan klien untuk memberikan respon yang adaptif dan efektif dalam menghadapi situasi-situasi yang bermasalah (Spiegler & Guevremont, 2013). Dalam penelitian yang dilakukan (Handley et al., 2015) terdapat peningkatan yang signifikan pada selfesteem setelah diberikan teknik CBT. Selain itu, penelitian lain yang dilakukan oleh (Salehyan & Aghabeiki, 2011) menunjukkan bahwa hasil rata-rata posttest self-esteem dalam kelompok eksperimen yang mendapatkan intervensi CBT meningkat secara signifikan dibandingkan kelompok kontrol yang tidak menerima intervensi. Hal ini menunjukkan bahwa teknik CBT cukup efektif dalam meningkatkan self-esteem. Selain itu dijelaskan pula oleh (Spiegler & Guevremont, 2013) apabila penerapan teknik cognitive behavior therapy ini dilakukan dengan secara konsisten maka akan meningkatkan kepercayaan dan harga diri (self-esteem) mereka terkait kondisi atau keadaan dirinya. Berdasarkan penjelasan diatas, oleh karena itu peneliti akan memberikan fokus penelitian untuk melihat apakah penggunaan teknik Cognitive Behaviour Therapy (CBT) efektif untuk meningkatkan self-esteem pada individu. Jenis penelitian yang dilakukan ialah meta-analysis, hal ini dikarenakan peneliti melihat adanya perbedaan penemuan dari literaturliteratur sebelumnya. Sehingga penelitian 233 meta-analysis ini perlu dilakukan untuk melihat effect size dari penelitian terdahulu yang serupa. METODE PENELITIAN Jenis penelitian yang akan digunakan ialah meta-analysis, yang mengikuti pedoman Prisma 2012 dan Meta-analysis Review Standart (MARS). Pengumpulan data penelitian terdahulu dilakukan dengan menggunakan Science Direct dan Google Scholar, dengan menggunakan kata kunci (1) Cognitive Behaviour Therapy for Self-esteem (2) Cognitive Behavioural Therapy Group for Self-esteem. Pencarian jurnal penelitian terdahulu ini dibatasi hanya untuk jurnal internasional dengan rentang waktu 20112020 (10 tahun terakhir). Hasil dari pencarian yang dilakukan, ditemukan 20 jurnal penelitian terdahulu yang dapat diolah. Namun penelitian ini hanya akan dibatasi untuk melihat efektivitas penerapan teknik Cognitive Behavioral Therapy (CBT) untuk meningkatkan selfesteem pada individu. Hasil penelitian terdahulu akan diolah dengan menggunakan Free Online Meta-Analysis Service (www.metamar.com) yang menggunakan indeks Hedges’g untuk mengetahui effect size. Pengolahan data dilakukan dengan melihat hasil mean (M), standard deviation (SD), dan jumlah sampel (N) pada posttest setelah diberikan intervensi. Inconsistensy (𝐼 2 ) akan digunakan untuk melihat keberagaman data penelitian 2 (heterogenitas), 𝐼 > 75% maka hasil penelitian semakin beragam. Menurut standar Hedges’g (Ellis, 2010) terdapat 3 kategori nilai effect size, yaitu (1) Small Dewi, R.S. & Yudiarso, A. Studi Meta-Analisis : Efektivitas Cognitive Behavioural Therapy effect size apabila nilai hedges’g 0,2; (2) Medium effect size apabila nilai hedges’g 0,5; (3) Large effect size apabila nilai hedges’g 0,8. 15.927 jurnal untuk hasil pencarian Cognitive Behavioral Therapy Group for self-esteem pada Science Direct 7.023 jurnal hasil untuk pencarian Cognitive Behavioral Therapy Group for self-esteem pada Science Direct setelah difokuskan tahun 2011 - 2020 Jurnal tidak menggunakan teknik intervensi CBT atau tidak menggunakan variable selfesteem 91 jurnal hasil penerapan Cognitive Behavioral Therapy pada self esteem Beberapa jurnal menggunakan korelasi dan one group (pretest & posttest) 20 jurnal eksperimen penerapan teknik CBT untuk meningkatkan self-esteem Gambar 1. Skema Alur Pencarian Jurnal Terdahulu Penerapan CBT Untuk Meningkatkan Self-Esteem No Peneliti 1 (Tylee et al., 2014) (Kothari et al., 2019) (Roghan chi et al., 2013) (Egan et al., 2014) (Premk umar et al., 2011) 2 3 4 5 6 Partisipan Eksperim en N = 171 M = 14.6 SD = 5.0 N = 31 M = 14.59 SD = 2.78 N = 24 M = 9.68 SD = 3.78 N = 16 M = 29.38 SD = 5.21 N = 25 M = -1.04 SD = 6.15 (Waite N = 11 et al., M = 122.73 2012) SD = 18.69 Tabel 1. Data 20 Jurnal Penelitian Terdahulu Metode Alat Effect Ukur Size (g) Kontrol N = 193 M = 12.9 SD = 4.8 N = 42 M = 13.15 SD = 1.49 N = 24 M = 6.78 SD = 3.84 N = 18 M = 27.87 SD = 4.60 N = 17 M = 2.25 SD = 3.84 N = 11 M = 84.81 SD = 18.86 SEg g_lower g_upper CBT fennell’s RSES model 0,346577 0,105 587 0,139627 0,553528 Internet RSES based (ICBT) 0,668106 0,240 712 0,19631 1,139902 REBT + art SEI therapy 0,756398 0,294 25 0,179668 1,333129 CBT face to RSES face 0,301166 0,337 458 -0,36025 0,962583 CBT for RSES psychosis (CBTp) + standard care CBT RSCQ -0,60367 0,315 385 -1,22183 0,01448 1,942989 0,504 055 0,955041 2,930937 234 Jurnal Diversita, 7 (2) Desember 2021: 231-239. 7 (Shu et al., 2019) N = 94 M = 16.71 SD = 3.80 N = 94 M = 16.73 SD = 5.08 8 (Meirong et al., 2019) (Sönme z et al., 2020) (Salehya n & Aghabei ki, 2011) (Shimot su et al., 2018) (Hazell et al., 2018) (Charkh andeh et al., 2016) (Handle y et al., 2015) (Joseph et al., 2016) (Tanoue et al., 2018) (Neac, 2013) N = 67 M = 28.43 SD = 4.91 N = 57 M = 27.78 SD = 5.14 N = 32 M = 27.1 SD = 5.9 N = 15 M = -5.6 SD = 1.55 N = 40 M = 30.60 SD = 8.80 N = 14 M = 15.07 SD = 5.37 N = 65 M = 3.09 SD = 1.44 9 10 11 12 13 14 15 16 17 18 19 20 N = 21 M = 28.44 SD = 0.36 N = 48 M = 18.65 SD = 4.93 N = 20 M = 32.06 SD = 10.46 N = 40 M = 19.22 SD = 6.23 (Hullu N = 46 et al., M = 37.57 2017) SD = 10.36 (Freema N = 15 n et al., M = 99.9 2014) SD = 21.6 (Tarrier N = 17 et al., M = 88.0 2014) SD = 27.0 Internet RSES based CBT for perfectionis m (ICBT-P) CBT SES 0,075487 0,145 328 -0,20936 0,36033 0,128764 0,179 269 -0,2226 0,480131 N = 31 M = 26.8 SD = 7.6 N = 15 M = -1.7 SD = 1.55 CBT + RSES Treatment as usual CBT RSES 0,043641 0,248 927 -0,44426 0,531538 -2,66957 0,494 975 -3,63972 -1,69942 N = 46 M = 29.26 SD = 9.10 N = 14 M = 10.71 SD = 5.37 N = 63 M = 3.97 SD = 1.70 CBT RSES 0,148182 0,214 554 -0,27234 0,568708 CBT + RSES Treatment as usual CBT CDI 0,78827 0,381 775 0,03999 1,53655 -0,55599 0,179 146 -0,90712 -0,20487 N = 21 M = 25.73 SD = 0.30 N = 52 M = 18.00 SD = 4.65 N = 23 M = 31.36 SD = 10.20 N = 40 M = 16.76 SD = 4.61 N = 35 M = 34.74 SD = 9.93 N = 14 M = 82.9 SD = 25.6 N = 19 M = 77.3 SD = 24.7 CBT for RSES perfectionis m (CBT-P) CBT RSES 8,024086 0,926 379 6,208384 9,839789 0,134761 0,198 853 -0,25499 0,524513 CBT RSES 0,066573 0,300 201 -0,52182 0,654966 Cignitive Behavioural Intervention CBT RSES 0,444559 0,224 221 0,005085 0,884033 RSES 0,275425 0,223 215 -0,16208 0,712927 CBT RSQ 0,699826 0,372 699 -0,03066 1,430315 0,405388 0,329 908 -0,24123 1,052007 Cognitive SERS Behavioural Prevention of Suicide in Psychosis protocol (CBSPp) (Sumber : www.meta-mar.com) 235 Dewi, R.S. & Yudiarso, A. Studi Meta-Analisis : Efektivitas Cognitive Behavioural Therapy kurang signifikan dalam meningkatkan self-esteem. Hasil dari pengolahan data 20 jurnal internasional menggunakan metamar online, dengan nilai random effect model 0,34 (small to medium), (95%CI = 0.11 hingga 0.3) serta nilai inkonsistensi (𝐼 2 = 88.0%) yang tergolong tinggi. Hasil tersebut menunjukkan bahwa CBT tidak dapat secara signifikan meningkatkan selfesteem. Selain itu juga menunjukkan adanya keberagaman data yang digunakan, baik dalam hal perbedaan alat ukur yang digunakan, jumlah partisipan penelitian, perbedaan jenis kelamin partisipan, perbedaan cara penerapan teknik CBT. HASIL DAN PEMBAHASAN Pada 20 jurnal internasional telah dipaparkan hasil effect size dengan cara mengolah nilai jumlah partisipan (N), mean (M), dan standar deviasi (SD), dari setiap kelompok eksperimen dan kelompok kontrol. Hasil 20 jurnal internasional menunjukkan bahwa penerapan teknik Cognitive Behaviour Therapy ini adalah sebuah teknik intervensi yang cukup dapat meningkatkan self-esteem yang dimiliki individu, meskipun tidak terlalu efektif. Hal tersebut nampak berbeda dari hasil perhitungan effect size pada penelitian ini, yang menunjukkan bahwa penerapan teknik Cognitive Behavioural Therapy Tabel . Summary Of Result - Fixed And Random Effect Models Hedges’g (SMD) 0.21 SEg 95%CI Fixed Effect Model 0.05 [0.11,0.3] Random Effect 0.34 0.156 [0.031,0.643] Model (Sumber Tabel: www.meta-mar.com) z score p value Heterogenity 4.078 4.5e-05 𝐼 =88.0%, 𝐶ℎ𝑖 2 = 158.08, 𝑑𝑓 = 19 2.156 0.03105 𝐼 2 =88.0%, 𝑇𝑎𝑢2 = 0.39 2 Gambar 2. Forestplot Random Effect Model (Sumber : www.meta-mar.com) Gambar 3. Funnel Plot Random Effect Model (Sumber : www.meta-mar.com) Self-esteem yang dimiliki setiap individu akan memengaruhi bagaimana seorang individu dapat tampil dengan maksimal dan kompeten pada berbagai macam bidang penting dalam kehidupannya, baik dalam bidang akademik atau pendidikan, keterampilan sosial maupun keadaan atau kondisi fisiknya (Santrock, 2012). Ketika seorang individu memiliki self-esteem yang rendah, maka ia 236 Jurnal Diversita, 7 (2) Desember 2021: 231-239. akan cenderung rentan mengalami berbagai macam gangguan klinis, seperti kecemasan, kesepian hingga mengalami gangguan makan. Selain itu, ketika berada dalam kondisi yang kurang baik, mereka akan memandang semuanya secara negatif dan hanya mengingat dan memikirkan perilaku-perilaku yang buruk saja (Myers, 2014). Terdapat dua hal yang dapat memengaruhi self-esteem pada individu, yaitu perasaan dan pikiran individu akan kemampuannya serta bahwa dirinya berharga serta dukungan sosial yang dimiliki individu, baik dari lingkungan keluarga, sosial, maupun teman sebaya (Nurhidayati, 2014). Berdasarkan pembahasan diatas, menunjukkan bahwa self-esteem sangatlah penting untuk kesejahteraan individu, oleh sebab itulah berbagai macam teknik intervensi dilakukan untuk dapat meningkatkan self-esteem seorang individu, diantaranya ialah cognitive behavioural therapy (CBT), group therapy, art therapy, rational emotive behavior therapy (REBT), dan berbagai macam alternatif lain yang diharapkan dapat meningkatkan selfesteem seorang individu. Namun, beberapa penelitian menyatakan bahwa Cognitive Behavioural Therapy (CBT) ialah salah satu teknik yang seringkali digunakan serta dapat meningkatkan self-esteem pada individu. Pada hasil penelitian ini menunjukkan bahwa CBT kurang efektif untuk dapat meningkatkan self-esteem. Hal ini ditunjukkan pada nilai hedges’g 0,34, dimana menurut standar Hedges’g nilai effect size tersebut tegolong dalam small effect size. Hasil tersebut dapat dipengaruhi oleh beberapa faktor, diantaranya ialah dukungan sosial yang diterima individu, baik dari lingkungan sosial maupun keluarga. Hal tersebut menunjukkan bahwa individu yang telah mendapatkan teknik Cognitive Behavioural Therapy (CBT), namun tidak mendapatkan dukungan baik dari keluarga, teman maupun lingkungan sosialnya, akan cenderung tetap memiliki self-esteem yang rendah. Selain itu ditemukannya keberagaman data pada hasil penelitian yang cukup besar, ditunjukkan pada nilai inkonsistensi (𝐼 2 = 88.0%). Nilai inkonsistensi yang tergolong tinggi tersebut dapat dipengaruhi oleh beberapa faktor, diantaranya ialah jumlah partisipan yang berbeda-beda pada tiap jurnal penelitian, perbedaan metode intervensi CBT yang digunakan pada setiap jurnal, perbedaan jenis kelamin partisipan, serta perbedaan alat ukur self-esteem yang digunakan pada setiap jurnal penelitian terdahulu. SIMPULAN Penelitian meta-analysis ini dilakukan untuk melihat efektivitas penerapan teknik Cognitive Behavioural Therapy untuk meningkatkan self-esteem. Teknik CBT sendiri merupakan teknik yang menggabungkan 2 model dasar, yaitu pikiran dan perilaku. Penerapan CBT ini diharapkan dapat merubah distorsi kognitif atau pemikiran yang tidak tepat pada individu dan merubahnya menjadi pemikiran yang lebih adaptif sehingga dapat memunculkan perilaku yang lebih adaptif dan efektif pula (Spiegler & Guevremont, 2013). Sehingga penerapan teknik CBT ini diharapkan dapat merubah pemikiran negatif individu terhadap dirinya sendiri serta dapat lebih menghargai dirinya sendiri, agar individu tersebut dapat lebih kompeten dan positif dalam menjalani kehidupannya, baik dalam lingkungan sosial maupun akademik. 237 Dewi, R.S. & Yudiarso, A. Studi Meta-Analisis : Efektivitas Cognitive Behavioural Therapy Namun hasil dalam penelitian metaanalysis ini ditemukan bahwa penerapan teknik CBT kurang efektif atau signifikan untuk meningkatkan self-esteem individu, hal ini dikarenakan selain mendapatkan terapi, individu juga seharusnya mendapat dukungan sosial yang cukup, baik dari keluarga, teman sebaya, maupun lingkungan sosialnya, agar self-esteem dapat meningkat (Nurhidayati, 2014). Hal ini juga mendapat dukungan dari penelitian lain yang menjelaskan bahwa belum dapat dipastikan apakah penerapan teknik CBT ini dapat merubah proses kognitif individu yang mendasari rendahnya harga diri (Kolubinski et al., 2018). Selain itu, hal ini juga dapat dipengaruhi oleh hasil perhitungan yang heterogen atau beragam, baik dari perbedaan alat ukur yang digunakan, perbedaan penerapan teknik CBT, jumlah partisipan, dan perbedaan jenis kelamin partisipan. Sehingga diharapkan untuk penelitian selanjutnya terkait topik ini dapat lebih memperhatikan metode dan partisipan yang digunakan, serta lebih dapat menggali literatur atau teknik intervensi lain yang mungkin dapat menjadi treatment tambahan untuk membantu meningkatkan self-esteem dalam diri seseorang. DAFTAR PUSTAKA Charkhandeh, M., Abu, M., & Jane, C. (2016). The clinical effectiveness of cognitive behavior therapy and an alternative medicine approach in reducing symptoms of depression in adolescents. Psychiatry Research, 239, 325– 330. https://doi.org/10.1016/j.psychres.2016.03.044 Egan, S. J., Noort, E. Van, Chee, A., Kane, R. T., Hoiles, K. J., Shafran, R., & Wade, T. D. (2014). 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"Getting Religion": Religion, Diversity, and Community in Public and Private Schools Citation Levinson, Meira, and Sanford Levinson. 2003. "Getting Religion": Religion, Diversity, and Community in Public and Private Schools. In Wrestling with Diversity, ed. Sanford Levinson, 90-123. Durham, NC: Duke University Press. Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:8454067 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility 

W4243708183.txt

Hindawi BioMed Research International Volume 2018, Article ID 3782512, 1 page https://doi.org/10.1155/2018/3782512 Corrigendum Corrigendum to (Evaluation of Plasma MicroRNAs as Diagnostic and Prognostic Biomarkers in Pancreatic Adenocarcinoma: miR-196a and miR-210 Could Be Negative and Positive Prognostic Markers, Respectively) Qi Yu,1 Changqing Xu,2 Wei Yuan,3 Chengfeng Wang ,4 Ping Zhao ,4 Lianzhen Chen ,5 and Jie Ma 6 1 Gastrointestinal Rehabilitation Center, Beijing Rehabilitation Hospital, Capital Medical University, Beijing 100144, China Department of Digestive Medicine, Qianfoshan Hospital, Shandong University, 16766 Jingshi Road, Jinan 250014, China 3 State Key Laboratory of Molecular Oncology, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China 4 Department of Abdominal Surgery, Cancer Hospital of Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China 5 Department of Pharmacy, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China 6 Department of Biotherapy, Beijing Hospital, National Center of Gerontology, Beijing 100730, China 2 Correspondence should be addressed to Ping Zhao; dr.zhaoping@263.net and Lianzhen Chen; yjkclz@163.com Received 1 August 2018; Accepted 2 August 2018; Published 6 September 2018 Copyright © 2018 Qi Yu 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. In the article titled “Evaluation of Plasma MicroRNAs as Diagnostic and Prognostic Biomarkers in Pancreatic Adenocarcinoma: miR-196a and miR-210 Could Be Negative and Positive Prognostic Markers, Respectively” [1], there were errors in the authors’ affiliations. The correct affiliation list is shown above. References [1] Qi Yu, Changqing Xu, Wei Yuan et al., “Evaluation of Plasma MicroRNAs as Diagnostic and Prognostic Biomarkers in Pancreatic Adenocarcinoma: miR-196a and miR-210 Could Be Negative and Positive Prognostic Markers, Respectively,” BioMed Research International, vol. 2017, Article ID 6495867, 10 pages, 2017. 

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Oncotarget, Vol. 6, No. 26 www.impactjournals.com/oncotarget/ MYC amplifications in myeloma cell lines: correlation with MYC-inhibitor efficacy Toril Holien1, Kristine Misund1, Oddrun Elise Olsen1, Katarzyna Anna Baranowska1, Glenn Buene1, Magne Børset1,2, Anders Waage1,3, Anders Sundan1,4 1  .G. Jebsen Center for Myeloma Research, Department of Cancer Research and Molecular Medicine, Norwegian University K of Science and Technology, Trondheim, Norway 2 Department of Immunology and Transfusion Medicine, St. Olav’s University Hospital, Trondheim, Norway 3 Department of Hematology, St. Olav’s University Hospital, Trondheim, Norway 4  EMIR (Centre of Molecular Inflammation Research), Department of Cancer Research and Molecular Medicine, Norwegian C University of Science and Technology, Trondheim, Norway Correspondence to: Toril Holien, e-mail: toril.holien@ntnu.no Keywords: oncology, carcinogenesis, molecular and cellular biology, signal transduction, cell cycle Received: March 09, 2015 Accepted: May 20, 2015 Published: June 02, 2015 ABSTRACT In multiple myeloma, elevated MYC expression is related to disease initiation and progression. We found that in myeloma cell lines, MYC gene amplifications were common and correlated with MYC mRNA and protein. In primary cell samples MYC mRNA levels were also relatively high; however gene copy number alterations were uncommon. Elevated levels of MYC in primary myeloma cells have been reported to arise from complex genetic aberrations and are more common than previously thought. Thus, elevated MYC expression is achieved differently in myeloma cell lines and primary cells. Sensitivity of myeloma cell lines to the MYC inhibitor 10058-F4 correlated with MYC expression, supporting that the activity of 10058-F4 was through specific inhibition of MYC. MYC activity has been shown to be associated with many features of cancer cells including cell metabolism and proliferation. [7] In multiple myeloma it was commonly thought that activation of MYC was a late-stage event. [8] The importance of MYC in myeloma disease progression has lately become clearer, and increased MYC activity has been implicated in progression from MGUS to fullblown myeloma. [9–12] Recently, two papers showed that complex rearrangements positioning MYC in the proximity of super-enhancers caused elevated MYC expression in primary myeloma cells. [13, 14] Moreover, MYC was shown to be the most frequent translocation partner in aberrations involving the immunoglobulin light chains. [15] Altogether, MYC rearrangements were found in nearly half of the myeloma patients leaving MYC the most commonly mutated gene in multiple myeloma. [13] We have earlier shown that MYC expression was important for in vitro survival of myeloma cells using different approaches for targeting MYC. [16–18] The question we wanted to address in this study was whether the vulnerability of multiple myeloma cells INTRODUCTION Multiple myeloma is the second most common hematological malignancy and accounts for about 2% of cancer-related deaths. The cancer cells arise from postgerminal center plasma cells and usually reside in the bone marrow. Myeloma is preceded by a benign condition termed monoclonal gammopathy of undetermined significance (MGUS), [1] and the annual risk of progression to myeloma is about 1%. [2] Treatment of multiple myeloma has improved during the last decades by the introduction of proteasome inhibitors such as bortezomib and carfilzomib as well as immunomodulatory drugs (IMIDs) such as thalidomide and lenalidomide. However; the median overall survival time from diagnosis is still no more than 5–7 years. [3, 4] The transcription factor c-MYC (hereafter termed MYC) and the related N-MYC and L-MYC oncogenes are involved in the development of up to 70% of all cancers. [5] Under normal conditions MYC increases cell proliferation and halts differentiation. [6] Abnormal www.impactjournals.com/oncotarget 22698 Oncotarget for MYC inhibition correlated to cellular levels of MYC. Pharmacological targeting of MYC activity has been challenging. One option is to use small molecule inhibitors that target MYC-MAX heterodimerization thereby preventing transactivation of MYC target genes. [19, 20] We found that the small molecule inhibitor of MYC, 10058-F4, suppressed proliferation and survival of myeloma cells, arguing for a distinct role of MYC in multiple myeloma. The importance of MYC was further supported by an inverse correlation between IC50 of the inhibitor and the level of MYC in myeloma cell lines. indicate that the main determinant of elevated MYC expression in myeloma cell lines is amplification of the MYC gene. We went on to investigate the variation in MYC gene copy numbers in myeloma patient samples by the same method as applied for cell lines. Interestingly, most of the primary samples (n = 21) had two copies of the MYC gene and the samples deviating from this (n = 7) had MYC gene copies varying from 1 to 4 (data not shown). The levels of MYC mRNA, on the other hand, showed remarkable variation (Figure 3A). Thus, in contrast to myeloma cell lines, MYC levels in primary cells apparently are not determined by the number of gene copies as measured here, but by other mechanisms. Interestingly, we originally compared MYC mRNA levels in cell lines and primary cells applying GAPDH mRNA as the only reference, getting higher MYC levels in primary cells than in myeloma cell lines. However, when comparing GAPDH mRNA levels in cell lines with primary cells using the Nanostring nCounter technology and using several genes as reference; it turned out that the difference in GAPDH mRNA was even greater than the difference in MYC mRNA. Thus, when comparing MYC mRNA levels in primary cells and cell lines, the patient cells as a group had lower MYC expression levels than cell lines (Figure 3B). Patients (n = 28) had a median MYC level of 2345, range 660–15969, whereas the cell lines (N = 11) had a median level of 8411, range 85–18859. Furthermore, the cell lines had a median GAPDH level of 33347, range 23653–59757, whereas the primary cells had a median GAPDH level of 8345, range 2297–15769 (Figure 3C). We also treated the primary myeloma samples with the MYC inhibitor to obtain IC50 values. However, the cell viability varied and only 18 out of 28 samples had a viability > 50% without treatment. Those samples were included in analysis (data not shown). When comparing IC50 values for 10058-F4 in the samples with baseline viability > 50% with MYC mRNA expression, we were not able to see the same correlation as in cell lines. However, analysis of only 18 samples may not be sufficient to see such correlations. There was no correlation between cell viability in the patient samples and 10058-F4 IC50 values (n = 28, data not shown). The primary cells studied were both from newly diagnosed, untreated patients (n = 13), as well as from treated patients (n = 15), i.e. patients at a later stage of disease. Clinical characteristics of the patient samples are found in Supplementary Table I. Interestingly, the MYC mRNA levels were not different between previously treated and untreated patients (Figure 3D), indicating that untreated patients had myeloma cells with as high MYC expression as patients in later stages of disease. These observations suggest that both untreated and treated patients may have plasma cells with high MYC expression that possibly could be targeted by MYC inhibition. RESULTS We have earlier shown that the small molecule MYC inhibitor 10058-F4 induces apoptosis in myeloma cell lines and primary cells. [17, 20] The inhibitor downregulated MYC protein and mRNA in a dosedependent manner in myeloma cells (Supplemenatry Figure 1A–1C). We wanted to find out if the baseline MYC expression could determine myeloma cell sensitivity to 10058-F4. A panel (n = 11) of myeloma cell lines were treated with increasing amounts of inhibitor for three days. The combined effects on cell proliferation and viability were determined using CellTiter Glo which measures the ATP content in cells (Supplementary Figure 2). IC50 values were determined from doseresponse curves and related to MYC transcript numbers measured by the nCounter Nanostring technology (Figure 1A, Supplementary Figure 3A) and protein levels using immunoblotting (Figure 1B, Supplementary Figure 3B, 3C). There was a negative correlation between IC50 values and mRNA (R2 = 0, 548) or protein (R2 = 0, 585) levels. Taken together, the correlation between MYC expression and sensitivity to the 10058F4 compound, supports that 10058-F4 is a relatively specific inhibitor of MYC activity. Secondly, the finding that the cell lines with the highest MYC concentration were the most sensitive suggests that cell lines expressing high levels of MYC are more dependent on the MYC expression for proliferation or survival than cell lines expressing lower amounts of MYC. Next, we measured MYC gene copy numbers in all 11 myeloma cell lines using PCR with primers for exon 3 (Supplementary Figure 3D) and correlated the copy numbers with MYC mRNA, as well as with protein levels (Supplementary Figure 3A, 3B and 3C). In cell lines, the MYC gene copy numbers varied from two to nine. The measured copy numbers were almost identical using primers that were specific for exon 1 and exon 2 (Supplementary Figure 3D), indicating the presence of the whole gene rather than fragments of the gene. Interestingly, the MYC gene copy numbers correlated well with both mRNA (R2 = 0.847) and protein (R2 = 0.607) levels (Figure 2A and 2B). The results thus www.impactjournals.com/oncotarget 22699 Oncotarget Figure 1: MYC gene copy numbers determine expression of MYC mRNA and protein in myeloma cell lines. In a panel of myeloma cell lines the levels of MYC gene copy numbers as measured by PCR was related to A. MYC mRNA measured using nCounter, and B. MYC protein levels measured using immunoblotting and normalized to GAPDH. The slope and R2-values are shown in the plots. www.impactjournals.com/oncotarget 22700 Oncotarget Figure 2: Expression of MYC in myeloma cell lines correlated positively with sensitivity to MYC inhibition. The IC50- values of the MYC inhibitor 10058-F4 calculated from the results shown in Supplementary Figure 2 was compared with A. MYC mRNA values or B. MYC/GAPDH relative protein levels. The slope and R2-values are shown in the plots. www.impactjournals.com/oncotarget 22701 Oncotarget Figure 3: MYC and GAPDH mRNA levels in primary myeloma cells. A. The levels of MYC mRNA in myeloma patient samples (n = 28), P01-P28, were measured using nCounter and the calculated mRNA copy numbers were plotted. Box plots comparing the levels of MYC B. and GAPDH C. mRNA in 28 isolated plasma cells from myeloma patients with 11 myeloma cell lines. D. The levels of MYC mRNA measured as in A were plotted for treated (n = 15) versus untreated (n = 13) patients in a box plot. The upper and lower borders of the box indicate upper and lower quartile, whereas the line inside the box indicates the median value. Whiskers indicate upper and lower adjacent values, whereas outside values are indicated by (o) and far out values are indicated by asterisks (*). DISCUSSION A major concern when applying small molecule inhibitors is the specificity of the inhibitor. The 10058-F4 drug has been proposed to be a specific inhibitor of MYCMAX heterodimerization at concentrations < 100 μM. [20, 21] However, even if 10058-F4 clearly inhibits the MYC-MAX interaction, the compound may also affect other molecules within a cell. The correlation between MYC expression and sensitivity for the 10058-F4 drug found in cell lines indicated that the activity of the drug was specifically directed towards MYC activity. As MYC is a factor that is functionally non-redundant it may be particularly attractive as a therapeutic target. [22] MYC could already indirectly be targeted in the clinic since many drugs, such as dexamethasone and lenalidomide, have been shown to kill myeloma cells concomitantly with partial suppression of MYC, [23, 24] albeit, these drugs clearly also have other effects. We found higher MYC mRNA levels in myeloma cell lines than in primary cells; although the range was approximately the same in these two groups. Nevertheless, gene copy number variations in MYC were only common in cell lines, indicating that primary cells had other ways of increasing MYC levels than cell lines. Indeed, recent publications indicate that different rearrangements involving MYC cause elevated MYC expression in nearly half of newly diagnosed patients. [13–15] MYC expression has been reported to affect cell proliferation and energy metabolism in rapidly proliferating cancer cells. [7] Amplification of the MYC gene seen in myeloma cell lines may be a consequence of the selection pressure for proliferation and survival in tissue culture flasks. Cell lines The most important data presented here is that there was a positive correlation between MYC expression and sensitivity to the MYC activity inhibitor, 10058-F4, in myeloma cell lines. That finding supports that MYC is important for myeloma cell survival and that the inhibitor specifically targets MYC. Secondly, although both myeloma cell lines and primary cells expressed high amounts of MYC, there are differences in the underlying mechanisms behind the expression. And thirdly, MYC expression was in the same range in plasma cells from untreated compared to treated patients indicating that MYC activation is an early oncogenic event in multiple myeloma. Cell lines expressing the highest levels of MYC were the most sensitive to MYC-inhibition, indicating that these cells were more dependent on MYC for survival and proliferation than low-expressors. Thus, we hypothesize that patients with myeloma cells expressing high levels of MYC could benefit from MYC inhibition. MYC protein and mRNA are very unstable and may be rapidly degraded. When isolating primary myeloma cells from bone marrow samples the procedure usually takes a few hours and there is a risk that MYC protein and mRNA levels are not maintained at the same levels as when the cells are situated in the bone marrow. Thus, the most reliable way to measure MYC levels in patients could be by quantitative in situ immunohistochemistry using bone marrow biopsies. Unfortunately, it was not possible for us to perform such experiments in this study. www.impactjournals.com/oncotarget 22702 Oncotarget are highly proliferating, whereas primary myeloma cells in the bone marrow in general proliferate very slowly, with a Ki-67 labelling index less than 5% for most patients. [25] Amplification of the MYC gene may be a simple way of achieving high MYC levels in cell lines that over years have been selected for cell proliferation in vitro. Another difference is that, unlike myeloma cell lines, primary cells depend on the bone marrow microenvironment and are unable to survive in tissue culture. There was also a difference in GAPDH expression levels between cell lines and primary cells that could reflect differences in utilization of glycolysis for ATP production. In patients, a negative relationship between MYC expression and progression-free and overall survival has been described, suggesting an important role for MYC in the regulation of tumor mass. [14, 26–28] We compared the MYC mRNA expression with clinical information on progression-free and overall survival in our patient samples, but could not find any relationship (data not shown). However, the number of informative patients was too low to draw any conclusions. Nevertheless, our findings that MYC mRNA is as high in untreated patients as in patients at later stages of disease suggest that MYC may be important during the whole course of disease. To summarize, our results suggest that many myeloma patients, but first of all patients with cells expressing high MYC levels, might benefit from MYC inhibition. If a specific and clinically applicable MYCinhibitor became available, it would be important to characterize which patients might benefit from inhibition of MYC. Norway) whereas KJON was maintained in 5% HS, all in RPMI and IL-6 (2 ng/mL). Cells were cultured at 37oC in a humidified atmosphere containing 5% CO2. For experiments 2% HS in RPMI was used as medium, with IL-6 (1 ng/mL) added for all IL-6 dependent cell lines. MATERIALS AND METHODS Real-time quantitative reverse transcription PCR (qRT-PCR) Primary cells Primary CD138+ myeloma cells were isolated from bone marrow specimens included in the Norwegian Myeloma Biobank using RoboSep automated cell separator and Human CD138 Positive Selection Kit (StemCell Technologies, Grenoble, France). The study was approved by the Regional Ethics Committee (approval # 2011/2029) and all patients had given informed consent. Peripheral blood mononuclear cells (PBMC) were obtained from EDTA-blood from healthy controls by density gradient centrifugation using Lymphoprep (AxisShield, Oslo, Norway). Nucleic acid extraction Genomic DNA and total RNA were extracted from frozen cell pellets using AllPrep DNA/RNA Micro Kit and Qiacube (Qiagen, Venlo, the Netherlands) following the manufacturer’s instructions. The concentration and quality of DNA and RNA was determined using NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and samples were stored at −80°C until further use. Cell lines and reagents Complementary DNA (cDNA) was synthesized from total RNA using the High Capacity RNA-to-cDNA kit (Applied Biosystems, Carlsbad, CA, USA). PCR was performed using StepOne Real-Time PCR System and Taqman Gene Expression Assays (Applied Biosystems). The comparative Ct method was used to estimate relative changes in gene expression using MYC Taqman assay (Hs00153408_m1) and GAPDH (Hs99999905_m1) as housekeeping gene. A panel of 11 myeloma cell lines was used. Four of the cell lines were in-house: OH-2, IH-1, URVIN and KJON, whereas 7 were from other sources: INA-6, CAG, JJN3 and ANBL-6 were kind gifts from Dr M. Gramatzki (University of Erlangen-Nurnberg, Erlangen, Germany), Dr J. Epstein (University of Arkansas for Medical Sciences, Little Rock, AR, USA), Dr J. Ball (University of Birmingham, UK), and Dr D. Jelinek (Mayo Clinic, Rochester, MN, USA), respectively, KMS-12-BM was obtained from DSMZ (Braunschweig Germany), and RPMI-8226 and U266 were from ATCC (Rockville, MD, USA). URVIN, INA-6 and ANBL-6 cells were grown in 10% heat inactivated fetal calf serum (FCS) in RPMI-1640 (RPMI) supplemented with interleukin (IL)-6 (1 ng/mL) (Biosource, Camarillo, CA, USA). CAG, JJN3, KMS12-BM, RPMI-8226 and U266 were grown in RPMI with 10, 10, 20, 20 or 15% FCS, respectively. OH-2 and IH-1 were maintained in 10% heat-inactivated human serum (HS) (Department of Immunology and Transfusion Medicine, St. Olav’s University Hospital, Trondheim, www.impactjournals.com/oncotarget Copy number variation (CNV) Real-time reverse transcriptase quantitative polymerase chain reaction (real-time qPCR) was performed using the TaqMan Copy Number Assay Hs01764918_cn (MYC, exon 3), Hs00834648_cn (MYC, exon 2), Hs02758348_cn (MYC, exon 1), and TaqMan Copy Number Reference Assay (RNAse P) for internal control (Applied Biosystems). All PCR reactions were performed in triplets with genomic DNA using a StepOnePlus PCR system (Applied Biosystems). CNV was 22703 Oncotarget Statistical analysis analyzed using CopyCaller Software (Applied Biosystems) and DNA isolated from PBMC was used for calibration. Statistical analysis and box plots were made in IBM SPSS Statistics 20 (IBM Corp., Armonk, NY, USA). Gene expression analysis ACKNOWLEDGMENTS For mRNA transcript counting the nCounter Human Cancer Reference Kit (cat.no GXA-CR1–12) and nCounter Technology (Nanostring Technologies, Seattle, WA, USA) was used. The standard mRNA Geneexpression experiment protocol provided by Nanostring was used, the only exception being that kit probes were diluted 1:2. Briefly, 100 ng total RNA from myeloma cell lines or patient samples was hybridized with reporter probes overnight at 65°C. The nSolver Analysis Software (Nanostring) was used for calculations of transcript numbers. Sample data was normalized against internal kit positive controls and the following housekeeping genes: CLTC, GAPDH, GUSB, HPRT1, PGK1, and TUBB. The authors are grateful for technical help provided by Lill-Anny Gunnes Grøseth, Solveig Kvam, Berit Størdal and Hanne Hella. FUNDING This work was funded by grants from the Norwegian Cancer Society, the Liaison Committee between the Central Norway Regional Health Authority and the Norwegian University of Science and Technology, and the KG Jebsen Foundation for Medical Research. The funding sources did not participate in study design, collection, analysis or interpretation of the data, writing of the paper or deciding to submit the paper for publication. Immunoblotting Cells were treated as indicated, washed with icecold phosphate-buffered saline (PBS) and lysed for 30′ on ice. The lysis buffer contained 1% NP40 (SigmaAldrich, St Louis, MO, USA), 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), a protease inhibitor cocktail (Roche, Basel, Switzerland), 1 mM Na3VO4 and 50 mM NaF. Samples were electrophoresed on pre-cast agarose gels and blotted onto nitrocellulose membranes using the NuPAGE system (Life Technologies, Carlsbad, CA, USA). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with 0.01% Tween 20 (TBS-T). The primary antibodies used were c-MYC (RRID: AB_2148606, Cat# 551102, BD Biosciences, Trondheim, Norway), and GAPDH (RRID:AB_2107448, Cat# Ab8245, Abcam, Cambridge, UK). The secondary antibody was horseradish peroxidase-conjugated goatanti-mouse (Dako Cytomation, Glostrup, Denmark). Positive bands were detected using the luminescence substrate SuperSignal West Femto (Thermo Fisher Scientific) and Odyssey Fc imager (LI-COR Biosciences, Ltd., Cambridge, UK). CONFLICTS OF INTEREST The authors state no conflicts of interest. REFERENCES 1. Landgren O, Kyle RA, Pfeiffer RM, Katzmann JA, Caporaso NE, Hayes RB, Dispenzieri A, Kumar S, Clark RJ, Baris D, Hoover R, Rajkumar SV. Monoclonal ­gammopathy of undetermined significance (MGUS) consistently precedes multiple myeloma: a prospective ­ study. Blood. 2009; 113:5412–5417. 2. 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Wang H, Hammoudeh DI, Follis AV, Reese BE, Lazo JS, Metallo SJ, Prochownik EV. Improved low molecular weight Myc-Max inhibitors. Molecular cancer therapeutics. 2007; 6:2399–2408. 11. Chng WJ, Huang GF, Chung TH, Ng SB, Gonzalez-Paz N, Troska-Price T, Mulligan G, Chesi M, Bergsagel PL, Fonseca R. Clinical and biological implications of MYC activation: a common difference between MGUS and newly diagnosed multiple myeloma. Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund. UK. 2011; 25:1026–1035. 22. Sodir NM, Evan GI. Finding cancer’s weakest link. Oncotarget. 2011; 2:1307–1313. 21. Gomez-Curet I, Perkins RS, Bennett R, Feidler KL, Dunn SP, Krueger LJ. c-Myc inhibition negatively impacts lymphoma growth. Journal of pediatric surgery. 2006; 41:207–211. discussion 207-211. 23. Chauhan D, Auclair D, Robinson EK, Hideshima T, Li G, Podar K, Gupta D, Richardson P, Schlossman RL, Krett N, Chen LB, Munshi NC, Anderson KC. Identification of genes regulated by dexamethasone in multiple myeloma cells using oligonucleotide arrays. Oncogene. 2002; 21:1346–1358. 12. Chiecchio L, Dagrada GP, Protheroe RK, Stockley DM, Smith AG, Orchard KH, Cross NC, Harrison CJ, Ross FM. Loss of 1p and rearrangement of MYC are associated with progression of smouldering myeloma to myeloma: sequential analysis of a single case. Haematologica. 2009; 94:1024–1028. 24. Lopez-Girona A, Heintel D, Zhang LH, Mendy D, Gaidarova S, Brady H, Bartlett JB, Schafer PH, Schreder M, Bolomsky A, Hilgarth B, Zojer N, Gisslinger H, et al. Lenalidomide downregulates the cell survival factor, interferon regulatory factor-4, providing a potential mechanistic link for predicting response. Br J Haematol. 2011; 154:325–336. 13. Affer M, Chesi M, Chen WD, Keats JJ, Demchenko YN, Tamizhmani K, Garbitt VM, Riggs DL, Brents LA, Roschke AV, Van Wier S, Fonseca R, Bergsagel PL, et al. 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Holien T, Vatsveen TK, Hella H, Rampa C, Brede G, Groseth LA, Rekvig M, Borset M, Standal T, Waage A, Sundan A. Bone morphogenetic proteins induce apoptosis in multiple myeloma cells by Smad-dependent repression of MYC. Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund. UK. 2012; 26:1073–1080. www.impactjournals.com/oncotarget 22705 Oncotarget 

W4225879952.txt

bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Asymmetric effects of acute stress on cost and benefit learning 2 Stella Voulgaropoulou1*, Fasya Fauzani1, Janine Pfirrmann1, Claudia Vingerhoets1, 2, Thérèse 3 van Amelsvoort1, Dennis Hernaus1 4 5 1 6 4-6, 6211 LK Maastricht, The Netherlands 7 2 8 Location AMC, Meibergdreef 5, 1105 AZ Amsterdam, The Netherlands. Department of Psychiatry & Neuropsychology, Maastricht University, Minderbroedersberg Department of Radiology & Nuclear Medicine, Amsterdam University Medical Centre, 9 10 Abstract 11 Stressful events trigger a complex physiological reaction – the fight-or-flight response – that 12 can hamper flexible decision-making. Inspired by key neural and peripheral characteristics of 13 the fight-or-flight response, here we ask whether acute stress changes how humans learn 14 about costs and benefits. Participants were randomly exposed to an acute stress or no-stress 15 control condition after which they completed a cost-benefit reinforcement learning task. 16 Acute stress improved learning to maximize benefits (monetary rewards) relative to 17 minimising energy expenditure (grip force). Using computational modelling, we demonstrate 18 that costs and benefits can exert asymmetric effects on decisions when prediction errors that 19 convey information about the reward value and cost of actions receive inappropriate 20 importance; a process associated with distinct alterations in pupil size fluctuations. These 21 results provide new insights into learning strategies under acute stress – which, depending on 22 the context, may be maladaptive or beneficial - and candidate neuromodulatory mechanisms 23 that could underlie such behaviour. 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 24 Introduction 25 Stress is ubiquitous in everyday life. From recurrent, brief, events (a work meeting, moving 26 to a new house) to major life events (armed combat, pandemic, financial crisis), humans are 27 continuously exposed to challenges in their daily environment. The immediate central and 28 peripheral physiological cascade triggered by such events, collectively termed the fight-or- 29 flight (or acute stress) response (Cannon, 1915), serves an allostatic role that enables 30 organisms to adequately respond to environmental demands (de Kloet, Joëls, & Holsboer, 31 2005). Although beneficial for survival, this allostatic process comes at a cost: stress-induced 32 redistributions of neural resources - e.g., towards vigilance or threat detection - may hamper 33 the deployment of strategies that support adaptive and optimal decision-making (Hermans, 34 Henckens, Joëls, & Fernández, 2014). 35 Optimal decisions essentially depend on the ability to rapidly learn from the positive 36 and negative outcomes of previous actions, also known as reinforcement learning (Niv, 37 2009). Considerable evidence now suggests that acute stress impairs aspects of reinforcement 38 learning (Carvalheiro, Conceição, Mesquita, & Seara-Cardoso, 2020; de Berker et al., 2016; 39 Raio, Hartley, Orederu, Li, & Phelps, 2017). Acute stress, among others, modulates the 40 impact of positive outcomes on future decisions - both positively and negatively - (Berghorst, 41 Bogdan, Frank, & Pizzagalli, 2013; Carvalheiro et al., 2020; Lighthall, Gorlick, Schoeke, 42 Frank, & Mather, 2013; Petzold, Plessow, Goschke, & Kirschbaum, 2010), likely driven by 43 changes in reward sensitivity and the signalling of reward prediction errors (RPEs) 44 (Berghorst et al., 2013; Carvalheiro et al., 2020; Huys, Pizzagalli, Bogdan, & Dayan, 2013); 45 putatively dopaminergic teaching signals that represent the mismatch between actual and 46 expected outcomes, which are used to flexibly adjust behaviour (Niv, 2009; Rescorla, 1972). 47 Alterations in the influence of RPEs on future decisions play a key role in the development of 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 48 motivational impairments, which are frequently observed in behavioural disorders associated 49 with repeated and/or prolonged stress exposure (Huys et al., 2013). 50 Intuitive as it is, the notion that the impact of acute stress on (potentially maladaptive) 51 decisions primarily involves changes in how reward value influences action may be 52 oversimplified. Decisions are not only motivated by appetitive properties; they equally 53 depend on the – cognitive (e.g., mental effort) or physical (e.g., energy) – cost associated 54 with actions (Hauser, Eldar, & Dolan, 2017; Pessiglione, Vinckier, Bouret, Daunizeau, & Le 55 Bouc, 2017; Schmidt, Lebreton, Cléry-Melin, Daunizeau, & Pessiglione, 2012). Expectations 56 about action costs are also updated according to a prediction error rule (Skvortsova, Degos, 57 Welter, Vidailhet, & Pessiglione, 2017; Skvortsova, Palminteri, & Pessiglione, 2014) 58 (henceforth “effort” prediction errors; EPEs), which due to the aversive and resource- 59 consuming nature of effort, optimal learners should utilize to minimize effort expenditure. 60 When decisions involve a potential cost and benefit, the former is subtracted from the latter 61 to compute a “net” or subjective decision value (i.e., effort-discounted reward value) (Klein- 62 Flügge, Kennerley, Friston, & Bestmann, 2016; Skvortsova et al., 2017; Skvortsova et al., 63 2014). Notably, stress exposure impairs cost-benefit decisions in rodents when learning is not 64 explicitly required (Friedman et al., 2017; Shafiei, Gray, Viau, & Floresco, 2012). Moreover, 65 in a reinforcement learning context, acute stress blocks the flexible updating of aversive 66 value (Raio et al., 2017), an inherent property of costly actions. These results suggest that 67 decisions during acute stress may involve a complex shift in reinforcement learning strategies 68 that serve to balance the cost versus benefits of decisions; a hypothesis that hitherto has 69 remained unexplored. 70 Although computationally similar in nature, distinct neural correlates of RPEs (e.g., 71 striatal subdivisions, ventromedial prefrontal cortex [vmPFC]) and EPEs (e.g., parietal 72 cortex, insula, dorsomedial PFC) can be observed in cost-benefit reinforcement learning 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 73 paradigms (Hauser et al., 2017; Skvortsova et al., 2014). The ascending dopaminergic (e.g., 74 RPEs, action cost, reward value) (Schultz, Dayan, & Montague, 1997; Skvortsova et al., 75 2017; Yohn et al., 2016), noradrenergic (e.g., mobilizing energy) (Pessiglione et al., 2017; 76 Varazzani, San-Galli, Gilardeau, & Bouret, 2015) and serotonergic (e.g., aversive value, 77 overcoming action costs) (H. E. den Ouden et al., 2015; Meyniel et al., 2016) 78 neuromodulatory systems, moreover, encode partly dissociable aspects of goal-directed 79 actions that involve learning about costs and benefits, which together support optimal 80 decision-making. These observations are noteworthy because the initial fight-or-flight 81 response triggers a large-scale reorganization of brain networks that is driven by alterations in 82 the firing mode of midbrain dopaminergic ventral tegmental area and noradrenergic locus 83 coeruleus neurons (Arnsten, 2015; Hermans et al., 2014); neurons that signal prediction 84 errors (Steinberg et al., 2013) and that are also responsive to reward value, action cost and 85 energy expenditure (Del Arco, Park, & Moghaddam, 2020; Varazzani et al., 2015). Thus, 86 catecholaminergic mechanisms that are recruited by the fight-or-flight response may 87 differentially impact cost and benefit reinforcement learning, resulting in a potential scenario 88 in which costs and benefits exert asymmetric influences on decisions. 89 As mentioned above, the central (i.e., neural) effects of acute stress trigger a shift in 90 cognitive strategies, including reinforcement learning. The peripheral counterpart of the acute 91 stress response, however, mobilizes the energy (i.e., adrenaline-mediated glucose release (de 92 Kloet et al., 2005; Russell & Lightman, 2019)) that is required to exert effortful actions 93 aimed at preserving homeostasis (Cannon, 1915). Therefore, decision-making and learning 94 policies regarding physical costs may be especially susceptible to stress: both via 95 computational (neural) mechanisms that support learning about and representation of action 96 cost, as well as peripheral mechanisms that co-determine the amount of available energy that 97 can be directed towards effortful actions. Indeed, preliminary evidence suggests that acute 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 98 stress alters the willingness to exert physical effort for rewards (Bryce & Floresco, 2016) and 99 reward-associated cues in a Pavlovian-instrumental transfer context (Pool, Brosch, 100 Delplanque, & Sander, 2015). 101 How acute stress impacts reinforcement learning involving costs and benefits has not 102 been investigated to date in humans. Based on the above considerations, we expect that 103 computationally frugal learning strategies, in concert with increased energy availability, 104 during acute stress should asymmetrically impact cost versus benefit learning. Using an acute 105 stress-induction paradigm, a cost-benefit learning paradigm and computational model of cost- 106 benefit reinforcement learning (Skvortsova et al., 2017; Skvortsova et al., 2014), we 107 demonstrate that acute stress asymmetrically prioritizes reward (maximization) learning over 108 physical effort (minimization) learning. Better benefit versus cost learning results from a 109 stress-induced change in the influence of RPEs versus EPEs on future decisions, and is 110 associated with altered pupil encoding of RPEs, EPEs, and subjective decision value. These 111 results reveal how neural and peripheral mechanisms that support the fight-or-flight response 112 may facilitate a shift in reinforcement learning strategies that confers strategic benefits during 113 acutely stressful situations (e.g., ignoring high action costs to achieve a desirable outcome), 114 yet might also give rise to maladaptive behaviour (e.g., stress-induced relapse in substance 115 users). 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 116 Results 117 Experiment design 118 Healthy human participants were randomly assigned to the acute stress (19 males/21 females; 119 age M=23.48, SD=3.94) or no-stress control condition (18 males/22 females; age M=23.80, 120 SD=4.23) of the Maastricht Acute Stress Task (MAST) (Smeets et al., 2012), a validated 121 psychological and physical stress-induction paradigm (see Materials and Methods). 122 Immediately post-MAST and within the confines of the acute stress response (Hermans et al., 123 2014), all participants completed a ~40 minute probabilistic cost-benefit reinforcement 124 learning paradigm, adapted from Skvortsova et al. (Skvortsova et al., 2017; Skvortsova et al., 125 2014), in which they learned to select stimuli with high reward value (20 Eurocents) and 126 avoid stimuli with high action cost (exerting grip force above a pre-calibrated individual 127 threshold of 50% maximum voluntary contraction for 3000ms), followed by a surprise test 128 phase. A detailed overview of the paradigm is provided in Figure 1 and the Materials and 129 Methods. Pupil size was continuously recorded while participants performed the task (see 130 Materials and Methods). 6 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 131 Figure 1 132 133 Reward maximisation/action cost minimization reinforcement learning task. 134 Visual depiction of the learning phase. Participants were presented with four distinct stimulus 135 pairs, and all stimuli were associated with a predetermined chance of a €0.20 monetary 136 reward (versus no reward) and a chance of having to exert physical effort (grip force) using a 137 dynamometer (versus no grip force required). Stimulus-outcome probabilities were yoked in 138 such a way that, for a given pair, two stimuli only differed in the probability of earning a 139 reward (“reward learning”, RL, left) or the probability of having to exert effort (“effort 140 learning”, EL, right). That is, for RL (left)/EL (right) pairs, reward/effort outcomes were 141 choice-dependent, respectively (see “Reward feedback” for RL and “Effort feedback” for EL 142 for outcome contingencies). For RL pairs, effort outcomes were independent of choice and 143 fixed (see “Effort feedback” for RL), while for EL pairs, reward outcomes were independent 7 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 144 of choice and fixed (see “Reward feedback” for EL). Percentages in blue and red refer to 145 outcomes for the Easy RL/EL and Hard RL/EL pair, respectively. 146 147 Acute stress manipulation 148 We first ascertained whether the acute stress manipulation was successful. Subjective stress, 149 physiological and neuroendocrine measurements are displayed in Figure 2. Acute stress and 150 no-stress control groups did not differ on physiological, subjective stress, or neuroendocrine 151 measurements pre-MAST (all p-values>0.05). We observed significant Condition-by-Time 152 interactions for subjective stress ratings [PANAS negative: F(1,78)=52.66, p<0.001, 153 n2G=0.10; PANAS positive: F(1,78)=9.82 p=0.002, n2G=0.02] and physiological measures 154 [systolic blood pressure (SBP): F(1,78)=15.50, p<0.001, n2G= 0.04; heart rate: F(1,78)=6.83, 155 p=0.011, n2G= 0.02]. Simple main effect analyses revealed that only the acute stress group 156 exhibited pre-to-post increases in negative affect [control pre-post: t(39)=4.21, p<0.001; 157 stress pre-post: t(39)=-6.17, p<0.001; control-stress post-MAST: t(55.1)=-5.78, p<0.001], 158 and greater pre-to-post decreases in positive affect [control pre-post: t(39)=4.09, p<0.001; 159 stress pre-post: t(39)=6.45, p<0.001; control-stress post-MAST: t(72.8)=2.53, p=0.014] 160 (Figure 2). Similarly, only the acute stress group exhibited stress-induced increases in SBP 161 [control pre-post: t(39)=1.60, p<0.117; stress pre-post: t(39)=-3.66, p<0.001; control-stress 162 post-MAST: t(69.1)=-3.27, p=0.002] and heart rate [control pre-post: t(39)=1.21, p=0.234; 163 stress pre-post: t(39)=-2.78, p=0.008; control-stress post-MAST: t(76.9)=-3.14, p=0.002] 164 (Figure 2a-d). 165 An expected Condition-by-Time interaction was found for salivary cortisol (sCORT) 166 responses [F(5,390)=18.05, p<0.001, n2G= 0.04], with the acute stress group displaying 167 greater sCORT levels 10 min post-MAST and onwards (all p-values<0.01). We additionally 168 observed a main effect of Condition on sCORT area-under-the-curve with respect to increase: 8 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 169 (AUCi) (Pruessner, Kirschbaum, Meinlschmid, & Hellhammer, 2003) (t(56.32)=-5.28, 170 p<0.001) and salivary alpha-amylase (sAA) AUCi (t(67.45)=-2.50, p=0.015; after excluding 171 one extreme outlier from the control group), suggesting greater sCORT and sAA levels in 172 response to acute stress (Figure 2e-g). These results confirm that the MAST robustly induced 173 stress on all levels of inquiry. 174 175 Figure 2 176 177 Neuroendocrine, physiological and subjective stress ratings. 178 PANAS negative (a) and positive (b) subscale sum scores, systolic blood pressure (mmHg: 179 millimetres of mercury; c) and heart rate (bpm: beats per minute; d) are displayed for no- 180 stress control (blue) and acute stress (red) groups separately for pre (light blue/red) and post 181 (dark blue/red) MAST time points. SCORT responses for both conditions across the 6 182 timepoints are displayed in panel e (“t+00” represents the first post-MAST measurement, and 183 the start of the reward maximization/action cost reinforcement learning paradigm; “t-10” 184 represent a baseline sample). Panel f and g show AUCi for sCORT (nmol/l: nanomoles per 185 litre) and sAA (U/mL: Units per millilitre) responses for both MAST conditions. Significant 9 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 186 differences are denoted by asterisks (*: p < 0.05, **: p < 0.01, ***: p < 0.001). In the upper 187 panel, the top line denotes a significant Condition-by-Time interaction; lower lines represent 188 simple main effects of Condition or Time. 189 190 Participants use reinforcement learning to optimize decisions 191 Next, we investigated whether participants in both conditions exhibited evidence of 192 reinforcement learning to optimize actions, which in this paradigm should be reflected by an 193 increased tendency to select stimuli with high reward value and avoid stimuli with high 194 action cost as a function of increasing number of stimulus pair presentations (i.e., “time”). 195 This intuition was confirmed by a main effect of Time on two distinct trial types: trials on 196 which participants could learn to accumulate frequent rewards while the probability of effort 197 (action cost) was kept constant (RL; selecting the stimulus more frequently associated with 198 €0.20) [control: F(2,78)=10.16, p<0.001, n2G= 0.06; stress: F(2,78)=20.44, p<0.001, n2G= 199 0.17] and trials on which participants could learn to frequently avoid effort while the 200 probability of reward was kept constant (EL; selecting the stimulus more frequently 201 associated with avoidance of physical energy expenditure) [control: F(2,78)=12.35, p<0.001, 202 n2G= 0.07; stress: F(2,78)=9.76, p<0.001, n2G= 0.05]. We additionally observed greater than 203 chance-level performance (≥0.5) on both trial types during the final part of the task 204 (presentation 21-30; all p-values<0.001 Figure Supplement 1). 205 206 Asymmetric cost-benefit reinforcement learning during acute stress 207 After having observed evidence for reward (maximization) learning and action cost 208 (minimization) learning, we tested our key assumption; that acute stress would induce a 209 reprioritization in learning to maximize reward value versus learning to minimize action cost. 210 Crucially, we observed a significant Condition-by-Trial Type interaction [F(1,78)=6.53, 10 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 211 p=0.013, n2G= 0.039] (Figure 3a) with pairwise comparisons indicating that the acute stress 212 group performed significantly better on RL than EL trials [t(39)=5.40, p<0.001], while the 213 no-stress control group performed similarly on both trial types [t(39)=1.01, p=0.320]. A main 214 effect of Condition on RL-EL accuracy difference scores [t(74.02)=-2.55, p=0.013] (Figure 215 3b) and one-sample t-tests revealed that RL-EL accuracy difference scores were significantly 216 greater than zero in the acute stress group but not in the no-stress controls. Simple main 217 effects of Condition on RL [t(65.9)=-1.75, p=0.085] and EL [t(77.5)=1.80, p=0.076] 218 performance showed numerical trends for group differences that failed to reach significance. 219 When we included participants that still performed at chance level at the end of the 220 learning phase (see Participants) in the Condition-by-Trial Type interaction analysis, the 221 interaction remained significant [F(1,91)=7.30, p=0.035, n2G= 0.04], with the acute stress 222 group displaying better RL vs. EL performance (t(46)=5.83, p<0.001), while no-stress 223 controls performed similarly on both trial types (t(45)=1.24, p=0.22). Participants in the acute 224 stress group outperformed participants in the no-stress control group on RL (t(91)=2.04, 225 p=0.04), but no simple main effect of Condition was observed for EL (t(91)=-1.67, p=0.1). 226 These participants were excluded for all other analyses reported below. 227 The use of different reinforcement probabilities for each RL and EL pair (Figure 1) 228 allowed us to discern whether the observed pattern of results reflected a specific change in 229 cost (EL) versus benefit (RL) reinforcement learning, or a more general impairment in 230 reinforcement learning for more difficult stimulus-response associations. We found no 231 evidence for the latter scenario in Condition-by-Trial Type-by-Difficulty [F(1,78)=1.05, 232 p=0.310] or Condition-by-Difficulty [F(1,78)=0.36, p=0.549; Figure Supplement 2] 233 interaction analyses. 234 235 When we investigated the use of win-stay (resampling a stimulus following a positive outcome) and lose-shift (switching to the other stimulus following a negative outcome) 11 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 236 strategies (Hanneke E. M. den Ouden et al., 2013), we observed no significant Condition-by- 237 Strategy interaction [F(1,78)=2.99, p=0.087, n2G= 0.03]. However, post hoc comparisons 238 revealed that participants in the acute stress condition [t(39)=-3.73, p<0.001] but not those in 239 the no-stress control condition [t(39)=-1.30, p=0.200], exhibited different win-stay compared 240 to lose-shift (difference) rates. Separate Condition (main effect) analyses indicated that acute 241 stress participants compared to no-stress controls were more likely to win-stay for rewards 242 (RL trials) than for avoidance of action cost (EL trials) [t(78)=-2.28, p=0.025], but not for 243 lose-shifting for reward omissions (RL trials) compared to exerting effort (EL trials) 244 [t(77.7)=-0.23, p=0.820]. Differences in win-stay rates for RL compared to EL trials (one- 245 sample t-test) were greater than zero for the acute stress [t(39)=4.91, p<0.001] but not the no- 246 stress control group [t(39)=1.68, p=0.101] (Figure 3c). 247 Taken together, our model-free results indicate that acute stress leads to a 248 reinforcement learning strategy that favours learning to maximise reward value over 249 minimisation of action cost, which based on analyses of win-stay/lose-shift rates, could be 250 attributed to increased sensitivity to positive reinforcement (i.e., reward delivery) compared 251 to negative reinforcement (i.e., avoidance of physical effort). 12 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 252 Figure 3 253 254 Acute stress leads to improved benefit versus cost learning. 255 Panel a: Average accuracy (choices of the optimal stimulus) for RL and EL trials, for each 256 condition separately. Panel b: RL-EL average accuracy difference scores. Panel c: 257 Win-stay (WSRL-WSEL) and lose-shift (LSRL-LSEL) difference scores for each condition 258 separately. Means ± SD, individual data points, distribution and frequency of the data are 259 displayed. In panel a, the top line indicates a significant Condition-by-Trial type interaction. 260 Significant differences are denoted by asterisks (*: p < 0.05, **: p < 0.01, ***: p < 0.001). 261 Source files of task performance data used for the analyses are available in the Figure 3 – 262 Source Data 1. 263 264 Asymmetric cost-benefit reinforcement learning biases actions in acute stress subjects 265 During a surprise 64-trial test phase (D. Hernaus, Gold, Waltz, & Frank, 2018), we asked 266 participants to discriminate original and novel combinations of stimuli on the basis of reward 267 value or action cost without receiving feedback (n=16 trials for original combinations; n=48 268 for novel combinations; see Materials and Methods). The surprise test phase allowed us to 13 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 269 assess learned choice tendencies without having to arbitrarily choose a given number of final 270 learning phase trials, during which participants may still learn. This approach also allowed us 271 to assess the degree to which learned tendencies would carry over to novel contexts. 272 First, both groups chose the optimal (most rewarding/effort avoiding) stimulus on 273 surprise test phase trials involving the original four pairs [one-sample t-test against chance; 274 controlRL: t(39)=8.73, p<0.001; controlEL: t(39)=3.72, p=0.002; stressRL: t(39)=13.54, 275 p<0.001; stressEL: t(39)=4.47, p<0.001], confirming that both groups had developed a 276 preference for the optimal stimulus. 277 Although we observed no Condition-by-Trial type (reward value, action cost 278 discrimination) interaction or main effects of Condition for novel stimulus combinations 279 [F(1,78)=1.10, p=0.298, n2G= 0.01; stress vs. controls reward value discrimination: t(75.9)= 280 0.15, p=0.878; stress vs. controls action cost discrimination: t(78)= 1.77, p=0.080], pairwise 281 comparisons revealed that the acute stress group performed better on reward discrimination 282 compared to action cost discrimination trials [t(39)=-2.23, p=0.032], while no-stress controls 283 performed similarly on both trial types [t(39)=-0.87, p=0.387]. These results provide some 284 evidence that a reward maximisation-over-action cost minimisation reinforcement learning 285 policy might bias future actions in novel contexts (Figure Supplement 3). 286 287 Computational cost-benefit reinforcement learning model: Model Fitting, Selection, 288 Demonstrations, and Simulations 289 To uncover latent mechanisms by which acute stress affects cost-benefit reinforcement 290 learning, we turned to computational cognitive modelling. Trial-by-trial choices of 291 participants were fit to all candidate models described in the Materials and Methods (see 292 Model Space). To calculate model fit, log likelihood was updated trial-wise by the log of the 293 probability of the observed choice, calculated via a softmax rule (see Materials and Methods, 14 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 294 equation 6), and best-fitting parameters were identified using fmincon in MATLAB v.2019B 295 (Mathworks, Natick, MA, USA). 296 Bayesian Model Selection (BMS; spm_BMS function in SPM12, 297 http://www.fil.ion.ucl.ac.uk/spm/software/spm12/) using the Akaike Information Criterion 298 (AIC) as a fit statistic that penalizes for the number of model parameters (Myung, Tang, & 299 Pitt, 2009), suggested that the 2LR_γ model was the most likely model, as indicated by the 300 protected exceedance probability (pxp, φ = 0.99) (Rigoux, Stephan, Friston, & Daunizeau, 301 2014) and expectation of the posterior [p(r|y), 0.70] (Figure 4a for p(r|y) of all candidate 302 models). We note that 2LR_γ remained the most likely model when we considered additional 303 models with greater redundancy and/or lesser biological plausibility (e.g., models with all 304 combinations of reward value/action cost discounting and weight parameters). 305 The 2LR_γ model contains separate learning rates that weight the importance of RPEs 306 and EPEs (αR, αE), an action cost discounting parameter (γ), and an inverse temperature 307 parameter (β), which in previous work could account for performance on a conceptually 308 similar cost-benefit learning task (Skvortsova et al., 2014). To demonstrate the effect of 309 changes in parameters values on choice preferences within the 2LR_γ architecture, we first 310 simulated choices from 50 artificial agents (averaged across 10 repetitions) performing the 311 reward maximization/action cost minimization reinforcement learning task using a range of 312 parameter values. As expected, greater values of αR and αE primarily impacted the speed of 313 RL and EL choice preferences, while low values of γ lead to asymmetric choice preferences 314 through discounting of action cost, and lower values of β lead to non-selective increases in 315 random sampling (Figure 4b). 316 In post hoc simulations, i.e., generating participant choices using the obtained 317 parameters, we additionally observed moderate-to-high correlations between simulated and 318 empirical RL/EL for the acute stress and no-stress control group [ρRL_control = 0.55, p < 0.01; 15 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 319 ρRL_stress = 0.84, p < 0.01; ρEL_control = 0.56, p < 0.01; ρEL_stress = 0.77, p < 0.01; see Figure 320 Supplement 4], although the canonical performance difference in RL versus EL accuracy was 321 not selective to the acute stress group [tcontrol(39)=-6.72, p<0.001; tstress(39)=-6.01, p<0.001]. 322 However, after we fixed β and γ to group-level averages, to better demonstrate the effect of 323 group differences in the learning rate parameters, we recovered a small but significant 324 simulated difference in RL versus EL performance for the acute stress group [t(39)=2.27, 325 p=0.029], which was not predicted in the no-stress control group [t(39)=0.91, p=0.367] 326 (Condition-by-Trial Type interaction: [F(1,78)= 0.77, p=0.38, n2G= 0.006]) (Figure 4c for 327 empirical versus simulated data, averaged across 100 repetitions per subject). 328 Importantly, even if a given model is the most likely one based on model fitting and 329 post hoc simulation results from the entire sample, there is still the possibility that different 330 models can better explain task performance in the no-stress control and acute stress 331 condition. When repeating BMS for each condition separately, 2LR_γ was the most likely 332 model in the no-stress control group [φ=0.99, p(r|y)=0.83], while for acute stress subjects 333 2LR_γ was not convincingly the most likely model [φ=0.47 p(r|y)=0.46]. Here, the 2LR 334 model (containing αR, αE, and β parameters) was equally likely to be the optimal model 335 [φ=0.53 p(r|y)=0.47]. Post-hoc simulations from the 2LR model also correlated with actual 336 data, both for no-stress control [ρRL = 0.65, p < 0.001; ρEL = 0.60, p < 0.001] and acute stress 337 participants [ρRL = 0.81, p < 0.001; ρEL = 0.75, p < 0.001]. 338 Similar to the 2LR_ γ model (results discussed in next section), the 2LR model 339 seemingly also explained stress-induced changes in cost-benefit reinforcement learning via 340 changes in learning rates; in the 2LR model, the acute stress group exhibited greater values of 341 αR versus αE (t(39)=2.65, p=0.01), while no-stress control subjects did not (t(39)=0.69, 342 p=0.50) (Condition-by-Learning Rate interaction: [F(1,78)=2.88, p=0.094, n2G= 0.01]. The 343 difference in learning rates between 2LR_γ (where αR and αE are similar for the acute stress 16 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 344 group, see next section) and 2LR (where αR > αE for the acute stress group) can be explained 345 by the absence of discounting parameter γ: 2LR is a special case of 2LR_γ, where γ=1, and 346 thus asymmetric effects of acute stress on reward value maximization and action cost 347 minimization can only be explained by dissimilarity in learning rates. 348 Although the effects of acute stress on reward value and action cost learning rates are 349 opposite in 2LR_ γ versus 2LR architectures, these results bolster our confidence in the 350 overall model space, as well as the interpretation that acute stress primarily impacts reward 351 value and action cost learning rates, and not discounting. The observations that I) 2LR_ γ fit 352 better in the entire group of participants, II) 2LR is fully contained within the 2LR_ γ model, 353 and III) 2LR_ γ displayed good recoverability (see below) motivated our choice to focus on 354 the 2LR_γ model. 355 In model recoverability analyses i.e., re-fitting the simulated data from the model to 356 all candidate models (Wilson & Collins, 2019), BMS confirmed that the simulated 2LR_γ 357 data (that is, simulations without fixed parameters) were most likely to be generated from 358 2LR_γ [φ=0.99, p(r|y)=0.71]. 359 To assess the stability of 2LR_γ parameters, we repeated model fitting using a 360 Bayesian hierarchical model fitting approach consisting of two steps, as described previously 361 (Daw, 2011; Frey, Frank, & McCabe, 2019). In the first step we fit the 2LR_γ model to trial- 362 wise choices to obtain subject-specific parameters; in a second step we again fit the model to 363 trial-wise choices, but this time we used the group-level average and covariance matrix of 364 every parameter as priors, thereby shrinking the parameter search space. Motivated by recent 365 work showing that group-specific priors, compared to a single prior for the entire sample, can 366 better account for between-group differences in task performance, as well as improve 367 parameter robustness and recoverability (Valton, Wise, & Robinson, 2020), we used separate 368 mean and covariance matrices for the acute stress and no-stress control groups. 17 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 369 Highly similar parameter estimates were obtained after hierarchical fitting (for 370 parameter estimates after Bayesian hierarchical model fitting see Figure Supplement 5). 371 Similar to post hoc simulations using parameters from the non-hierarchically fit 2LR_γ 372 model, we observed moderate-to-high correlations between empirical and simulated data 373 using parameters obtained from the hierarchically fit model [ρRL_control = 0.65, p < 0.01; 374 ρRL_stress = 0.84, p < 0.01; ρEL_control = 0.37, p =0.02; ρEL_stress = 0.78, p < 0.01; see Figure 375 Supplement 6]. All in all, these results confirm parameter stability within the 2LR_γ 376 architecture. 377 In light of model fitting results, post-hoc simulations, model and parameter 378 recoverability analyses, we used parameters and trial-by-trial predictions of the non- 379 hierarchically fit 2LR_ γ model in all analyses reported below. 18 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 380 Figure 4 381 382 Model selection, demonstrations, and post hoc simulations of the winning model. 383 Panel a: Expectation of the posterior for all candidate models. Panel b: Model 384 demonstrations. To demonstrate how different parameter values within the 2LR_γ 385 architecture impact choice preferences for the optimal stimulus (“accuracy”), αR, αE, and γ 386 were set to 0.01/0.33/0.66/0.99, while β, a non-linear parameter, was set to 0.1/5/10/20. 387 Parameter effects were always demonstrated for a single parameter (columns), while all other 388 parameter values were kept constant (αR and αE=0.25, γ=1, β=25). Greater values of αR and 389 αΕ selectively increase the speed with which the agent develops a preference for the optimal 390 RL and EL stimulus, respectively. Lower values of γ produce an asymmetric decision- 391 making policy that emphasises reward value over action cost, leading to better performance 392 on RL versus EL trials, while greater values of γ correct this asymmetric choice bias. Finally, 393 greater β values lead to more deterministic sampling of optimal stimuli. Panel c: Post-hoc 19 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 394 simulations after fixing β and γ to group-level averages. Coloured lines represent mean ± SD. 395 Dashed lines denote chance level (0.5). *: p < 0.05, **:p < 0.01, ***: p < 0.001. 396 397 Acute stress selectively reduces the difference between reward and action cost learning 398 rates 399 Comparing 2LR_γ parameters between conditions, we observed a significant Condition-by- 400 Learning Rate (αR, αE) interaction [F(1,78)= 6.42, p=0.01, n2G= 0.03; 95% highest density 401 interval (HDI) for Bayesian mixed ANOVA = -0.405 to -0.023, mean= -0.219]. with greater 402 EPE relative to RPE learning rates in no-stress control participants [t(39)=-4.75, p<0.001], 403 while learning rates in the acute stress group did not significantly differ [t(39)=-1.61, 404 p=0.116]. No between-group differences in αR and αE or in the other parameters (γ, β) were 405 observed (all p-values>0.05) (Figure 5). 406 Paradoxically, symmetric reward value and action cost learning rates in the presence 407 of lower values of γ will lead to more efficient RL compared to EL. This is because lower 408 values of γ bias decisions towards reward value (via greater discounting of action cost) and 409 similar absolute values of αR/αE will not counteract this bias. Asymmetric learning rates 410 (αE>αR) in combination with lower values of γ, however, will lead to more symmetric 411 performance on RL and EL trials via more efficient updating of action cost versus reward 412 expectations. This interpretation is supported by our demonstration of model parameters 413 (Figure 4b) and post hoc simulations (Figure 4c), as well as the observation that lower values 414 of γ (i.e., greater action cost discounting) were associated with greater learning rate 415 asymmetry (αE>αR; more efficient EL) in no-stress controls (ρ=-0.40, p=0.040), who 416 displayed similar RL and EL performance. These results demonstrate that, in a context where 417 all decisions involve a potential cost and benefit, acute stress selectively reduces the 418 difference between EPE and RPE learning rates, while leaving action cost discounting and 20 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 419 choice stochasticity unaffected. The direction of the change in learning rates (i.e., greater 420 similarity) implies a stress-induced failure to modulate learning rates in the service of 421 overcoming an asymmetric choice bias that emphasises reward value. 422 In analyses using posterior parameters obtained from the hierarchically fit model, we 423 recovered the key Condition-by-Learning Rate interaction (95% HDI for Bayesian mixed 424 ANOVA = -0.406 to -0.128, mean=-0.269) [and acute stress and no-stress control subjects 425 differed from each other on αE (95% HDI = 0.0841 to 0.281, mean=0.183) but not αR (95% 426 HDI = -0.186 to 0.0102, mean=-0.0872)] (Figure Supplement 7). Similar to the non- 427 hierarchically fit parameters, acute stress and control subjects did not differ on posterior 428 estimates of γ (95% HDI = -0.0914 to 0.139, mean=0.0182) and β (95% HDI = -0.0331 to 429 0.213, mean=0.0895) . 430 21 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 431 Figure 5 432 433 Acute stress reduces the difference between reward and effort prediction error learning 434 rates. 435 Free parameters (αR, αE, γ, β) of the winning 2LR_ γ model for both groups. Black lines 436 denote means ± SD, dots represent individual data points, and the violin-like shape denotes 437 distribution and frequency of the data. *: p < 0.05, **: p < 0.01, ***: p < 0.001. 438 439 Pupil size fluctuations track asymmetric cost-benefit reinforcement learning during 440 acute stress 441 We employed pupillometry to better understand whether task-relevant computational 442 processes may be encoded by fluctuations in pupil dilation, which are thought to be 443 controlled by ascending midbrain modulatory systems that play a role in value-based 444 decision-making and the acute stress response (Arnsten, 2015; Hermans et al., 2011; Joshi, 445 Li, Kalwani, & Gold, 2016). 22 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 446 In model-free analyses – that is, comparing bins of pupillometry data between 447 conditions - we observed no main effect of Condition on pupil size fluctuations during 448 choice, effort outcome, and reward outcome epochs, suggesting that acute stress was not 449 associated with more general changes in pupil size (all bin-level p>0.05; Figure 6, a. Model- 450 free; Figure Supplement 8 for all effort trials). 451 Next, we conducted model-based pupillometry analyses (Lawson, Bisby, Nord, 452 Burgess, & Rees, 2020) to understand how trial-wise estimates of computational processes of 453 interest were encoded by fluctuations in pupil size. These analyses revealed effects of 454 Condition on pupil encoding of subjective decision value, EPEs and RPEs (Figure 6, b. 455 Model-based) in a manner commensurate with task performance results. First, immediately 456 prior to the stimulus choice, acute stress reduced pupil encoding of subjective decision value, 457 as evidenced by the absence of an association between pupil size and subjective decision 458 value (control>stress; stress n.s., control>0). Second, briefly after the presentation of effort 459 avoidance outcomes, both groups exhibited different pupil size-EPE associations, with no- 460 stress controls showing a non-significant numerically positive association between pupil size 461 and action cost prediction errors (control>stress, both groups n.s. different from 0). In model- 462 based analyses using all effort outcome trials, we were not able to uncover group differences 463 in pupil encoding of EPEs, which were likely eclipsed by prominent grip force-related effects 464 on pupil size (Figure Supplement 8). Third, during the reward outcome phase, acute stress 465 participants exhibited greater positive associations between pupil size and RPEs compared to 466 no-stress controls (stress>control, stress>0, control n.s.). The average pupil size-RPE slope 467 for bins in which no-stress control and acute stress participants differed (Figure 6b) correlated 468 significantly with stress-induced changes in SBP [ρstress(38)= -0.41, p(permutation)=0.019] 469 and PANAS negative affect changes [ρstress= -0.46, p(permutation)=0.005] in the stress group. 23 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 470 Crucially, group differences in pupil encoding of subjective decision value, EPEs, and 471 RPEs imply that the ascending neuromodulatory systems may have facilitated a stress- 472 induced shift in asymmetric cost versus benefit learning. 473 474 Figure 6 475 476 Model-based analysis reveals altered pupil encoding of prediction errors and decision 477 value during acute stress . 478 a. Model-free analyses of pupil size during choice, effort outcome, and reward outcome 479 phase revealed no main effect of Condition (no-stress control, acute stress). b. Model-based 480 analyses revealed a stress-induced shift in pupil encoding of subjective decision value (left), 481 action cost prediction errors (middle) and reward prediction errors (right). Black line 482 indicates significant main effect of Condition; blue and red line indicate significance against 483 zero for no-stress control and acute stress groups, respectively (cluster and bin level 484 apermute<0.05, 2000 permutations). Group differences in pupil encoding of action cost and 485 reward prediction errors were observed at similar times (note the x-axis differences for effort 24 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 486 outcome and reward outcome epochs). Source files of pupillometry data used for the analyses 487 are available in Figure 6 – Source Data 2. 488 489 Discussion 490 Stress-induced alterations in adaptive decision-making are commonly studied using 491 paradigms that isolate positive and negative reinforcement, such as the receipt of a reward or 492 avoidance of a loss. However, it remains poorly understood how acute stress affects the 493 complex process that entails learning about costs and benefits, a critical and pervasive feature 494 of everyday decisions. Participants completed a paradigm in which all actions (stimulus 495 choices) contained a potential cost (exerting physical effort) and a financial benefit (€0.20). 496 Crucially, acute stress induced a shift in reinforcement learning strategies that improved 497 maximization of monetary rewards relative to minimisation of energy expenditure. When 498 presented with novel stimulus arrangements and in the absence of feedback, individuals in 499 the acute stress condition, moreover, exhibited better discrimination of stimulus reward value 500 compared to action cost. 501 Relative improvements in reward versus action cost learning align well with previous 502 reports of enhanced reward learning during acute stress (Byrne, Cornwall, & Worthy, 2019; 503 Lighthall et al., 2013; Petzold et al., 2010), although such effects may depend on stressor 504 timing (Joëls, Pu, Wiegert, Oitzl, & Krugers, 2006), stressor type (Carvalheiro et al., 2020), 505 and/or sample characteristics (Evans & Hampson, 2015; Morris & Rottenberg, 2015). While 506 reports on action cost learning during acute stress are scarce, acute exposure to stress in 507 rodents impairs cost-benefit decisions via a selective change in sensitivity to physical effort, a 508 process mediated by corticotropin-releasing factor and dopamine (Bryce & Floresco, 2016). 509 Our analyses of win-stay/lose-shift rates indicate that asymmetric cost-benefit learning can be 25 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 510 driven by a relative increase in the sensitivity to monetary gains compared to the avoidance 511 of costly deterrents. 512 How might maximization of reward value take precedence over minimisation of 513 action cost? Acute stress leads to a redistribution of finite cognitive resources (Hermans et 514 al., 2014): this process limits the availability of computationally intensive strategies, 515 including working memory (Otto, Raio, Chiang, Phelps, & Daw, 2013; Qin, Hermans, van 516 Marle, Luo, & Fernández, 2009) and goal-directed instrumental actions (Lars Schwabe & 517 Wolf, 2011). Assuming that acute stress does not merely increase random responding - which 518 we verified via the choice stochasticity model parameter - a computationally cheap heuristic 519 in our task should present itself as better learning for one modality over the other. Increased 520 energy availability (Hermans et al., 2014), insensitivity to aversive stimuli (Timmers et al., 521 2018), and impaired aversive value updating (Raio et al., 2017) under stress may have 522 reduced the ability – or urgency – to dedicate cognitive resources to strategies that minimize 523 action cost. Importantly, effort expenditure increases the perceived value of rewards 524 (Hernandez Lallement et al., 2014; Inzlicht, Shenhav, & Olivola, 2018). Thus, frequent 525 expenditure of physical effort, due to suboptimal action cost learning, may increase the 526 perceived value of rewards, and thus tilt learning towards the maximization of reward value. 527 Using a computational model of reinforcement learning (Skvortsova et al., 2017; 528 Skvortsova et al., 2014), we confirmed that biased cost-benefit learning can arise when 529 inappropriate (i.e. more similar) importance is afforded to teaching signals that convey 530 information about reward value (RPEs) and action cost (EPEs). Humans display presumably 531 instinctive biases, such as more efficient learning from better-than-expected outcomes 532 (Lefebvre, Lebreton, Meyniel, Bourgeois-Gironde, & Palminteri, 2017) (compared to worse- 533 than-expected outcomes) and asymmetric “Go”/approach learning (Guitart-Masip et al., 534 2012) (compared to “No-Go”/avoidance learning), the latter being a bias that is also 26 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 535 modulated by acute stress (de Berker et al., 2016). From this perspective, no-stress controls, 536 who assigned greater importance to EPEs than RPEs, may have used a computationally 537 costly learning strategy that provides counterweight to a decision-making policy that is 538 biased towards the reward value of actions (captured by action cost discounting parameter γ). 539 Paradoxically, when decisions are by default tilted towards reward value, similar reward and 540 action cost learning rates will facilitate reward learning but hamper action cost learning. 541 Reduced learning rate asymmetry in the presence of action cost discounting may therefore 542 represent a computational reformulation of a heuristic that is employed when cognitively 543 demanding learning strategies are unavailable and the policy towards energy expenditure is 544 more liberal, such as during acute stress. 545 Importantly, stress-induced changes in task performance may crucially depend on the 546 release of catecholamines in neural circuits that support motivation and learning. Dopamine’s 547 actions at D1 and D2 receptors in the basal ganglia mediate approach and avoidance learning 548 (Frank, Seeberger, & Reilly, 2004), and acute stress can improve associative learning by 549 augmenting reward-evoked DA bursts in selective striatal subdivisions (Stelly, Tritley, 550 Rafati, & Wanat, 2020). Dopamine’s enhancement via L-DOPA administration, moreover , 551 improves reward but not action cost learning (Skvortsova et al., 2017). To the degree that 552 pupillometry can be considered a proxy measure of activity of ascending neuromodulatory 553 systems, these findings are consilient with greater encoding of RPEs by pupil size 554 fluctuations during acute stress. Negative correlations between SBP and PANAS negative 555 affect and RPE-pupil size slopes suggest that primarily moderately stressed participants 556 displayed a preference for maximizing reward value, which might be consistent with an 557 inverted U-shape relationship between cognitive performance and DA transmission which is 558 modulated by stress (Arnsten & Goldman-Rakic, 1998; Baik, 2020). Noradrenaline, however, 559 mobilizes available energy to complete effortful actions and locus coeruleus neurons track 27 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 560 energy expenditure (Varazzani et al., 2015). Stress-induced sAA concentrations, increased 561 heart rate, and group differences in the association between pupil size fluctuations and EPEs 562 all point to the involvement of the noradrenaline system. Thus, our model-based pupillometry 563 and stress-induction results hint at stress-sensitive dopaminergic and noradrenergic 564 mechanisms that may regulate cost and benefit learning, which could be explored in future 565 work using targeted pharmacological approaches. 566 The results presented here may improve understanding of stress-related 567 psychopathology. While asymmetric cost-benefit learning during acute stress may be 568 beneficial to reach a desired goal state (e.g., safety) despite high action cost, such strategies 569 could also be maladaptive. For example, stress exposure can lead to drug or smoking relapse 570 (L. Schwabe, Dickinson, & Wolf, 2011), a context in which reward value and action cost 571 may be misaligned. Cost-benefit reinforcement learning may provide a useful framework to 572 test hypotheses regarding stress-related impairments in learning and decision-making. 573 Some study limitations need to be acknowledged. First, pupil dilation associated with 574 effort expenditure greatly reduced our power to detect robust associations between EPE 575 encoding and pupil size fluctuations. Future studies should, therefore, consider a temporal 576 delay between effort outcome and effort expenditure phases. Second, while our 577 computational model was able to recover overall task performance patterns in both groups, 578 such effects were subtle and dependent on the contribution of other (non-learning) 579 parameters, which may highlight the importance of interindividual differences in model 580 parameters. 581 To summarize, we present evidence of asymmetric effects of acute stress on cost 582 versus benefit reinforcement learning during acute stress, which computational analyses of 583 task behaviour explain as a failure to assign appropriate importance to RPEs versus EPEs, 584 and our model-based pupillometry tentatively link to activity of ascending midbrain 28 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 585 neuromodulatory systems. These results highlight for the first time how learning under acute 586 stress can be tilted in favour of acquiring good things and away from the avoidance of costly 587 things. 588 589 Materials and Methods 590 Participants 591 Adult participants were recruited via paper and online advertisements. All participants were 592 screened for a DSM-5 psychiatric and/or neurological disorder, substance use, endocrine 593 and/or vascular disorder, abnormal BMI (>40 or <18), smoking and drinking (>10 594 cigarettes/units per week), psychotropic medication use (lifetime) and hormonal 595 contraceptive use (current; female participants only). All participants completed the ~2-hour 596 experiment between 12:00h and 18:00h to minimize diurnal cortisol fluctuations (Bailey & 597 Heitkemper, 2001). Participants were instructed to refrain from alcohol (starting the evening 598 before the day of the experiment), smoking, food, caffeine intake, strenuous physical activity 599 and brushing their teeth (all >2 hr prior to experiment), which was verified verbally at the 600 start of the session. Four participants were excluded due to an equipment failure (n=4). Three 601 participants quit during stress-induction (n=2) or task procedures (n=1). Because chance- 602 level performance on reinforcement learning tasks might indicate a successful manipulation, 603 a lack of motivation, or a failure to comprehend the task instructions, participants that 604 performed at or below chance level (0.5) on both RL and/or both EL pairs near the end of the 605 experiment (final 10 presentations) were excluded (n=13; 6 acute stress, 7 no-stress control). 606 Including these participants did not alter our key finding that acute stress was associated with 607 asymmetric cost versus benefit learning (see Results). Pupillometry and neuroendocrine data 608 were not processed further for these participants. The study was approved by the ethics 609 committee of the Faculty of Psychology and Neuroscience, Maastricht University (ERCPN- 29 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 610 197_03_08_2018) and carried out in accordance with the Declaration of Helsinki. 611 Participants were remunerated in gift vouchers or research participation credits. Task 612 earnings were paid out in gift vouchers. 613 614 Acute stress induction 615 The MAST is a validated stress-induction paradigm combining both psychological and 616 physiological stressors, and robustly increases neuroendocrine, physiological, and subjective 617 indices of acute stress (Smeets et al., 2012). During a 5-min preparation phase, participants 618 are instructed about the upcoming task via oral and visually displayed instructions, followed 619 by a 10-min stress-induction phase consisting of alternating blocks of cold-water immersion 620 (non-dominant hand; 2oC) and backward counting in steps of 17 (while receiving negative 621 evaluative feedback from an experimenter), with a (non-recording) camera continuously 622 directed at the participant’s face, which was displayed to the participant on a second display. 623 During the MAST no-stress control condition, participants immerse their hand in lukewarm 624 water (36oC) and perform simple mental arithmetic, e.g., counting from 1 to 25, without 625 receiving feedback or fake camera recordings. 626 627 Neuroendocrine, physiological and subjective stress measurements 628 sCORT and sAA were collected to measure stress-induced increases in hypothalamic- 629 pituitary-adrenal (HPA) axis and sympathetic-adrenal-medullary (SAM) axis activity, 630 respectively (Dickerson & Kemeny, 2004; Koh, Ng, & Naing, 2014). Saliva samples were 631 obtained using synthetic Salivette® devices (Sarstedt, Etten-Leur, the Netherlands) during 3- 632 min sampling periods at 6 time points. A baseline sample was collected 10 min prior to the 633 MAST (baseline: t1 = t-10) and five samples post-MAST (t2= t+00, t3= t+10, t4= t+20, t5= t+30, t6= 634 t+40). SAA assessments were obtained only for t1-t4, due to the rapid decay of sAA post-stress 30 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 635 induction (Dennis Hernaus, Quaedflieg, Offermann, Casales Santa, & van Amelsvoort, 2018; 636 Nater et al., 2005). For all participants, t2 marked the starting point for the reward value 637 maximisation/action cost minimisation task. Samples were stored at -20oC immediately after 638 completion of each session. SCORT and sAA levels were determined using a commercially 639 available luminescence immune assay kit (IBL, Hamburg, Germany) and kinetic reaction 640 assay (Salimetrics, Penn State, PA, USA), respectively. 641 Systolic blood pressure (SBP) and heart rate (HR) as an index of autonomic nervous 642 system (ANS) arousal (Schubert et al., 2009; Wright, O'Brien, Hazi, & Kent, 2014) were 643 assessed at t1 and t2 using an OMRON M4-I blood pressure monitor (OMRON Healthcare 644 Europe B.V., Hoofddorp, The Netherlands). Subjective affect ratings were assessed at t1 and 645 t2 using the 20-item Positive and Negative Affect Scale (PANAS) (Watson, Clark, & 646 Tellegen, 1988). 647 648 Reward maximization versus action cost minimization reinforcement learning task 649 All participants completed a probabilistic stimulus selection paradigm during which they 650 learned to select stimuli with high reward value (20 Eurocents) and avoid stimuli with high 651 action cost (exerting force above a pre-calibrated individual threshold for a duration of 652 3000ms). This reinforcement learning task is conceptually similar to a previously-validated 653 probabilistic action selection task that has been employed to study the neural signatures of 654 reward and effort prediction errors and dopaminergic drug effects on reward-effort 655 computations (Skvortsova et al., 2017; Skvortsova et al., 2014). The paradigm was designed 656 in PsychoPy v3.0.0b11 (Peirce et al., 2019) and presented on a 24″ monitor (iiyama ProLite 657 b2483HSU). Physical effort (in mV/kgf) was registered using a hand-held dynamometer in 658 combination with a transducer amplifier (DA100C) and data acquisition system (MP160; all 659 manufactured by BIOPAC Systems, Inc). Individual effort thresholds used throughout the 31 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 660 task were obtained by calculating 50% of each participant’s maximal voluntary contraction 661 (MVC) (Le Heron et al., 2018) reached over three calibration trials by squeezing the 662 dynamometer with the dominant hand. 663 On each of 120 trials, participants chose between two paired distinct black-and-white 664 images (“stimuli”) that were probabilistically associated with both the receipt of a monetary 665 reward and exertion of physical effort (see Figure 1 for a graphical overview). At trial onset, 666 a fixation cross flanked by two images was presented; participants chose one image by 667 pressing the V/B button for the left/right option, respectively. A 440Hz/600Hz tone for 668 left/right choice (200ms) was presented to confirm the participant’s choice. Next, a 669 thermometer with the command “SQUEEZE” or “DON’T SQUEEZE” was displayed. If 670 participants were required to exert effort, they were instructed to squeeze the dynamometer 671 until the mercury level reached the top. The mercury bar only moved if participants exerted 672 above-threshold levels of force and stopped moving if exerted force fell below. The 673 cumulative above-threshold time was 3000ms. If no effort production was required, an 674 animation of a rising mercury bar was displayed (3000ms). Finally, a screen was presented 675 showing either a €0.20 coin or a crossed-out coin, indicating no reward (3000ms). 676 Participants learned to choose the optimal (most reward or most effort avoiding) 677 stimulus for four distinct image pairs, 30 presentations each, with yoked reward and action 678 cost contingencies. For 2/4 pairs, participants could regularly acquire rewards by selecting 679 one (optimal) stimulus over the (suboptimal) other (henceforth, “reward learning”/RL pairs), 680 while the probability of having to exert effort was identical for both stimuli. For the other two 681 pairs, choices of one stimulus were more frequently followed by the avoidance of effort 682 (“effort learning”/EL pairs), while the probability of reward was kept constant between both. 683 For all pairs, the probability of the stimulus property that was kept constant (reward/effort) 32 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 684 was set to a 33.3% chance of positive outcome upon selection (reward/ effort avoidance) and 685 66.6% chance of negative outcome (no reward/effort). 686 To assess whether any acute stress effects on reward maximization (measured using 687 RL pairs) and effort cost minimization (measured using EL pairs) learning were potentially 688 mediated by task difficulty, we employed different difficulty levels for each RL and EL pair. 689 That is, for one RL and one EL (“easy”) pair, a choice for the optimal stimulus was followed 690 by a positive outcome in 83% (vs. 17% negative outcome) of all trials (83% negative/17% 691 positive outcome for suboptimal stimulus); for the other RL and EL (“hard”) pair a choice for 692 the optimal stimulus was followed by a positive outcome in 70% (vs. 30% negative outcome) 693 of all trials (and 70% negative/30% positive outcome) for the suboptimal stimulus. This 694 approach allowed us to disentangle whether acute stress primarily impacted domain-specific 695 (RL vs. EL) or general (easy vs. hard) reinforcement learning (the latter which also might 696 involve other cognitive skills that might be beneficial to performance and sensitive to change 697 under stress, such as working memory (Schoofs, Wolf, & Smeets, 2009). The task 698 contingencies described above were based on extensive pilot tests to identify a reinforcement 699 schedule that would enable us to detect stress-induced improvements and decreases in task 700 performance. We selected task contingencies based on pilot sessions involving a no-stress 701 control condition and chose a reinforcement schedule associated with non-ceiling/floor 702 performance on RL and EL trials. 703 Following the learning phase, participants completed a surprise test phase, similar to 704 previous work (D. Hernaus et al., 2019; D. Hernaus et al., 2018). This phase consisted of 64 705 trials in which participants were presented with the original four, as well as six novel, 706 stimulus combinations. Participants were asked to choose the stimulus with the highest 707 reward value or the lowest action cost - depending on a coin or thermometer image presented 708 in the middle of the screen - and received no choice feedback. This allowed us to assess 33 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 709 acquired choice tendencies, as well as generalizability of this information to novel situations. 710 The four original pairs were presented four times (total n=16) during which we only asked 711 participants to discriminate on the basis on the reward value (for RL) or action cost (for EL). 712 For novel stimulus combinations, we only presented stimuli that differed in reward 713 value/action cost if reward value discrimination/action cost discrimination was assessed (total 714 n=48: n=4 presentations for the 6 combinations). 715 For every participant, stimuli were randomly assigned to pairs, optimal/suboptimal 716 stimulus orientation was balanced (50% of all optimal stimulus presentations occurred on the 717 left-hand side) and misleading outcomes (e.g., negative outcomes for optimal stimuli) were 718 equally spaced out across the thirty presentations (and balanced for left/right side). Trial 719 presentation order was pseudo-randomized such that I) a given pair would never be presented 720 more than twice in a row and II) the gap between two presentations of a given pair was never 721 greater than four trials. 722 Prior to performing the actual task and prior before acute stress/no-stress control 723 procedures, participants received standard verbal instructions and completed a 16-trial 724 practice round of the learning phase. Participants were not informed about stimulus-outcome 725 contingencies; they were only advised to accrue as much money as possible and avoid 726 exerting unnecessary effort. A 60% accuracy performance threshold was used to confirm that 727 participants understood the general task procedure. The practice round was repeated if 728 participants failed to reach 60% accuracy. To prevent learning, we used deterministic 729 stimulus-outcome probabilities and different stimuli. 730 731 Computational cost-benefit reinforcement learning model: model space 732 In an attempt to uncover latent mechanisms by which acute stress affects reward 733 maximization and/or action cost minimization, we turned to cognitive computational 34 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 734 modelling. We employed a modified reinforcement learning framework based on Rescorla 735 and Wagner (Rescorla, 1972), and used in Skvortsova et al. (Skvortsova et al., 2017; 736 Skvortsova et al., 2014) to investigate whether acute stress impacted learning about 737 sensitivity to, and/or discounting of reward value and action cost. We first describe the model 738 space. 739 Various reinforcement learning models assume that choice preferences of an agent are 740 updated via the prediction error, i.e., the mismatch between outcome and expectation 741 (equation 1A, 1B) and the critical quantity that drives learning (Rescorla, 1972): 742 743 RPE(t) = r(t) – QR(t)(s, a) (1A) 744 EPE(t) = e(t) – QE(t)(s, a) (1B) 745 746 Here, QR(t)(s, a) and QE(t)(s, a) represent the expected reward value and action cost 747 (i.e., effort), where s reflects the given pair and a refers to the more abstract action of 748 selecting a stimulus (not to be confused with action selection), r(t) and e(t) represent the reward 749 and effort outcome for the chosen stimulus at trial t. RPE(t) and EPE(t), thus, represent the RPE 750 and EPE at trial t, respectively. 751 In order to allow for the possibility that humans do not calculate the prediction error 752 against the actual outcome but, rather, what the outcome “feels” like (Huys et al., 2013) , we 753 considered a scenario in which reward and effort outcomes are first multiplied by a free 754 parameter that captures the weight that reward and effort outcomes receive (“WR” and WE” in 755 equation 2A and 2B). As the value of these parameters approaches 1, rewards are 756 increasingly valued more positively, and effort more negatively. These parameters, therefore, 757 control the maximum size of the prediction error. 758 35 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 759 RPE(t) = (r(t)*WR) – QR(t)(s, a) (2A) 760 EPE(t) = (e(t)*WE) – QE(t)(s, a) (2B) 761 762 In various formulations of reinforcement learning, such as Q-learning (Watkins & 763 Dayan, 1992) and the actor-critic framework (Niv, 2009; Rescorla, 1972), the degree to 764 which prediction errors update choice preferences is represented by α, the learning rate 765 (equation 3A), which determines how current prediction errors update choice preferences on 766 the subsequent trial. High values of α allow for rapid updating of choice preferences, while a 767 low α implies that choice preferences are updated at a slower pace and are thus co- 768 determined by outcomes further into the past. 769 770 QR(t)(s, a) = QR(t-1)(s, a) + αR*RPE(t-1)(s, a) (3A) 771 QE(t)(s, a) = QE(t-1)(s, a) + αE*RPE(t-1)(s, a) (3B) 772 773 Extensive evidence suggests that organisms use different learning systems for different types 774 of information, including reward value and action cost (Palminteri & Pessiglione, 2017; 775 Skvortsova et al., 2017; Skvortsova et al., 2014) (equation 3A/B). Thus, the use of separate 776 learning rates for RPEs and EPEs allows for asymmetrical learning about these types of 777 information. 778 While the learning rate controls the speed at which choice preferences are updated, 779 learning rate (nor reward/effort weight) alone does not explain how learned estimates of 780 reward value and action cost may compete at the decision stage (i.e., when participants 781 choose between two stimuli). Agents weight costs against benefits to calculate a subjective 782 decision value (Pessiglione et al., 2017; Skvortsova et al., 2017), which is used to guide 783 choices (equation 4). 36 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 784 785 Q(t)(s, a) = QR(t)(s, a) - QE(t)(s, a) (4) 786 787 In its simplest form, Q, the subjective decision value of a stimulus is represented by the 788 difference between the expected reward and action cost value at trial t (equation 4) 789 (Skvortsova et al., 2014). However, this particular operationalization of subjective value does 790 not take into account the observation that humans tend to discount or prioritize certain types 791 of information in their decisions (Apps, Grima, Manohar, & Husain, 2015; Inzlicht et al., 792 2018). We, therefore, allowed for variation in the calculation of subjective decision value via 793 action cost discounting (equation 5). While discounting rates can be linear or hyperbolic 794 (Hartmann, Hager, Tobler, & Kaiser, 2013), here we only considered linear discounting in 795 light of previous work using a similar task design (Skvortsova et al., 2017; Skvortsova et al., 796 2014). As the value of γ approaches zero, action cost discounted increases leading the agent 797 to ignore action cost/only utilize reward value to make a decision. 798 799 Q(t)(s, a) = QR(t)(s, a) - γ*QE(t)(s, a) (5) 800 801 Once the subjective decision value has been computed, the degree to which 802 participants deterministically sample the optimal stimulus is captured by a softmax decision 803 function (equation 6). 804 805 pr(s, a) = exp(Q(t)(s, a)) / sum(exp(β*Q(t)(s))) (6) 806 807 808 Here, pr is the probability of selecting an action, β is the inverse temperature parameter that among others captures the balance between exploration and exploitation (Nassar & 37 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 809 Frank, 2016), Q(t)(s, a) is the net value of the chosen option and Q(t)(s) represents the net 810 values of both stimuli in the pair. 811 Within the above-described model space our predictions of acute stress effects on reward 812 maximization and action cost minimization could, thus, be explained by changes in 813 sensitivity to reward value and/or action cost (WR, WE), changes in how much weight RPEs 814 and EPEs are afforded (i.e., learning rates, αR, αE), and/or changes in the discounting of 815 reward value by action cost (γ). If acute stress leads to more random responses, such effects 816 should be captured by β. 817 Based on our predictions and the obtained pattern of results (most notably asymmetrical 818 RL/EL performance in the acute stress condition), we considered six candidate models that 819 could capture these various scenarios: I) a model with 2 distinct learning rates for reward and 820 effort (αR, αE) [2LR]; II) a model with 2 learning rates (αR, αE) and a discounting parameter 821 (γ) (2LR_ γ); III) a model with 2 learning rates (αR, αE), a reward weight (WR) and an effort 822 weight parameter (WE) (2LR_WR_WE), IV) a model with a single learning rate (α), reward 823 weight (WR), effort weight (WE), and a discounting (γ) parameter (LR_ WR__WE_ γ); V) a 824 model with 2 learning rates (αR, αE), a reward weight (WR), and a discounting (γ) parameter 825 (2LR_WR_γ); VI) a model with 2 learning rates (αR, αE), a reward weight (WR), effort weight 826 (WE) and discounting (γ) parameter (2LR_ WR_ WE_ γ). 827 Lower/upper bounds for all parameters were set to [0,1] and all models contained a β 828 parameter. Consistent with previous work (Skvortsova et al., 2017; Skvortsova et al., 2014), 829 reward and action cost outcomes were set to [0,1 for no/yes reward] and [-1,0 for no/yes 830 effort avoidance], respectively. 38 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 831 Pupillometry 832 Fluctuations in pupil diameter were continuously measured using an SR-Research Eyelink 833 1000 Tower Mount infrared eye tracker while participants performed the reward 834 maximization/action cost minimization reinforcement learning task (1000Hz sampling rate, 835 except for three participants, whose data were obtained at 500Hz). Participants placed their 836 head on an adjustable chin rest and against a forehead bar to minimize motion. Eye-tracker 837 calibration was performed at the start of the paradigm, and subsequently every 10 min. 838 Stimulus luminance was matched using the SHINE toolbox (Willenbockel et al., 2010) in 839 MATLAB (v. 2014B; The MathWorks, Inc., Natick, Massachusetts, United States). Due to 840 the COVID-19 pandemic, pupillometry data were not collected for the final eight 841 participants. Three participants, moreover, failed the quality control for eye-tracking data (2 842 no-stress control/1 acute stress) leaving a final sample of 69 participants with eye-tracking 843 data (34 no-stress control/35 acute stress). 844 Eye-tracking data were pre-processed using an open source pre-processing toolbox 845 (Kret & Sjak-Shie, 2019) and in accordance with previous work (Jackson & Sirois, 2009). 846 Blinks and other invalid samples, due to dilation speed, deviation from the trend line, and 847 extreme values (Kret & Sjak-Shie, 2019) were removed, interpolated, smoothed (4Hz low- 848 pass filter, fourth-order Butterworth filter) (Jackson & Sirois, 2009), z-scored and down- 849 sampled to 50hz (i.e., 20ms). Bins with fewer than 80% valid samples were removed 850 (Lawson et al., 2020). For analyses, we considered three epochs of interest: choice (-1500ms 851 pre-choice - 15000ms post-choice), effort outcome (0-1000ms post-outcome), and reward 852 outcome (0-2000ms post-outcome). We reduced the duration of the effort outcome epoch to 853 1000ms to minimize force exertion-related effects on pupil size (see below). Recent work has 854 shown that expectation violations (prediction errors) are encoded by pupil size fluctuations 855 within this timeframe (Lawson et al., 2020). Given that we observed large grip force- 39 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 856 associated effects on the pupillometry signal (see Figure Supplement 8 middle row, for a 857 comparison between effort and effort avoidance trials), we limited effort outcome analyses in 858 the main text to effort avoidance trials, although we also report analyses involving all effort 859 outcome trials in Figure Supplement 8. 860 861 Statistical analyses 862 Statistical analyses were conducted using R, version 3.6.2 (Team, 2020) and, where 863 applicable, results were visualised using Raincloud Plots (Allen, Poggiali, Whitaker, 864 Marshall, & Kievit, 2019). Acute stress measurements were analysed using mixed ANOVAs 865 involving Condition (between-factor condition: no-stress control, acute stress induction) and 866 Time (within-factor: 2 pre/post-MAST or 6 levels for sCORT). 867 For the reward maximisation/action cost minimisation reinforcement learning task, an 868 accuracy score was calculated by dividing the number of optimal stimulus choices by the 869 total trial amount (n=30 per pair). Mixed ANOVAs involving Condition, Trial Type (RL, EL) 870 and Difficulty (Easy, Hard pairs) were carried out. For analyses involving Time effects (i.e., 871 repeated presentations of stimulus pairs), accuracy scores were averaged per bin of ten 872 presentations (presentation 1-10, 11-20, and 21-30). To better understand whether acute 873 stress effects on task performance were primarily driven by changes in sensitivity to positive 874 or negative outcomes, win-stay (repeating a choice following a positive outcome) and lose- 875 shift (choosing the other stimulus following a loss) rates were calculated for RL and EL trials 876 (Hanneke E. M. den Ouden et al., 2013). For RL trials, we calculated win-stay/lose-shift rates 877 using reward outcomes (yes/no reward); for EL trials we used effort outcomes (yes/no effort). 878 We refer to the 2-level factor representing win-stay/lose-shift rates as “Strategy”. For surprise 879 test trials involving the original four pairs (n=4 presentations per pair), we investigated final 880 choice tendences using a one-sample t-test against chance level (0.5). Participants’ ability to 40 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 881 discriminate stimuli based on reward value and action cost in novel stimulus arrangements 882 (n=48, 24 reward value and 24 action cost discrimination trials) were investigated using 883 mixed ANOVAs involving Condition and Trial Type. 884 Group differences in model parameters from the non-hierarchically fit model were 885 investigated using Condition-by learning rate (αR, αΕ) mixed ANOVAs and independent 886 samples t-tests. Given that we used separate priors for the two groups, we report the Bayesian 887 analogue of a t-test and mixed-ANOVA (Kruschke, 2014) - a more robust test of group 888 differences - for posterior parameters obtained from the hierarchically fit model (for 889 reference, we also report these analyses for the non-hierarchical data). 890 Post hoc (simple) main effect analyses for all ANOVAs were conducted using 891 independent sample (Condition), paired-samples (Time, Trial Type, Strategy), and one- 892 sample t-tests (≠ 0 or 0.5). Greenhouse–Geisser-corrected statistics were reported when 893 sphericity assumptions were violated. We report statistical significance as p<0.05 (two- 894 sided), but we note that most main and interaction effects involving Condition survived at a 895 more stringent threshold (p<.01), except for some strategy and surprise test phase effects, 896 which should be interpreted with caution. In case of statistically significant results, 897 generalized eta square (ges; n2G) was reported, with n2G values of 0.02, 0.06, and 0.14 898 representing a small, medium, and large effect size, respectively (Lakens, 2013). 899 With respect to pupillometry, we conducted model-free and model-based analyses. In 900 model-free analyses, we investigated group differences in pupil size during the choice, effort 901 outcome, and reward outcome stage, for every bin of interest. To better understand how 902 putative activity of ascending neuromodulatory systems may drive stress-induced changes in 903 computational strategies that support reward maximisation/action cost minimisation learning, 904 we conducted model-based pupillometry analyses using computational parameters from the 905 winning (2LR_ γ) model (Lawson et al., 2020). First, we used linear regression to estimate 41 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 906 beta weights for the association between pupil size and computational estimates of task- 907 related behaviour for every participant, for every epoch, for every bin. For the choice phase, 908 we regressed trial-wise measures of pupil size against trial-wise estimates of the subjective 909 decision value (i.e., effort-discounted reward value) of the chosen stimulus. For the effort and 910 reward outcome phase, trial-wise EPEs and RPEs were the primary predictors of interest, 911 respectively. Trial number (1-120) and presented images/pair (RL_easy, RL_hard, EL_easy, 912 EL hard) served as additional predictors of interest for all models. Additional epoch-specific 913 variables of interest were included for the choice (optimal choice yes/no), effort (action cost 914 of chosen stimulus, effort avoidance yes/no), and reward (reward value of chosen stimulus, 915 reward yes/no, effort avoidance yes/no) outcome phase. Similar results were obtained when 916 repeating the analyses with more elaborate GLMs (e.g., the addition of yes/no most likely 917 outcome based on reward/effort outcome probabilities [“surprise”] and reward/action cost for 918 EL/RL trials). Secondly, in group-level GLMs, we compared the resulting beta weights I) 919 against zero (for the no-stress control/acute stress condition separately), to investigate when 920 the pupil encoded the computational process of interest, and II) between groups, to assess 921 stress-induced changes in associations between pupil size and computational processes. To 922 control the false positive rate, we conducted permutation tests at the bin- and cluster-level 923 (2000 permutations, apermute=0.05). All correlations were performed using Spearman’s ρ 924 correlations. Permutation tests were also conducted for correlation analyses involving acute 925 stress measures and pupil encoding of predictions errors. 42 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 926 Figure 3 – Source Data 1 927 Source files for task performance data. 928 This link contains all task performance data used for the analyses shown in Figure 3. Raw 929 data can be found under “task_performance”. 930 https://osf.io/ydv2q/ 931 932 Figure 6 – Source Data 2 933 Source files for pupillometry data. 934 This link contains pupillometry data used for the analyses shown in Figure 6. Raw data can 935 be found under “pupillometry”. 936 https://osf.io/ydv2q/ 937 938 Acknowledgements 939 We are indebted to Drs. Conny Quaedflieg, Edwin S. Dalmaijer. and Ross D. Markello for 940 advice on stress-induction procedures and paradigm development. We thank Dr. Michael 941 Frank for advice on hierarchical computational modelling procedures. We thank Katya Brat- 942 Matchett for involvement in participant recruitment and Truda Driessen for administrative 943 support. 944 945 Competing interests 946 D.H. has received financial compensation as a consultant for P1vital Products Ltd. These 947 activities were unrelated to the work presented in this manuscript. 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Behav 1142 Neurosci, 123(5), 1066-1075. doi:10.1037/a0016980 1143 Schubert, C., Lambertz, M., Nelesen, R. A., Bardwell, W., Choi, J. B., & Dimsdale, J. E. 1144 (2009). Effects of stress on heart rate complexity--a comparison between short-term 1145 and chronic stress. Biological psychology, 80(3), 325-332. 1146 doi:10.1016/j.biopsycho.2008.11.005 1147 1148 1149 Schultz, W., Dayan, P., & Montague, P. R. (1997). A neural substrate of prediction and reward. science, 275(5306), 1593-1599. doi:10.1126/science.275.5306.1593 Schwabe, L., Dickinson, A., & Wolf, O. T. (2011). Stress, habits, and drug addiction: a 1150 psychoneuroendocrinological perspective. Exp Clin Psychopharmacol, 19(1), 53-63. 1151 doi:10.1037/a0022212 1152 Schwabe, L., & Wolf, O. T. (2011). Stress-induced modulation of instrumental behavior: 1153 from goal-directed to habitual control of action. Behavioural brain research, 219(2), 1154 321-328. 1155 Shafiei, N., Gray, M., Viau, V., & Floresco, S. B. (2012). Acute stress induces selective 1156 alterations in cost/benefit decision-making. Neuropsychopharmacology, 37(10), 2194- 1157 2209. doi:10.1038/npp.2012.69 1158 Skvortsova, V., Degos, B., Welter, M.-L., Vidailhet, M., & Pessiglione, M. (2017). A 1159 selective role for dopamine in learning to maximize reward but not to minimize effort: 1160 evidence from patients with Parkinson's disease. Journal of Neuroscience, 37(25), 1161 6087-6097. 1162 Skvortsova, V., Palminteri, S., & Pessiglione, M. (2014). Learning to minimize efforts versus 1163 maximizing rewards: computational principles and neural correlates. Journal of 1164 Neuroscience, 34(47), 15621-15630. 52 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1165 Smeets, T., Cornelisse, S., Quaedflieg, C. W., Meyer, T., Jelicic, M., & Merckelbach, H. 1166 (2012). Introducing the Maastricht Acute Stress Test (MAST): a quick and non- 1167 invasive approach to elicit robust autonomic and glucocorticoid stress responses. 1168 Psychoneuroendocrinology, 37(12), 1998-2008. doi:10.1016/j.psyneuen.2012.04.012 1169 Steinberg, E. E., Keiflin, R., Boivin, J. R., Witten, I. B., Deisseroth, K., & Janak, P. H. 1170 (2013). A causal link between prediction errors, dopamine neurons and learning. 1171 Nature neuroscience, 16(7), 966-973. doi:10.1038/nn.3413 1172 Stelly, C. E., Tritley, S. C., Rafati, Y., & Wanat, M. J. (2020). Acute Stress Enhances 1173 Associative Learning via Dopamine Signaling in the Ventral Lateral Striatum. The 1174 Journal of Neuroscience, 40(22), 4391. doi:10.1523/JNEUROSCI.3003-19.2020 1175 Team, R. C. (2020). R: A language and environment for statistical 1176 computing. R Foundation for Statistical Computing. Retrieved from https://www.R- 1177 1178 project.org/ Timmers, I., Kaas, A. L., Quaedflieg, C., Biggs, E. E., Smeets, T., & de Jong, J. R. (2018). 1179 Fear of pain and cortisol reactivity predict the strength of stress-induced hypoalgesia. 1180 Eur J Pain, 22(7), 1291-1303. doi:10.1002/ejp.1217 1181 Valton, V., Wise, T., & Robinson, O. J. (2020). Recommendations for Bayesian hierarchical 1182 model specifications for case-control studies in mental health. arXiv preprint 1183 arXiv:2011.01725. 1184 Varazzani, C., San-Galli, A., Gilardeau, S., & Bouret, S. (2015). Noradrenaline and 1185 dopamine neurons in the reward/effort trade-off: a direct electrophysiological 1186 comparison in behaving monkeys. J Neurosci, 35(20), 7866-7877. 1187 doi:10.1523/jneurosci.0454-15.2015 1188 1189 Watkins, C. J. C. H., & Dayan, P. (1992). Q-learning. Machine Learning, 8(3), 279-292. doi:10.1007/BF00992698 53 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1190 Watson, D., Clark, L. A., & Tellegen, A. (1988). Development and validation of brief 1191 measures of positive and negative affect: the PANAS scales. J Pers Soc Psychol, 1192 54(6), 1063-1070. doi:10.1037//0022-3514.54.6.1063 1193 Willenbockel, V., Sadr, J., Fiset, D., Horne, G. O., Gosselin, F., & Tanaka, J. W. (2010). 1194 Controlling low-level image properties: The SHINE toolbox. Behavior Research 1195 Methods, 42(3), 671-684. doi:10.3758/BRM.42.3.671 1196 1197 1198 Wilson, R. C., & Collins, A. G. (2019). Ten simple rules for the computational modeling of behavioral data. Elife, 8, e49547. Wright, B. J., O'Brien, S., Hazi, A., & Kent, S. (2014). Increased systolic blood pressure 1199 reactivity to acute stress is related with better self-reported health. Scientific reports, 1200 4, 6882-6882. doi:10.1038/srep06882 1201 Yohn, S. E., Errante, E. E., Rosenbloom-Snow, A., Somerville, M., Rowland, M., Tokarski, 1202 K., . . . Salamone, J. D. (2016). Blockade of uptake for dopamine, but not 1203 norepinephrine or 5-HT, increases selection of high effort instrumental activity: 1204 Implications for treatment of effort-related motivational symptoms in 1205 psychopathology. Neuropharmacology, 109, 270-280. 1206 doi:10.1016/j.neuropharm.2016.06.018 1207 54 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1208 1209 Supplementary Figures Figure Supplement 1 1210 1211 Evidence of reward and action cost reinforcement learning. 1212 Optimal stimulus choices (“accuracy”) on reward learning (RL) and effort learning (EL) 1213 (rows) trials for both conditions (columns). Trials were binned into groups of 10 1214 presentations. Participants performed significantly better than chance level in all bins. Means 1215 ± SD. Significant differences are denoted by asterisks (*: p < 0.05, **: p < 0.01, ***: p < 1216 0.001). 55 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1217 Figure Supplement 2 1218 1219 Acute stress does not affect difficulty learning. 1220 Easy and hard pairs collapsed across RL/EL trials depicted for each condition separately. 1221 While all participants sampled the optimal choices more frequently for Easy vs. Hard pairs, 1222 no significant Condition-by-Difficulty interaction or between-group differences were 1223 observed. Means ± SD, individual data points, distribution and density of the data are 1224 displayed. Significant differences are denoted by asterisks (*: p < 0.05, **: p < 0.01, ***: p < 1225 0.001). 56 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1226 Figure Supplement 3 1227 1228 Surprise test phase performance. 1229 The acute stress group performed better on reward than action cost discrimination trials. 1230 Means ± SD, individual data points, distribution and density of the data are displayed. 1231 Significant differences are depicted with asterisks (*: p < 0.05, **: p < 0.01, ***: p < 0.001). 57 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1232 Figure Supplement 4 1233 1234 Correlations between empirical and simulated 2LR_γ choices. 1235 Actual and post hoc simulated choices for RL and EL (rows) were highly correlated both for 1236 no-stress control and acute stress subjects (columns). Simulations were averaged across 10 1237 repetitions per subject. Solid and shaded lines represent mean ± CI95%. Dots represent 1238 individual data points. Horizontal dashed lines indicate chance level (0.5). 58 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1239 Figure Supplement 5 1240 1241 Parameter estimates after Bayesian hierarchical model fitting. 1242 Hierarchical model fitting reproduced the overall pattern of parameter estimates (Figure 5 for 1243 comparison) . 59 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1244 Figure Supplement 6 1245 1246 Correlations between empirical and simulated 2LR_γ choices after Bayesian 1247 hierarchical model fitting. 1248 Correlations between actual and post hoc simulated choices for RL and EL (rows) for no- 1249 stress control and acute stress subjects (columns). Simulations were averaged across 10 1250 repetitions per subject. Solid and shaded lines represent mean ± CI95%. Dots represent 1251 individual data points. Horizontal dashed lines indicate chance level (0.5). 60 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1252 Figure Supplement 7 1253 1254 Bayesian estimation analysis to evaluate group differences in posterior parameter 1255 distributions 1256 Panel A. Bayesian estimation (mixed-ANOVA) using posterior parameters (following 1257 hierarchical fitting) revealed evidence for a credible Condition-by-Learning Rate interaction. 1258 The observed mean difference from zero that falls outside the 95% HDI suggests that the 61 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1259 difference between αE and αR was greater in no-stress controls compared to acute stress 1260 subjects. Panel B. Both groups did not differ in the magnitude of αR, as indicated by a 95% 1261 HDI that included 0. Panel C. Acute stress compared to no-stress control subjects exhibited a 1262 lower value of αE, as indicated by a 95% HDI that falls well above zero. 62 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1263 Figure Supplement 8 1264 1265 Pupillometry analyses using all effort outcome trials. 1266 Left: Model-free analyses of pupil size using all effort outcome trials. Middle: Pupil size 1267 differences during effort/effort avoidance outcomes in the entire sample; force exertion was 1268 associated with large effects on pupil size and these trials were therefore excluded from 1269 analysis. Right: Model-based action cost prediction error analyses using all effort outcome 1270 trials. 63 

W1532739614.txt

We are IntechOpen, the world’s leading publisher of Open Access books Built by scientists, for scientists 6,900 186,000 200M Open access books available International authors and editors Downloads Our authors are among the 154 TOP 1% 12.2% Countries delivered to most cited scientists Contributors from top 500 universities Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI) Interested in publishing with us? Contact book.department@intechopen.com Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com 8 Appendicitis in Children Ngozi Joy Nwokoma Addenbrooke’s Hospital, Cambridge University Hospitals, Cambridge United Kingdom 1. Introduction Abdominal pain is a common clinical problem in children. The challenge is to determine which could be secondary to serious pathology. For the paediatric surgeon, the evaluation of a child with abdominal pain is often to ascertain if there is a surgically amenable pathology. The first clinical report of appendicitis in 1711 is credited to a German surgeon called Lorenz Heister (Ramsted et al., 1993). Appendicitis is the commonest acute childhood surgical abdominal emergency in developed countries. The peak incidence of acute appendicitis in children is in the second decade of life, at about 12years of age (Pearl et al., 1995; Tsze et al., 2011). It is uncommon in children less than 5years old, rare in infants and neonates, slightly more frequent in males than females with an incidence ratio of 1:1.5. The overall lifetime risk of appendicitis is 7%, slightly higher in females. 2. Embryology The appendix develops as a true diverticulum of the caecum and becomes visible at the eighth week of gestation. It becomes more distinct as the inferior border of the caecum fails to enlarge as rapidly as the rest of it (Swain, 2005). As the proximal colon enlarges the caecum undergoes a downwards displacement into the right iliac fossa region of the abdomen. In certain congenital anomalies the final position of the appendix is outside the right lower quadrant. In situs inversus, the orientation of the intra-abdominal organs is reversed so that left sided organs are on the right and vice versa. The thoracic organs may also be involved in situs inversus totalis. In this condition, the appendix ends up in the left lower quadrant. In developmental arrest of the normal rotation of the midgut, the appendix may lie in the subhepatic region or towards the left side of the abdomen. 3. Anatomy 3.1 Position The base of the appendix is located in the posteromedial aspect of the caecum; below and within 3cm of the ileocaecal junction. Though the base of the appendix assumes a relatively fixed position the final position of the appendix body and tip is variable (Figure 1). It commonly lies behind the caecum (retrocaecal: 64%) or crossing the pelvic brim into the pelvic cavity (pelvic: 32%). It could also lie posterior to the proximal colon (retrocolic), posterior to the terminal ileum (retroileal), anterior to the terminal ileum (preileal), just below the caecum (subcolic), along the lateral border of the caecum and colon www.intechopen.com 134 Appendicitis – A Collection of Essays from Around the World (paracolic/precaecal) or it may be an obturator appendix crossing over the obturator internus muscle (Moore & Dalley, 2006; Standring et al., 2005). Rarely, the appendix may lie on the right kidney or duodenum with a retroperitoneal tip and has been reported to ulcerate into the duodenum (Ellis & Mahadevan, 2010). Fig. 1. Positional variation of appendicular body and tip: A. retrocaecal; B. Pelvic; C. retrocolic; D. retroileal; E. preileal; F. subcolic; G. paracolic/precaecal; H. obturator. The superficial landmark of the base of the appendix corresponds to the level of the first segment of the sacral vertebrae (S1) at the McBurney’s point. The McBurney’s point is the junction of the outer and middle thirds of an imaginary line running from the right anterior superior iliac spine to the centre of the umbilicus. However, the appendix is located within 5cm of the McBurney’s point less than 50% of the time (Karim et al., 1990). 3.2 Innervation The midline development of the intra-abdominal viscera and associated innervation results in visceral pain being perceived in the midline. The level of pain may also be different from the level of the organ from which the pain stimulus arises due to the cranial migration of the nervous system. In line with the foregoing, epigastric pain is typically associated with pathology or irritation of the organs that originate from the foregut, periumbilical pain relates to midgut organs while infraumbilical or suprapubic pain relates to disease in the www.intechopen.com Appendicitis in Children 135 hindgut. The midgut stretches from the second part of the duodenum to the midpoint of the transverse colon. Being a midgut-originated structure, the initial pain sensation from the appendix is felt in the periumbilical region. Perception of abdominal pain occurs when the nociceptors in the respective organ or region of the abdomen have been stimulated by appropriate agents. Appendicitis represents inflammation of a magnitude great enough to stimulate these nociceptors. The nerve supply to the appendix is derived from the autonomic nervous system and has fibres that respond to stretch rather than pain which explains the poorly localised symptoms until the parietal peritoneum becomes involved. The sympathetic nerve supply is from the superior mesenteric plexus while the parasympathetic nerve supply is from the Vagus nerve. 3.3 Structure The appendix is commonly referred to as the vermiform appendix because of its worm-like tubular structure. The length of the appendix is variable ranging from 2 – 25cm but can be up to 31cm. It is longer in children, than in adults probably due to age-related atrophy. The external diameter could range from 3 – 8mm and the luminal diameter between 1 – 3mm (Williams & Myers, 1994; Petras & Goldblum, 1996). The maximum transverse diameter of the appendix is attained by the age of 4 years. It progressively narrows with age with increasing fibrosis after 40 years. The three taeni coli of the proximal colon converge at the base of the appendix. The anterior taenia colon is commonly used as a landmark to identify the base of the appendix. In the neonate, the characteristic haustration of the large bowel are absent appearing within the first 6months and the taenia coli are thin (Standring et al., 2005). The appendicular wall consists of four main layers: mucosa, sub-mucosa, muscularis propria and the serosa. The mucosa is similar to the colonic mucosa and consists of the epithelial lining, the lamina propria and the muscularis mucosa. The epithelial lining is a single layer of surface epithelial cells including columnar cells with basally located nuclei, goblet cells, apical mucin and absorptive cells as well as scattered paneth and endocrine cells. The lamina propria contains crypts of Lieberkühn. The muscularis mucosa of the appendix is poorly developed unlike the rest of the gastrointestinal tract. The sub-mucosa contains a rich network of arterioles, venules, capillaries and lymphatics in a connective tissue framework. It also contains a plexus of nerves, the Meissner’s plexus. The neurosecretory cells in the submucosa are few till the age of 9years. The age-related increase in the number of these cells is thought to explain the increase in number of carcinoid tumours in older patients. The muscularis propria contains muscles which are arranged in a similar pattern as those of the small intestine. The outer longitudinal muscle fibres aggregate into the taenia coli to become continuous with them at the base of the appendix. The inner circular muscles are thicker. Between these muscle layers is the myenteric or Auerbach’s plexus of nerves which is morphologically similar to the Meissner’s plexus in the submucosa, unlike the rest of the gastrointestinal tract where the Meissner’s plexus is thinner. 3.4 Lymphatics The appendix belongs to the group of lymphatic organs called the Mucosa Associated Lymphatic Tissue which also includes the intestinal Peyer’s patches, the tonsils and the www.intechopen.com 136 Appendicitis – A Collection of Essays from Around the World lymphoid follicles in the walls of the bronchi. They are thought to protect the gastrointestinal tract and the respiratory tract from recurrent infections from foreign matter and organisms entering these body cavities (Snell, 2004b). However, its role in immune protection in the gastrointestinal tract is unclear. The submucosa of the appendix contains prominent lymphoid tissue similar to that in the terminal ileum; this feature differentiates the appendix from the colon. These may become hypertrophic in the presence of inflammation and may obstruct the lumen in acute appendicitis. Lymphoid hyperplasia is at its peak during the second decade of life. This has been postulated to be the reason behind the high incidence of appendicitis in this age group. Lymphoid hyperplasia is thought to be responsible for 60% of acute appendicitis and occurs mainly in children. The appendicular lymphatic vessels drain into the lymph nodes in the mesoappendix, the anterior ileocolic lymph nodes which often become enlarged during acute appendicitis and then into the right para-aortic lymph nodes. 3.5 Vasculature The appendicular artery arises from the inferior branch of the ileocaecal artery and the vein drains through the ileocaecal vein into the portal venous system. The meso-appendix connects the appendix to the ileal mesentry. The artery enters the mesoappendix a short distance from the appendicular base where it gives off the recurrent branch which anastomosis with a branch of the posterior caecal artery. It is common to find accessory arteries associated with the appendix (Standring et al., 2005). These must be handled carefully to limit blood loss during appendicectomy. The appendicular artery runs through the meso-appendix along its free edge and lies on the appendix wall in its distal aspect. The anastomosis at the base gives rise to a good blood supply but it is an end artery from the midpoint to distal appendix where its close proximity to the appendix makes it susceptible to thrombosis as the appendix enlarges during acute inflammation. 4. Aetiology of appendicitis The aetiology is multi-factorial and may involve interplay of factors including obstruction, infections, ischaemia and hereditary factors. Obstruction from lymphoid hyperplasia is a common causal factor and this has been addressed in detail elsewhere in this chapter. A faecolith is a small stone-like mass of stool. Its formation starts with entrapment of vegetable fibre. Like the colonic mucosa, the appendix mucosa is well equipped for water absorption resulting in concentration of its contents with mucous entrapment. Several layers of deposits eventually result in increase in diameter and a faecolith diameter of 1cm leads to appendicular obstruction. Faecoliths are less common in children than in adults; 7.7% versus 42% (Gillick et al., 2001). A primary neoplasm of the appendix is found in 0.5-1.0% of specimens removed for appendicitis. The neoplasm could be mucinous adenoma, mucinous adenocarcinoma, colonic type adenocarcinoma, non-Hodgkins lymphoma, classical carcinoid tumour, or goblet cell carcinoid tumour. 30-50% of patients with carcinoid present with acute appendicitis, being associated with obstruction of the appendix in 25% of cases. An appendicular diameter greater than 15mm should raise suspicion as to the presence of an appendicular tumour (Pickhardt et al., 2002). Carcinoid tumours mostly are located in the distal tip of the appendix, taking the form of a bulbous solid tumour of about 2-3cm diameter. In children it is usually of a diameter of less than 2cm. 75% is at the tip; 20% mid- www.intechopen.com Appendicitis in Children 137 appendix and 5% at the base. The incidence of carcinoid tumours in surgical specimens is about 0.08-0.7%; 0.2-0.5% in children. It is the most frequent tumour of the gastrointestinal tract in childhood and adolescence. It occurs more in white females. A mucocele is a dilated appendix filled with mucinous substance. It may present as an obstructed appendix containing insipissated mucin or be a consequence of mucinous cystadenoma or mucinous cystadenocarcinoma. Bacterial and fungal infections can also lead to appendicitis. The bacteria involved are usually of a mixed aerobic and anaerobic population; most commonly Bacteroides fragilis and Escherichia coli. Others include Streptococcus milleri (associated with a seven-fold increased risk of abscess formation) and Campylobacter jejuni (Feneglio-Preser et al., 2008). Infections may further lead to fibrin thrombi which can block the small appendicular vessels leading to secondary ischaemia. The appendix is particularly prone to ischaemic insult because the appendicular artery is an end artery beyond the base of the appendix. Torsion of the appendix may occur resulting in ischaemic appendicitis; but, this condition is rare (Fenoglio-Preiser et al., 2008). Familial aggregation of appendicitis suggests polygenic inheritance and the appendicitis usually manifests before the age of 10years. The hypothesis of appendicitis being associated with low fibre diet is weakened by the finding in Africa that populations on high fibre diet did not have a lower appendicitis rate (Naaeder & Archampong, 1998). 5. Pathophysiology The human appendix secretes up to 2ml of clear fluid containing mucin, amylase and proteolytic enzymes, which may be produced by bacteria each day. The appendicular aperture is guarded by semilunar mucosal folds which give it a valve effect. The basal intraluminal caecal pressure is approximately 5cm of water while the appendicular intraluminal pressure ranges from 15 – 25cm of water creating a pressure gradient of about 10cm of water. This is believed to keep gut contents from entering the appendicular lumen. Experimental studies have shown that the obstruction of exteriorised human appendices can raise the intra-luminal pressures to an extent that exceeds the perfusion pressure in the vascular plexus within the wall of the appendix. The distal end of the appendix is most vulnerable to this reduction in blood flow. Electrical stimulation of the appendix has been demonstrated to cause closure of the ileocaecal valve (Williams and Myers, 1994). This may be a contributing factor to the nausea and vomiting associated with acute appendicitis. The peritoneum consists of a continuous visceral and parietal layer. Both layers are of mesodermal origin, but develop separately with independent nerve supplies. The visceral layer covers the intra-abdominal organs and is supplied by autonomic nerves. The parietal peritoneum lines the under surface of the abdominal wall and is supplied by somatic nerves. Pathways for pain differ in each layer and so also the quality of pain. Visceral pain has a dull aching character, often crampy and may be associated with nausea and sweating. Parietal pain on the other hand is mostly sharp, severe and persistent in nature. Visceral organs have limited response to pain stimulus but the stretching of the mesentry and irritation of the parietal peritoneum produces severe pain. Visceral afferent fibres carrying sensation of distension and pressure are responsible for the initial pain of appendicitis, poorly localised initially and referred to the periumbilical region. Afferent nerve fibres from viscera enter the dorsal horn of the spinal cord along with afferent nerve fibres from cutaneous structures of the corresponding dermatome. These two groups of nerve fibres overlap at the synaptic junctions in the dorsal horn leading to the www.intechopen.com 138 Appendicitis – A Collection of Essays from Around the World phenomenon of referred pain whereby pain is perceived by the brain as arising from the corresponding cutaneous structures. Nerve fibres decussate and travel up to the thalamus along the lateral spinothalamic tract and then onwards to the cerebral cortex. Increased intravisceral pressure by stretch, distension or contraction of the viscus especially against an obstruction leads to visceral pain. The dermatomal distribution associated with the midgut relates to the umbilical region, with nerves entering the spinal column at the tenth thoracic spinal segment (T10). The midgut extends from the second part of the duodenum to the midpoint of the transverse colon. Therefore, pain arising from the midgut is felt initially in the umbilicus before the parietal peritoneum becomes involved (Klish, 2006). In 1886, the American pathologist - Reginald Fitz became the first person to describe the pattern of the pathophysiological basis of appendicitis in literature. He noted that the condition started with onset of inflammation, followed by perforation, abscess formation and peritonitis (Morrow & Newman, 2005). Appendicitis is commonly secondary to luminal obstruction which is demonstrable in 50-80% of cases (Turner, 2010). As stated previously, the commonest cause of luminal obstruction in children is lymphoid hyperplasia or hypertrophy which mostly results from dehydration and viral infection. Faecoliths take several years to form. They are commoner in older children and may cause direct focal or diffuse mucosal ulceration. The stasis that results creates an environment which favours bacterial proliferation and also causes ischaemic injury. The fore-going results in inflammatory changes including oedema, neutrophilic infiltration of the lumen, muscular wall and periappendicular soft tissue. In early appendicitis, subserosal vessels become congested and perivascular neutrophilic infiltrate develops within all the layers of the wall leading to loss of lustre which gives the appendix a dull granular erythematous appearance. Therefore, the histological diagnosis of acute appendicitis must demonstrate neutrophilic infiltration of the muscularis propria not just within the lumen (Turner, 2010). Figure 2 illustrates the sequence of events that follow appendicular obstruction. Luminal obstruction  Increased mucous production and bacterial overgrowth │ ↓ Dilatation/swelling of the appendix │ ↓ Impaired venous drainage  worsening congestion and oedema Impaired lymphatic drainage Increasing oedema and turgidity Loss of the appendicular lustre │ ↓ Further appendicular swelling Reduction in arterial blood flow Thrombosis of appendicular vessels Necrosis of the appendicular wall. Fig. 2. Sequence of events that follow appendicular obstruction www.intechopen.com Appendicitis in Children 139 More severe inflammation results in prominent neutrophilic exudate which generates serosal fibrino-purulent reaction that gives the appendix the creamy yellow appearance associated with this stage of the inflammatory process. If the inflammatory process is not curbed at this stage, it progresses to formation of focal abscesses within the appendicular wall; this is acute suppurative appendicitis. Progressive increase in the intraluminal pressure leads to venous flow compromise. Laplace law suggests that the wall tension of a tubular structure is directly proportionate to the thickness of the wall divided by the square of the radius. Further increase in the wall tension culminates in necrosis of the appendix. Further inflammation leads to the formation of large areas of haemorrhagic ulceration with gangrenous necrosis that extends to the serosal layer; this is acute gangrenous appendicitis. Rupture of the appendix follows with suppurative peritonitis (Turner, 2010). The risk of perforation of the appendix rises with the duration of symptoms being about 30% for <24hours and greater than 70% in >48hours (Swain, 2005). The perforation rate also varies with the age of the child. The average rate is 30-45% which may be as high as 80% in those under 5years and nearly 100% in those under 2years (Morrow & Newman, 2005; Stevenson, 2003). The normally glistening serosal and peritoneal surface becomes dull and lustreless; serous or slightly turbid fluid begins to accumulate within 2-4hours of the onset of inflammation. With progression of the inflammatory process creamy suppurative material with increasing viscosity accumulates. At this point, the process can take the form of localisation by the omentum and viscera to be controlled in a small area of the abdominal cavity, or become widespread filling the entire abdomen. The cellular response results in the formation of dense collections of neutrophils and fibrinopurulent debris that coat the viscera and abdominal wall at the site of the inflammation (Turner, 2010). The greater omentum is smaller in children and only at the level of the umbilicus in the neonate containing small amount of fat and therefore providing limited omental protection (Standring et al., 2005). Inflammation of the peritoneum and surrounding intra-abdominal organs follows with peritonitis. Bowel obstruction may result from the adhesive inflammatory process. Irritation of the rectosigmoid may lead to enteritis manifesting with frequent loose stools. Irritation of the bladder by the inflammatory process may cause dysuria, increased frequency of micturition and urgency simulating urinary tract infection. Severe inflammation may lead to haemorrhagic cystitis. The inflammatory process may also be accompanied by increased tissue porosity or permeability with bacterial translocation. Peritonitis from bacterial translocation across the porous inflamed wall of the appendix may still occur in the absence of obvious perforation or faecal contamination. 6. Histopathological features In the acute phase, serosal injection leads to loss of the normal appendiceal lustre; if inflammation progresses purulent exudate forms on the surface of the appendix followed by perforation. There may be fecolith within the appendix lumen; or the lumen may be distended with pus or mucous. Enterobius vermicularis may be present in the lumen and sometimes within the mucosa where they may induce a granulomatous reaction. They can be identified on microscopy by their lateral spines evident on the cross section of the transected worms (Sebire et al, 2010). www.intechopen.com 140 Appendicitis – A Collection of Essays from Around the World Histological features of acute appendicitis include  Acute transmural inflammatory aggregation of neutrophils and eosinophils  Hyperplasia of mucosal lymphoid tissue  Haemorrhagic changes in the mucosa  Pus within the appendicular lumen  Mucosal ulceration  Acute serosal inflammation  Haemorrhagic necrosis of the appendicular wall  Adenovirus inclusions may be seen in the epithelial cell nuclei  There may be vasculitic changes with or without thrombi within the vessels in the wall  Following interval appendicectomy, there may be chronic inflammatory changes with fibrosis of the wall with or without occlusion of the appendicular lumen (Sebire et al, 2010). Inflammation without mucosal ulceration is of uncertain significance. In acute intraluminal appendicitis, there is increased neutrophil presence in the appendicular lumen with no evidence of mucosal infiltration. Similar findings have been documented in incidentally removed appendix specimens (Feneglio-Preser et al., 2008). The issue of sending normal appearing appendix for histological analysis is supported by the fact that certain conditions may present in the appendix with macroscopically normal appearance. These include polyarteritis nodosa, tuberculosis, amoebiasis, parasitic infestations including bilharzisis, schistosomiasis, trichuriasis, ascariasis and clonorchiasis, actinomycosis as well as epithelial tumours. 2 – 5% of macroscopically normal appendices may have significant unsuspected pathological condition (Williams & Myers, 1994). Furthermore, neurogenic appendicopathy may appear macroscopically normal and can only be diagnosed with certainty on histological analysis (Zaupa et al., 2011). 7. Microbiological perspective Peri-appendicular abscesses may occur from bacteria usually present in the bowel including Escherichia coli, Proteus species, other enterobacteriaceae, Bacteroides species, anaerobic cocci and other anaerobes. Infections are therefore commonly polymicrobial. The resultant secondary peritonitis commonly yields Escherichia coli and other enterobacteriaceae and anaerobes from intra-operative peritoneal pus swabs. Some authors argue that the precise value of peritoneal swabs in many cases of secondary peritonitis is difficult to assess because the bacteriology seldom influences antibiotic treatment which is given empirically on clinical grounds for short duration, often ending within 2-3days before the full bacteriology results become available (Baker et al, 2004). The gastrointestinal tract like other portals of entry into the human body, has a normal flora that helps protect it against pathogenic micro-organisms. The flow rate reduces from the small intestine to the large intestine giving the bacteria more time to colonise and reproduce leading to higher concentration of the organisms. The amount of flora increases in number and varies in type as the gastrointestinal tract progresses from the oral cavity to the anorectum. About one thousand species of micro-organisms are present in the large intestine. Approximately 20% of the volume of faecal matter in the healthy person consists of bacteria, most of which come from the colon. The terminal ileum flora is similar to colonic flora. More than 90% of these are anaerobes, mostly Bacteroides, Fusobacterium, Eubacterium and Clostridium. Others include E. coli, enterococci, yeasts and numerous www.intechopen.com Appendicitis in Children 141 others (VanMeter et al, 2010). Organisms commonly isolated from peritoneal microbiological tests in secondary peritonitis are mostly anaerobes which outnumber aerobes in the bowel by a thousand fold (Forbes et al, 2007). Enterobius vermicularis is the most common nematode infection in humans and can be found in up to 3% of appendices in the USA. Schistosomiasis of the appendix is rare. Strongiloides stercoralis infection results in eosinophilic appendicitis. Viral appendicitis has been associated with measles in the prodromal phase. Other viruses that may cause appendicitis are adenovirus and gastrointestinal cytomegalovirus infection. Acute infectious mononucleosis and Epstein Barr virus infection rarely may give rise to abdominal pain (Petras & Goldblum, 1996). 8. Diagnosis 8.1 Clinical presentation Making a diagnosis of acute appendicitis in children can be a difficult task even for the experienced paediatric surgeon. Negative appendicectomy rate was found to be higher among children operated upon in district general hospital than in a specialist paediatric centre, 20% versus 4% (Whisker et al., 2009). Chang et al., (2010) found that approximately 12-15% of paediatric appendicitis were missed at the first visit to the emergency department with the rate of perforation of 73.1% versus 49% in those diagnosed at first presentation. The duration of symptoms was longer in the former group and the rate of perforation higher the longer the duration of symptoms. Generally, clinical symptoms are the patient’s report of the manifestation(s) of dysfunction in the normal body physiology. Thus, the younger the child, the less accurate the report of symptoms can be expected to be. Neurodevelopmental immaturity precludes accurate understanding, interpretation and description of symptoms by children particularly those younger than eight years of age. Not surprisingly, this is the age group that commonly presents late with advanced appendicitis. Furthermore, parents of infants often ascribe febrile illness and vomiting to “teething” and do not seek medical evaluation early. The clinical symptoms of appendicitis are often secondary to luminal obstruction leading to colicky abdominal pain at onset which progresses to constant pain with progression of the inflammatory process. Nausea and vomiting are commonly present. Clinical signs are discussed in detail later in this chapter and often include tenderness in the right lower quadrant of the abdomen. Advanced appendicitis is often associated with delayed presentation especially in children below the age of five years and also with retroileal, retrocolic or pelvic appendicitis. Irritation of pelvic structures may produce symptoms and signs suggestive of urinary tract infection or enteritis. 8.2 History Rigorous pursuit of a detailed history is invaluable in the diagnosis of appendicitis. In children, patience is an indispensable virtue and a rushed history increases the risk of misdiagnosis. Possession of the clinical skills required for eliciting appropriately focused and chronologically accurate history from the child and parent is key to early diagnosis. The surgeon therefore has to make the most of open-ended and direct questioning at appropriate key moments of the history taking applying sensitivity to the emotional climate. www.intechopen.com 142 Appendicitis – A Collection of Essays from Around the World Background information of the child’s usual status of health should be obtained. The onset of the current symptoms should be carefully ascertained. Site of onset of abdominal pain and its present location may suggest migration of pain which may be associated with acute appendicitis; usually starting in the peri-umbilical region, the pain later localises to the region of the right iliac fossa. In addition it is often preceded by nausea or vomiting. Characterising the abdominal pain is key to accurate diagnosis of its source. The onset of the pain associated with acute appendicitis is often gradual, progressively worsening. It may be intermittent initially then sharp and constant within a few hours. Children may not give this typical presentation; even the older ones may become very quiet and distracted by other issues including pain, fear, strange environment with unfamiliar people or even psychosocial circumstances in the family. The usual duration of symptoms at presentation is 24 – 36hours. There may be a history of pain being made worse by road bumps on the way to the hospital. This suggests the presence of rebound tenderness. Enquiry into the presence of associated factors should be made. Nausea and vomiting may be present in up to 90% of patients. Diminished appetite may be absent in children with appendicitis. Diarrhoea may suggest irritation of the anorectum by inflamed tissue in the rectovesical pouch. The sigmoid colon is often redundant in children, with a tendency to loop into the pelvis. Consequently, it may come in contact with an inflamed appendix manifesting as diarrhoea. Care must be taken not to mistake this for gastroenteritis. Dysuria may be associated with appendicitis from irritation of the urinary bladder by an inflamed pelvic appendix. The history should also explore other possible causes of abdominal pain. Symptoms of upper respiratory tract infection may suggest mesenteric adenitis. Cough may suggest pneumonia with referred abdominal pain as a diagnosis. Vulvovaginal irritation with or without vaginal discharge may suggest pelvic inflammatory disease. Abdominal pain may also be referred from an acute scrotal condition and older boys in particular do not offer this important information without direct questioning. Constipation may produce symptoms in children that may imply the presence of pathology and should be considered. In addition, enquiry should be made about any previous history of abdominal pain, previous abdominal surgery, recent foreign travel, current or recent medications as well as the presence of similar condition in other family members or pupils in the same school. 8.3 Differential diagnosis of acute abdominal pain in children The cause of acute abdomen in children may vary according to sex and age. Possible causes are presented below. The list is by no means exhaustive and not in order of frequency. In addition, some conditions may co-exist. 8.3.1 Infants Viral enteritis Intussusception Pyelonephritis/ other urinary tract infection (UTI) Gastro-oesophageal reflux Bacterial enterocolitis Chest infection Appendicitis Pyloric stenosis www.intechopen.com Appendicitis in Children Strangulated hernia of the anterior abdominal wall Testicular torsion Mesenteric cysts Ruptured abdominal tumour Pancreatitis Meckel’s diverticulitis Hirschsprung’s disease with or without enterocolitis Poisoning Trauma Non-accidental injury 8.3.2 Children aged between 2-10years old Meckel’s diverticulitis Cystitis Pyelonephritis Viral enteritis Bacterial enterocolitis Appendicitis Non-specific abdominal pain Crohn’s disease Abdominal trauma, including non-accidental injury Chest infection Mesenteric adenitis Neutropenic enterocolitis Pancreatitis Ruptured intra-abdominal tumours Poisoning 8.3.3 Children above 11years old Viral enteritis Bacterial enterocolitis Appendicitis Non-specific abdominal pain Mesenteric adenitis Pelvic inflammatory disease Tubo-ovarian cysts Tubo-ovarian abscess with or without rupture Torsion of an ovarian cyst Haemorrhage in an ovarian cyst Endometriosis Mittleschmerz Crohn’s disease Pancreatitis Neutropenic enterocolitis Chest infection Haematocolpos www.intechopen.com 143 144 Appendicitis – A Collection of Essays from Around the World Peptic ulcer disease Psychosomatic condition Trauma Ectopic pregnany Dysmenorrhoea Gall stone disease including cholecystitis, biliary colic, cholangitis Urinary tract infections Neuronal abdominal wall pain including shingles, spinal or nerve root problem, iatrogenic peripheral nerve injury Spontaneous rectus sheath haematoma 8.4 Physical examination A thorough physical examination would compliment the clinical suspicion formed from the reported symptoms. A general examination of the child with abdominal pain is imperative and requires experience in identifying the sick child. The child’s appearance should be noted – body habitus, facial expression, position, willingness or reluctance to move, alertness, pallor and whether the child is flushed or sweaty. Assess the child’s pulse for volume and rate. The temperature in early appendicitis may be normal or mildly raised. A temperature above 38◦C should prompt further investigation or evaluation to exclude other causes. Ears, throat and lymph nodes should be evaluated. Tachypnoea, recessions, shallow breathing and flaring of the alar nasi may suggest a respiratory tract problem or be secondary to circulatory system contraction. An antalgic gait, leaning to right side, limping on the right leg and slow motion are all cues to the presence of abdominal pathology. Flexion at the hip suggests abdominal wall discomfort with or without peritoneal irritation. Younger children typically poorly localise pain. Most of the under five-year-olds point to their umbilical region as the site of all pain; perhaps because the umbilicus is a central feature with a unique appearance that sets it out as a point of focus which captures the child’s attention. The demonstration of certain clinical signs may further qualify the pain but atypical abdominal pain is seen in about 40 – 45% of patients. One should beware of the child who is on antibiotic therapy for other presumed infection who presents with attenuated features of appendicitis. The child’s anxiety should be taken into consideration and reducing the number of people in the room or creating a distraction may help. Distraction may be accomplished with the help of a paediatric play therapist. Building a rapport with the parents gains the child’s trust and allays anxiety. A warm child-friendly environment is desirable and is common practice in many paediatric specialist centres. Focused examination of the abdomen should commence with inspection for distension, abdominal wall excursions with respiratory activity, hernia orifices, external signs of trauma, scars or visible peristalsis. Percussion of the abdomen may reveal the presence of rebound tenderness suggesting peritonism. Palpation of the abdomen should in the first instance be superficial and general, starting away from the site of pain. This gives the examiner the opportunity to explore all the quadrants of the abdomen and improves the chance of identifying non-appendicular pathology. This gentle approach reassures the child and allows him or her to trust the examiner, and also to relax the abdominal wall. Guarding may be present as well as tenderness. Depending on the child’s level of development and co-operation, he or she may be encouraged to cough, laugh, distend the abdomen or retract the abdominal wall. Rebound tenderness may be present if www.intechopen.com Appendicitis in Children 145 these activities elicit pain. This is followed by deep palpation to explore the presence of an intra–abdominal mass. The character of any palpable mass should be evaluated – soft, firm, mobile or fixed to surrounding structures, regularity of its palpable surface and possible organ of origin. Due to the variability of position of the appendicular tip as previously discussed, the parietal pain may be related to the right upper quadrant, right loin or pelvis. The practice of gently rocking the pelvis from side to side is still practised by some surgeons and may elicit rebound tenderness. Auscultation should evaluate bowel sounds but is generally not very useful. Bowel sounds may be absent or diminished in advanced inflammation. However, the presence of normal bowel sounds does not exclude advanced appendicitis. For the tense anxious child, using the stethoscope as a palpation tool can help with the evaluation of the abdomen. Also palpating over the child’s hand can play the same role. An auscultation of the chest is part of the evaluation for probable appendicitis to rule out or confirm chest pathology. In the presence of positive chest signs, the abdomen should still be carefully evaluated for the presence of possible co-existing intra-abdominal pathology. Children are not good at responding to the question – “Does this hurt?” The young child is very likely to respond in the affirmative when asked such questions. Conversely, beware of the older child who denies any pain for fear of being admitted into the hospital. The child with acute appendicitis would often be reluctant to move and may express discomfort by facial grimace or tears rather than verbally. Therefore careful observation of the child’s facial expression and non-verbal responses is paramount to the interpretation of clinical signs. Right lower quadrant pain, tenderness and rebound tenderness should be elicited. The traditional method of eliciting rebound tenderness by suddenly withdrawing the hand following a deep palpation, is not advisable in children. It results in sudden severe pain which may make the child loose confidence in the doctor. Rather, rebound tenderness is usually tested for by asking the child to increase the intra-abdominal pressure by coughing (Dunphy sign). This brings the inflamed appendix or surrounding tissues to the anterior abdominal wall manifesting as rebound tenderness. Similarly, the abdominal pain may also be exaggerated by attempting to move the abdominal wall outwards – “blowing out the abdomen” or moving the abdominal wall inwards – “sucking the abdomen inwards”. McBurney’s sign is the presence of maximum tenderness over the McBurney’s point. This was first described by McBurney who was the first to recommend appendicectomy for treatment of appendicitis (Morrow, 2005). Rovsing sign is positive if there is perception of pain in the right lower quadrant on palpation of the left lower quadrant. Obturator sign may be positive. To elicit this sign, the patient lies supine with the right knee and hip flexed to 90degrees. The examiner, holding the patient’s right ankle in the right hand, places the left hand on the knee. Outward rotation of the flexed right knee causes internal rotation of the right hip which causes the obturator internus to become tense. The test is positive if pain in the right lower quadrant is elicited; usually in appendicitis in the pelvic or obturator positions where the appendicular tip lies over the obturator fascia covering the obturator internus muscle. The iliopsoas comprising of the powerful hip flexors – iliacus and psoas major, can become inflamed in appendicitis which is retrocaecal and therefore retroperitoneal giving a positive psoas or iliopsoas sign. This can be evaluated by two approaches. With the patient lying supine, the examiner’s hand is placed just above the right knee and the patient asked to flex the right hip against resistance. Eliciting pain means positive psoas sign. An alternative method is to have the patient lie on the left side, if hyper-extension on www.intechopen.com 146 Appendicitis – A Collection of Essays from Around the World the right hip elicits pain, the sign is positive. A psoas abscess from a different cause would elicit similar pain (Liu & McFadden, 2003). The introduction of the algometry for the diagnosis of acute abdominal pain in children has been welcomed by many paediatric surgeons (Vajcner et al., 2011). This device is used to predict acute appendicitis by observing the abdominal tenderness threshold which is the minimum pressure applied to the anterior abdominal wall to produce discomfort. With regards to diagnosing acute appendicitis, when combined with other clinical findings, it was found to have a sensitivity of 82% specificity of 73% and positive likelihood ratio of 3.03. This new innovation may become popular in the future but it needs evaluation through appropriately designed clinical trials. 9. Investigations In cases where the clinical history or physical signs are atypical and inconclusive for the diagnosis of acute appendicitis, various investigations may be used to complement the clinical findings, strengthen the diagnosis and exclude the presence of alternative pathology. They may also aid the peri-operatively management of the patient. These may be bed-side, laboratory, radiological or laparoscopic investigations. There is no one investigation that can accurately diagnose appendicitis every time. The clinical value and economic benefit of laboratory investigations for the diagnosis of appendicitis has been the cause of much debate (Liu and McFadden, 2003). The general rule to the selection of an investigation is that it would:  Complement the history and examination  Determine what other clinical tests may be required  Alter treatment approach. An ideal diagnostic test should offer the following benefits:  High level of accuracy: high sensitivity and specificity  Capable of assessing the extent of disease  Cost effective: cost of investigation should be less than the consequences of treatment without the benefit of the information derived from the examination  Short length of study  Quick and easy access to result or diagnostic information  Non-invasive  Suited to local needs, resources and available expertise (Hernanz-Schulman, 2010) 9.1 Bedside investigations Bedside investigations can be done alongside the initial evaluation. Urinalysis with urine dipstix may suggest urinary tract irritation or infection, diabetes or pregnancy-related conditions. A bedside blood sugar test is a quick check for possible diabetic ketoacidosis. 9.2 Laboratory investigations Laboratory tests commonly used to evaluate acute abdominal pain include full blood count, electrolyte studies, C-reactive protein (CRP), urine microscopy and culture, liver function tests and serum amylase level. Approximately two-thirds of the patients would have elevated white blood cell (WBC) count with neutrophilia but this is not specific to www.intechopen.com Appendicitis in Children 147 appendicitis. The relative neutrophil count may be above normal range even in the presence of a normal total white blood count. Serum levels of inflammatory markers may not be raised in early appendicitis. Repeating the investigations at least 6hours after the initial test may increase the diagnostic yield (Wu & Fu, 2010). Laboratory urine microscopy would assess for presence of pyuria and micro-organisms. Urinalysis may be abnormal in up to 48% of patients with acute appendicitis. This may show microscopic haematuria, pyuria or proteinuria (Rothrock & Pagane, 2000). Pyuria may arise as a consequence of irritation of the urinary bladder or the ureter by the inflamed appendix or surrounding inflamed tissue. Serum or urine βhCG tests should be performed in young women of child-bearing age and if positive, an ectopic pregnancy should be excluded by further evaluation involving the gynaecologist. Moreover, pregnancy and appendicitis can co-exist. Serum electrolytes and creatinine levels should be requested and any abnormalities corrected appropriately. Blood glucose should be obtained and any abnormality appropriately managed. It must be borne in mind that diabetic ketoacidosis may present as acute abdomen. The CRP is an acute phase reactant synthesized in the liver which is often raised within 12hours of an acute inflammatory process. It may be raised in 50-90% of patients with acute appendicitis but again it is non-specific. Serum levels of inflammatory markers including CRP and WBC count cannot be reliably used to distinguish between acute appendicitis and other causes of abdominal pain (Dalal et al., 2005). They are more effective in supporting a clinical diagnosis of appendicitis than excluding the diagnosis (Birchley, 2006). 9.3 Radiological investigations Radiological investigations should be tailored to the specific presentation and possible differential diagnoses. A chest radiograph may be useful in the presence of clinical suspicion of lower respiratory tract infection or complications there from. 9.3.1 Plain abdominal radiograph Plain abdominal radiographs are not commonly used in the evaluation of abdominal pain in children particularly when appendicitis is felt to be a likely cause. There are several reasons for this stance, one being that children present with abdominal pain commonly and obtaining an abdominal radiograph each time may lead to a significant amount of radiation. Extremes of age are more sensitive to radiation and it should be avoided as much as possible. The average plain abdominal radiograph exposes the patient to a typical effective radiation dose of 0.7millisieverts (mSv), equivalent to 4months of natural background radiation which is equal to 35 chest radiographs (Hampson and Shaw, 2010). It is of limited use in the evaluation of abdominal pain in children but it may be useful in atypical presentation where no obvious diagnosis can be made after adequate history, examination and laboratory investigations. It is noteworthy that only 10% of patients with an acute abdomen have abnormalities on plain radiographs. A study in the adult population demonstrated that the specificity of abdominal radiograph for acute appendicitis can be as low as 0% (Hampson & Shaw 2010). An adequate abdominal radiograph should include the diaphragm and pelvis; anteroposterior and lateral shoot through views may be required if the patient is unable to sit up. The preperitoneal fat often gives rise to a fine line of fat on a plain abdominal radiograph. Inflammation of a retrocaecal appendix may be associated with infiltration of the preperitoneal fat and lead to a focal absence of this fine line of fat. In addition, a mass www.intechopen.com 148 Appendicitis – A Collection of Essays from Around the World between the preperitoneal fat and ascending colon, gas in the appendix lumen, a faecolith above the anterior superior iliac spine combined with haustral irregularity of the ascending colon can raise the suspicion of appendicitis on plain abdominal radiograph. As stated earlier faecoliths are uncommon in children. Retrocaecal extraperitoneal gas suggests perforation. Extraluminal gas on radiograph from a perforated appendicitis may be demonstrable in 1% of perforated cases. Loss of shadow of the right psoas suggests advanced appendicitis with retroperitoneal inflammation. An abdominal radiograph may also demonstrate dilated loops of bowel suggesting obstruction or extraluminal gas in perforation of abdominal viscus. Bowel obstruction in the absence of features of peritonism may be secondary to adhesive obstruction. It has a significant role in the evaluation of the neonate with suspected intra-abdominal pathology where it may demonstrate radiological features of necrotising enterocolitis as clinical signs would not conclusively demonstrate perforations. In addition, it may demonstrate the renal outline with a huge outline being suggestive of obstructive uropathy. B A Fig. 3. Plain abdominal radiograph of a 2year old showing: A. Faecolith; B. Focal absence of fine line of preperitoneal fat (uninterrupted on the left side). Note also, the absence of bowel gas in the same region. www.intechopen.com Appendicitis in Children 149 9.3.2 Ultrasonography Where clinical observation by an experienced paediatric surgeon over a 48hr period still reveals equivocal diagnosis and suspicion of appendicitis persists, imaging is recommended, mainly by way of abdominal ultrasonography (Lander, 2007). Ultrasonography for the evaluation of appendicitis was introduced by Puylaert in 1986. It is a useful investigation in the further evaluation of abdominal pain with atypical and inconclusive findings. Some authors suggest that its specificity and sensitivity may be higher in children than in adults (Rothrock & Pagane, 2000). This is particularly relevant to peripubertal and older girls where ovarian pathology may mimick appendicitis. Even a left pedunculated ovarian cyst could present with right-sided symptoms if it assumes a right lower quadrant position. Abdominal ultrasonography can usually be performed without any sedation and the sonographer can communicate with the child and ask where the pain or tenderness is maximal. However, this may be distracting in children who localise pain poorly. The closeness is reassuring to the child and also allows the sonographer to observe the child’s facial expression or reaction to contact with the examination probe. Appropriate application of the probe relies heavily on co-operation from the patient and the graded compression can be limited by the presence of guarding. In addition, ultrasound is operator dependent and has reduced efficacy in obese patients. It can achieve up to 98.5% sensitivity, 98.2% specificity, 98.0% positive predictive value and 98.7% negative predictive value in experienced hands (Strouse, 2010). A repeat ultrasound in case of persisting clinical borderline suspicion may increase diagnostic yield (Schuh et al., 2011). Ultrasonographic features suggestive of appendicitis include: 1. Rigid non-compressible appendix 2. Tenderness on attempted compression 3. Non-peristalsing appendix 4. Appendicular wall thickness of > 6mm 5. Distension of the appendicular lumen 6. Presence of abscess in the peri-appendicular region 7. Increased amount of intraperitoneal fluid 8. Inflammatory changes in surrounding tissues 9. Discontinuity of the appendicular wall 10. Extruded faecolith 11. Thickening of ileum or caecum which may represent part of the inflammatory mass around the inflamed appendix but may also suggest a diagnosis of Crohn’s disease. 9.3.3 Computed Tomography (CT) CT has been demonstrated to be more effective than ultrasonography in the diagnosis of appendicitis and evaluation of abdominal pathology in general. The radiation load from an abdominal CT remains a hindrance to its widespread application in children. The typical effective radiation dose from a CT of abdomen/pelvis is 10 mSv (Hampson and Shaw, 2010). For a single abdominal CT study in a 5 year old child, the life time risk of radiation induced malignancy would be 26.1/100 000 in girls and 20.4/100 000 in boys. Reported CT sensitivity is 79-98%, increased with intravenous contrast. Luminal contrast may further improve its sensitivity (Theoni and Thornton, 2007). Kaiser et al., (2002) www.intechopen.com 150 Appendicitis – A Collection of Essays from Around the World demonstrated that compared to graded compression ultrasound in acute childhood appendicitis, CT sensitivity is 97%, with accuracy of 95%, negative predictive value of 92% while ultrasound sensitivity was found to be 80%, accuracy of 89% and negative predictive value of 88%. CT is also preferable in obese patients and those with significant ileus or bowel gas. It was found to lead to a reduction in negative appendicectomy rates in children. The negative appendicectomy rate without imaging was found to be 14%, 17% with ultrasound but reduced to 2% with CT. No difference was observed in perforation rate (Theoni and Thornton, 2007). Lower abdominal CT should be performed with intravenous contrast where possible. Features suggesting appendicitis include (Theoni and Thornton, 2007):  Appendicular diameter of more than 6mm  Presence of inflammatory changes in the peri-appendicular area combined with a dilated or thickened appendix  Inflammatory changes extending to the psoas muscle  A calcified faecolith may be seen  There may be free fluid with or without an enhancing rim suggesting abscess  Thickened caecum and terminal ileum with inflamed appendix  Periappendicular fat stranding  Air in the appendix wall, retroperitoneum or abdomen associated with inflammatory changes in the area around the appendix  Advanced appendicitis may give CT findings of pericaecal phlegmon or abscess  The right lower quadrant may demonstrate free air which suggests perforation. Early appendicitis may not be distinguishable from normal appendix because the features mentioned above would be absent. Consequently, failure to visualise the appendix radiologically does not rule out acute appendicitis. It is noteworthy that air within the appendix lumen may be normal in the absence of other features of periappendicular inflammation and the appendix may not be demonstrable in the presence of focal inflammatory changes of the appendix. Thickening of the wall of the appendix observed on axial images as three concentric rings or as single thick ring of enhancement with or without periappendicular soft tissue stranding may be the only feature present. Disadvantages of CT include the following:  Risk of radiation.  CT costs more to perform  Patients are at risk of allergic reaction to the contrast agent  It takes longer to perform  It may have a lower sensitivity in patients with low body fat (Rothrock & Pagane, 2000). 9.3.4 Radionuclide scanning Radionuclide scanning using 99mTc-hexamethylpropyleneamine oxime (HMPAO) labelling of patient’s leucocyte or technetium-99m-labelled antigranulocyte antibodies can be used to evaluate abdominal pain in children presenting with equivocal clinical and laboratory findings. Accumulation of the radionuclide material in the right lower quadrant of the abdomen indicates positivity for appendicitis. The sensitivity is between 91-94% and specificity is 82-94%. The disadvantages to its use include the issue that it is not universally available, takes long to perform and interpretation of the scan is operator dependent (Sarosi & Turnage, 2002 ). www.intechopen.com Appendicitis in Children 151 9.3.5 Contrast studies A contrast enema is not usually employed in children for the diagnosis of acute appendicitis because it is unpleasant to the child, may require sedation, involves contrast going through probably inflamed bowel and may not contribute much to the evaluation following the use of other radiological investigations. If it is done, it may show failure of the appendix to fill with contrast. However, 10-20% of normal appendixes do not fill during contrast study. False negative result may be caused by distal appendicitis at the tip without proximal obstruction or partial obstruction in early appendicitis. It may demonstrate right colonic or terminal ileal mucosal changes secondary to infective enterocolitis e.g from Yersinia enterocolitica, Salmonella spp. Shigella spp. Campylobacter spp. Bacteroides spp. Escherichia coli, as well as changes due to Crohn’s disease or non-specific inflammatory bowel disease. It may compliment CT and US in equivocal cases, particularly in recurrent abdominal pain. An upper gastrointestinal contrast study may be used to evaluate the rotational status of the midgut in such cases. Contrast studies offer advantages of being 1. Simple 2. Safe 3. Readily available where ultrasound and CT are not available Disadvantages include:  Up to 40% of barium studies may be equivocal where CT and US have been equivocal (Liu and McFadden, 2003)  In the presence of perforation, contrast may extravasate into the peritoneal cavity  It takes time to set up  It may require sedation. 9.3.6 Laparoscopy Up to 59% of patients with right lower quadrant pain may have appendicitis confirmed at laparoscopy for suspected appendicitis. 35% of the females with suspected appendicitis may be found to have gynaecological pathology at laparoscopy (Liu and McFadden, 2003). Laparoscopy also offers the advantages of direct inspection of all the intra-abdominal organs as well as the opportunity to treat the identified pathology where appropriate. 9.4 Clinical scoring systems Several scoring systems have been put forward to facilitate the diagnosis of appendicitis. Unfortunately, paucity of validation studies limits their clinical application. It should be borne in mind however that achieving a maximum score in any of the scoring systems may still lead to a negative appendicectomy. Two of these are discussed. The Paediatric Appendicitis Score (PAS) for the evaluation of children aged between 415years with probable appendicitis is based on scores assigned to the clinical history, presenting signs and laboratory results. A score of ≤5 implies the diagnosis is unlikely to be appendicitis; ≥ 6 is compatible and 7-10 indicates a high probability of appendicitis. PAS has been advocated and shown to reduce the rate of normal appendicectomy to less than 5% giving a mean score of 3.1 ± 1.1 in non-appendicitis cases and 9.1 ± 0.1 in appendicitis (Samuel, 2002). Samuel (2002) also demonstrated that the PAS had a sensitivity of 100%, specificity of 87%, positive predictive value of 90% and negative predictive value of 100%. Table 1 shows the details of the scoring system. www.intechopen.com 152 Appendicitis – A Collection of Essays from Around the World Diagnostic indicator Tenderness with cough or percussion or hopping Anorexia Pyrexia Nausea/ vomiting Tenderness in right lower quadrant Leucocytosis ≥ 10,000 (109/L) Neutrophilia Migration of pain PAS (maximum 10) 2 1 1 1 2 1 1 1 Table 1. Paediatric Appendicitis Score Similarly, the Alvarado score (Table 2) employs clinical and laboratory values in predicting the possibility that the cause of abdominal pain is acute appendicitis. Shreef et al., (2010) in their review of 350 children demonstrated that with an Alvarado score of ≥6, the sensitivity of the scoring system could be as high as 100%, specificity 84.4%, positive predictive value of 83% and accuracy of 91.1%. Diagnostic indicator Tenderness in right iliac fossa Rebound tenderness in right iliac fossa Anorexia Pyrexia >37.3 Nausea/Vomiting Leucocytosis Neutrophilia (>75%) Migration of pain Alvarado score(maximum 10) 2 1 1 1 1 2 1 1 Table 2. Alvarado Score 10. Treatment 10.1 Suspected appendicitis Where a definite diagnosis is not reached following history taking and examination in a child with significant symptoms, admission for observation should be undertaken. The child should be managed according to symptoms with analgesia and rehydration therapy where appropriate. The gastric emptying in children with inflammatory intestinal problems is delayed, therefore, these patients should be kept on clear liquid diet to avoid aggravating the condition and also to minimise the risk of aspiration during induction of anaesthesia should this subsequently becomes necessary. Surana et al., (1995) demonstrated that active observation of children with suspicion of appendicitis was not associated with a significant increase in complication rate; 5.5% vs. 4.2% in those diagnosed at presentation. Moreover, after the inflammation reaches the submucosa, it progresses quickly to involve the rest of the appendix (Fenglio-Preiser et al., 2008). Therefore, hospital admission and active observation is recommended with regular review of the patient at intervals of 4-6hours. www.intechopen.com Appendicitis in Children 153 10.2 Immediate treatment The immediate management of a child with presumed acute appendicitis should include resuscitation, analgesia +/- abdominal decompression with a nasogastric tube. The child’s clinical status should be evaluated to determine the appropriate level of care most suitable for the individual child. Some children would require level 2 intensive care nursing, or higher, before and/or after surgical treatment. Fluid resuscitation should be commenced and the child should be well-hydrated to ensure safe surgery. Broad spectrum antibiotics should be administered once the diagnosis of acute appendicitis has been made and surgery planned. There is evidence that commencing antibiotics at least 4hours before surgery reduces the risk of post-operative wound infection particularly when the duration of symptoms is longer than 48hours (Krukowski et al., 1987; Lander et al., 1992). Using a protocol involving adequate fluid resuscitation and a minimum of two pre-operative doses of antibiotics (Coamoxiclav +/- Gentamicin), Cleeve et al., (2011) demonstrated a complication rate of 6% in children with advanced appendicitis. The choice of antibiotics should cover the micro-organisms expected at the site of infection as described in the microbiology section of this chapter. Commonly, a third or fourth generation cephalosporin is used with or without a penicillin. An aminoglycoside, often Gentamicin, should be added where there are features suggesting advanced appendicitis. In the supine position, the lowermost levels of the peritoneal cavity are the right subphrenic space and the pelvic cavity. In peritonitic patients the rate of absorption of toxins from the intraperitoneal infection can be reduced by keeping them in the 45˚ position to encourage gravitation into the pelvis where the rate of toxin absorption is slow (Snell, 2004). 10.3 Conservative treatment Delayed diagnosis is associated with higher rate of perforation, pelvic abscess, longer duration of hospital stay, delayed return to normal activities and greater risk of adhesive bowel obstruction. Up to 30% of children under 3years of age present with appendix mass with a duration of symptoms usually longer than 4-5days (Stevenson, 2003). In cases with long duration of symptoms, ultrasound should be performed before planning surgery if the clinical status of the abdomen precludes adequate palpation, or if the presence of a mass cannot be excluded. In the presence of a clinically palpable or radiologically identified appendicular mass and absence of gross peritonitis, conservative management with broad spectrum intravenous antibiotics can be safely undertaken. Hoffman et al., (1984) demonstrated that up to 80% of patients successfully managed with antibiotics for an appendix mass required no further treatment, 14% of these presented with recurrent abdominal pain not related to appendicitis; 20% had recurrent appendicitis and 66% of these occurred within 2 years of initial treatment. Swain et al., (2005) also demonstrated that an appendix-related abscess of ≤ 2cm can be successfully treated conservatively. Larger abscesses should be drained whenever this can be safely undertaken either by radiologyguided approach or surgically using laparoscopy or into the rectum. Careful monitoring of physical signs, both local and systemic should be undertaken at regular intervals. The temperature, heart rate, respiratory rate, abdominal tenderness and size of inflammatory mass should be observed. Laboratory investigations should be used to compliment clinical findings. Repeat radiological investigations may also be required. The resolution may take a few days to become evident though generally a definite improvement should be noticed after 48 hours. If the acute appendicitis settles, interval appendicectomy www.intechopen.com 154 Appendicitis – A Collection of Essays from Around the World should be performed within 6 weeks to 3 months. For those who show persistent or worsening clinical signs, early appendicectomy should be undertaken to avoid more serious complications. 10.4 Definitive surgery Complications of appendicitis include pyelophlebitis, portal venous thrombosis, cholangitis, liver abscesses and bacteraemia. Also, fistula formation may result from appendicitis including enteroenteric, enterovaginal, enterocutaneous and enterovesical fistulae. Therefore, in the presence of strong suspicion of appendicitis, it is less of a clinical risk to undertake the removal of a normal appendix than expose the patient to the significant morbidity associated with advanced appendicitis. A negative appendicectomy rate of 5-10% can be expected (Stevenson, 2003). Oyetunji et al., (2011) observed a reduction in the negative appendicectomy rate over the years from 8.1 % in 2000 to 5.2% in 2006, being higher in rural areas, younger children, and girls. Of patients with negative appendicectomy, 12% may have a different surgical condition, 18-20% may have nonsurgical pathology and 60% may have no identifiable pathology. Complication rate for negative appendicectomy may range from 5-15% including wound related problems, pulmonary complications, urinary tract infection and small bowel obstruction (Sarosi & Turnage, 2002). Following induction of anaesthesia, palpation of the abdomen should be undertaken. In the presence of a clearly defined mass which was not identified earlier, further management would involve two main secondary options: to continue with the planned surgery, or, to defer the operation and further evaluate the child with treatment using intravenous antibiotics. The latter view was strongly expressed by Surana and Puri (1995). Gillick et al., (2001) found that children who had a palpable mass under anaesthesia, which was not diagnosed clinically earlier, had a shorter duration of symptoms (mean 2days) than those with clinically palpable or ultrasound diagnosed mass (mean 4days). In their series, half of the children aged ≤ 2years and one-third of those ≤ 3years had an appendix mass present at the time of first evaluation. 15.8% of their patients failed to settle with conservative management, 41.5% of whom had abscess drainage followed by appendicectomy, while 26% required early appendicectomy; 50% of these had post-operative complications. 10% of those who settled with conservative management had recurrent appendicitis. Considering the short duration of symptoms associated with a mass that was not palpable before anaesthesia, the author recommends that surgical treatment should proceed in these cases; having commenced antibiotic therapy at least 4hours before surgery where the duration of symptoms was longer than 48hours as suggested above. This recommendation is also given by Stevenson, (2003) and adopted by many paediatric surgeons in the United Kingdom. 10.4.1 Anaesthetic considerations Appendicitis is usually an acute illness in otherwise healthy persons. It is often associated with gastroparesis and a patient who is admitted for observation for a probable diagnosis of appendicitis should be given fluid diet if not nil per oral as the stomach may not empty as well as in other conditions. Intraoperative precautions should be observed as for patients with a full stomach with rapid sequence intravenous induction of anaesthesia (Oberhelman & Malott, 2004). Once anaesthetised, the stomach should be promptly emptied with a nasogastric tube. The presence of associated peritonitis and abdominal distension may lead www.intechopen.com 155 Appendicitis in Children to splinting of the diaphragm which in turn reduces the functional lung volume. Respiratory impairment may be present especially in very young children. Tachypnoea may be a manifestation of respiratory embarrassment, pain, dehydration or sepsis. The circulatory system may be affected by hypoperfusion from associated fever, vomiting, diarrhoea or nausea with resultant reduced oral intake. This may manifest as increased heart rate and end organ signs including increased capillary refill time, reduced peripheral temperature, dry mucous membranes and reduced urine output. Preoperative correction of any hypovolaemia is mandatory for safe anaesthesia. There may be coexistent electrolyte imbalance and this also needs to be appropriately corrected preoperatively (Oberhelman & Malott, 2004). Muscle relaxation is required to facilitate surgery whether open approach where muscle splitting is applied or laparoscopy which requires adequate exposure by pneumoperitoneum using the lowest possible intra-abdominal pressure. The physiological challenges posed by the pneumoperitoneum required for laparoscopic surgery needs careful attention from the anaesthetists (Nwokoma & Tsang, 2011). 10.4.2 Laparoscopic approach Since the description of laparoscopic appendicectomy by the German gynaecologist Kurt Semm in 1983 (Semm, 1983), this approach to appendicectomy has continued to gain wide acceptance. With the advances in laparoscopic surgery in recent years, it has become common practice in many centres to have laparoscopic approach to appendicectomy in the absence of contraindications (Table 3). Patient unsuitable for open surgery Uncontrolled bleeding or coagulation problems Multiple previous abdominal surgery Table 3. Contraindications to paediatric laparoscopy Where the child presents with features of advanced appendicitis with bowel obstruction, this may constitute a relative contraindication to the use of laparoscopy due to increased risk of injury to the dilated bowel loops. Previous abdominal surgery predisposes the patient to intra-abdominal adhesions which increase the risk of bowel injury and bleeding but this risk is less if the previous surgery was performed laparoscopically (Nwokoma et al., 2009b). Laparoscopic approach has been safely used to treat advanced appendicitis in children with results similar to that in open approach. We demonstrated that laparoscopic approach offered significant advantages with better outcomes than open approach in paediatric advanced appendicitis with less wound-related complications: 8.6% versus 17.6% (Nwokoma et al., 2009a), and a conversion rate of 0%. Brügger et al., (2011) and Garg et al., (2009) drew similar conclusions from their studies. Brügger et al., (2011) further demonstrated the rate of wound infections (0.50% vs. 6.98-7.97%), post-operative ileus (0.15% vs. 0.33%), urinary complications (0.13% vs. 0.66%) and pulmonary complications (0.18 vs. 1.19%) to be lower in their group of laparoscopically treated appendicitis than data from large studies using the open approach. www.intechopen.com 156 Appendicitis – A Collection of Essays from Around the World The age-long principles of safe surgery include quick and adequate access, adequate target organ visualisation and minimal tissue trauma. In children, access can be quite a challenge because of the smaller height/width ratio of the abdomen particularly observed in those under 8years of age. In many cases, however long the incision, gaining access to the target organ or indeed to the four quadrants of the abdomen and pelvis, can be very difficult. Laparoscopy offers the paediatric surgeon the advantage of been able to visualise these areas while reducing the trauma usually consequent upon use of large abdominal wall incisions (Nwokoma & Tsang, 2011). There is growing evidence that laparoscopy has more advantages and benefits to offer children than was earlier presumed to be the case. These benefits have been widely reported (Table 4) and significantly outweigh any concerns regarding the technical difficulties (Table 5) which are largely overcome with increasing experience and further developments in the laparoscopic equipment. Reduced wound size Reduced wound trauma Less wound infection Less incisional hernia Less wound dehiscence Less wound pain Early mobilisation Less bleeding Less heat loss from tissue Wider field of vision Less postoperative adhesions Less postoperative ileus Earlier return to usual activities Earlier commencement of chemotherapy Less respiratory complications Less risk of thromboembolism Reduction in nerve entrapment Table 4. Advantages of laparoscopy Loss of tactile sensation Loss of spatial and depth orientation Two-dimensional imaging Difficulty with control of bleeding Difficulty with extraction of resected tissue or organ Table 5. Technical difficulties of laparoscopy A 10mm primary port should be inserted using the Hasson’s open technique either in the suprapubic region, half way between the symphysis pubis and the umbilicus making sure that the urinary bladder is not in the path of entry or in the umbilical region – centrally or www.intechopen.com Appendicitis in Children 157 infraumbilically. Two secondary 5mm ports should be inserted under laparoscopic guidance in the left lower quadrant for instruments. Alternatively, with an umbilical primary port, each of the two secondary ports can be placed on either side in the left and right lower quadrants. Single port transumbilical laparoscopy-assisted appendicectomy is gaining popularity and has been demonstrated to give results comparable to standard laparoscopic appendicectomy for uncomplicated appendicitis (Guanà et al., 2010). It has been successfully used to treat uncomplicated appendicitis as day case procedures (Alkhoury et al., 2011). Local anaesthetic injection into the port sites is advisable. Safe pneumoperitoneum should be established with 5-8mmHg in the newborn, 10-12mmHg in infants and <15mmHg in older children (Nwokoma & Tsang, 2011). Pus can be obtained with the suction device for microbiological analysis. The appendix is dissected free, the appendicular vessels divided by diathermy cauterisation or between endoclips. Ligation of the appendix should be carried out with three endosurgical loops; two proximally and one distally, as close to the base as possible to avoid the complications of stump appendicitis and enterocutaneous fistula (Lintula et al., 2002). Stump appendicitis which can occur following open or laparoscopic appendicectomy may occur in residual appendix as small as 6mm (Waseem & Devas, 2008) and is associated with significant morbidity. Cauterisation of the appendicular stump may prevent later formation of a mucocele. All incisions ≥ 5mm should be closed with absorbable sutures to the deep fascia and subcutaneous tissue to avoid port site hernia. Advanced appendicitis poses a significant challenge for the paediatric surgeon and many opt for the open approach if this is suspected preoperatively. This is because the abdomen in children is shorter in height and relatively wider than in adults, especially children younger than eight years of age which is the group that commonly present with advanced appendicitis. However, as we demonstrated above, advanced appendicitis can be safely managed laparoscopically in children with outcome comparable to those of open approach. An inflammatory mass may be present during surgery and this can be drained laparoscopically with good vision of all four quadrants of the abdomen. Following laparoscopic drainage of the abscess, liberal peritoneal lavage should be performed as appropriate and the inflammatory mass should be assessed with regards to safety of continuing with the operation. Where the tissues are very friable, it is preferable to postpone the appendicectomy and treat with intravenous antibiotics with a view of performing interval appendicectomy safely at a later date. It is preferable to place the patient in a reverse Trendelenburg position and drain the purulent material from the pelvic cavity before putting the patient in the Trendelenburg position required for good access for the appendicectomy. This practice should reduce the risk of post-operative subphrenic, subhepatic and parasplenic abscesses. Following laparoscopic appendicectomy in 7446 cases (age range between 12 and 100years), Brügger et al. (2011) observed an overall complication rate of 8.63% with individual complications detailed in Table 6. 10.4.3 Open appendicectomy The open approach to appendicectomy has been established for over a century. The first recorded appendicectomy was performed by Claudius Amyand in 1735 at St. George’s Hospital in London (United Kingdom) where he removed an appendix containing a calcified mass around a pin in a patient presenting with inguinal hernia. Lawson Tait performed the first successful appendicectomy for appendicitis in 1880 (Williams and Myers 1994). www.intechopen.com 158 Appendicitis – A Collection of Essays from Around the World Complication Intraoperative complications % frequency 1.88 Haematoma/intra-abdominal bleeding Haematoma/ abdominal wall bleeding Injury to intra-abdominal organ Injury to stomach/ bowel Vascular injury Inadvertent bowel puncture by trocar Inadvertent puncture by Veress needle Other intraoperative complications 0.6 0.28 0.13 0.08 0.07 0.07 0.01 0.63 Postoperative Complications 6.75 Surgical postoperative complications Abscess Peritonitis Paralytic ileus Haematoma/intra-abdominal bleeding Haematoma/abdominal wall bleeding Haematoma/bleeding requiring transfusion Wound infection Obstructive ileus Intestinal perforation Stricture Other surgical complications 4.24 0.98 0.59 0.56 0.50 0.34 0.13 0.50 0.15 0.04 0.01 0.44 General postoperative complications Cardiac complication Pulmonary embolism Urinary tract infection Jaundice Pneumonia Deep vein thrombosis Stroke Nerve compression Other general postoperative complications 2.51 0.36 0.15 0.13 0.05 0.03 0.01 0.01 0.01 1.75 Table 6. Complication rates following laparoscopic appendicectomy The commonly applied incision in children is the Lanz incision. It is an almost transverse incision in the right lower quadrant, about 2cm above and medial to the anterior superior iliac spine with its centre in the McBurney’s point. The Lanz incision is more popular in www.intechopen.com Appendicitis in Children 159 children than the gridiron incision which also has its centre as the McBurney’s point but runs perpendicular to an imaginary line between the anterior superior iliac spine and the pubic tubercle. This is because the Lanz incision has better cosmesis and healing, being along the Langerhan’s lines. It also offers the surgeon the qualities of a good incision including easy and quick access to the abdominal cavity, extendable if required and easy to close. It crosses less dermatomal regions making the post-operative pain less and easier to control. As mentioned earlier, the abdomen of young children is relatively wider than its height on the longitudinal axis. Therefore access to the abdominal organs during surgery is best achieved by an incision that can go across the abdomen; the Lanz incision offers this advantage. Some authors advocate palpating the abdomen just before induction of anaesthesia and placing the incision just below the point of maximum tenderness. The problem with this is that the point of maximum tenderness usually marks the appendicular tip and may be far from the base. This may result in a longer than necessary incision. For example a pelvic appendix tip may give maximum tenderness suprapubically and a retrocaecal appendix may give maximum tenderness in the right upper quadrant. An incision over the region of the base of the appendix works best with various positions of the appendix body and tip. In certain situations, lengthening of the incision is necessary to perform a four-quadrant examination and drainage of associated pus. The Lanz incision allows such an extension of the incision to be undertaken safely and effectively. Muscle splitting is preferable to the muscle cutting approach because the reduced tissue trauma is associated with reduced risk of bleeding, infection and post-operative pain. The peritoneum should be entered between clips, avoiding damage to the underlying bowels by ensuring bowel clearance from the edges of the clips. A microbiological swab of the peritoneal fluid should be taken, preferably from the appendix itself to increase microorganism yield. Pus is the creamy-yellow viscid fluid present in infected tissues which consists of bacteria – living and dead, dead polymorphonuclear leukocytes, extravasated plasma and damaged host cells or tissue debris (Eykyn, 1998). If purulent material is present, a sample of it should be sent for analysis as well as a swab sample. The caecum should be identified and the anterior taenia followed inferomedially to the appendix base. Any inflammatory adhesion should be carefully released by blunt digital dissection. The peritoneal folds along the lateral and inferior borders of the caecum may need to be divided to adequately mobilize the caecum and deliver the appendix into the wound, especially so when it lies retroperitoneally. The mesoappendix is narrowest at the tip and widest at the base with the appendicular vessels within its edge. Ligation of the vessels usually commences from the tip towards the base. This is the antegrade dissection. In some cases retrograde dissection from the base may be required for safe appendicectomy. The appendix base should be crushed with a straight clamp as close to the caecum as can be safely achieved. Reapplying the clamp just above the crushed portion, the appendix should be transfixed and ligated with strong absorbable suture material, then cut above this. Cauterisation of the appendicular stump reduces risk of formation of a mucocele. Inversion of the appendicular stump with a purse string suture or a Z- stitch anchored within the taenia coli on the caecum adjacent to the base is still common practice. Taking too much caecum into the purse string suture or Z-stitch may lead to the development of a mucocele or become a lead point for intussusception (Swain, 2005). With local purulent peritonitis, local irrigation is preferable to wide spread lavage in other to minimise any dissemination of infective agents. On the other hand, if free pus is present, liberal peritoneal lavage is www.intechopen.com 160 Appendicitis – A Collection of Essays from Around the World recommended. The addition of Betadine (Povidone iodine) or antibiotic agent(s) to the lavage fluid is widely practiced. However, it is noteworthy that Schneider et al., (2005) reported no significant advantage from the use of adjuvant peritoneal Taurolidine lavage in children with appendicitis associated with localised peritonitis. Local anaesthetic injection into the wound at this point compliments immediate post-operative analgesia. The abdominal wall should be closed carefully with absorbable suture material in layers or as mass closure. Subcuticular absorbable sutures should be used to close the skin. Pauniaho et al., (2010) demonstrated a reduced incidence of wound-related complications in acute appendicitis using subcuticular absorbable sutures than with the use of non-absorbable sutures. The complication rate following open appendicectomy in children varies with age and severity of the appendicitis. Intra-abdominal abscesses may complicate up to 20% of perforated appendicitis; wound abscess <5%; faecal fistula <1% and wound haematoma <0.5%. Other complications include intestinal obstruction, missed bowel injury and bleeding. Mortality for non-perforated appendicitis is <0.1% and for perforated appendicitis this rises to up to 2% (Oberhelman & Malott, 2004). 10.5 Post-operative management Careful monitoring of the patient in the post-operative period should follow the principles of management of the critically ill surgical patient. As stated earlier, level 2 (or higher) nursing care may be required. Careful management of respiratory and cardiovascular system should be continued. In the very young patient a urinary catheter may be a useful adjunct to fluid management and opiate analgesia may make the child prone to urinary retention. A nasogastric tube may be required if features of bowel obstruction are present. A peripherally inserted central line may be inserted intra-operatively if prolonged antibiotics or significant delay to return of bowel function is anticipated. Post-operative analgesia may initially be administered as a patient or nurse-controlled intravenous opiate analgesia. Where advanced appendicitis has necessitated a wide incision and laparotomy, an epidural analgesia may be preferable. If epidural analgesia is used, a urethral catheter should be placed. Oral analgesia should be introduced when gastrointestinal function returns. The administration of antibiotics for any reason can potentially upset the balance of the normal gastrointestinal flora. This may create an environment that is favourable for the multiplication of exogenous pathogens as well as the overgrowth of select pathogenic strains. Antibiotic-related complications are common with use beyond 5 days (Mui et al., 2005). Principles for the selection of antibiotic therapy (Raftery, 2002) are as follows;  There should be clinical evidence of infection  Best guess antibiotics to cover known likely infective micro-organism(s)  Where possible, remove infected tissue or foreign body  Appropriate specimen collection from the site of infection for microbiology examination  Cheapest and most effective drug or drug combination with known effectiveness over known likely organisms  Monitoring of clinical response to treatment  Appropriate route to achieve therapeutic levels of drug at site of infection www.intechopen.com Appendicitis in Children 161  Duration of administration should cover acute infection period, avoiding prolonged antibiotic treatment  Re-evaluate clinical response with microbiology result and change antibiotics if clinically indicated. Appendicectomy creates a contaminated wound with an infection risk of 12%. In the presence of pus or a perforation, a dirty wound results, with infection risk of 25% (Raftery, 2002). Perioperative antibiotics administration should follow local sensitivities, usually – Amoxicillin/ Gentamicin/ Metronidazole or a cephalosporin given instead of Amoxicillin. The former combination is used in our institution. Antibiotics should be given at least one hour before the skin incision is made to ensure adequate therapeutic plasma levels. Further antibiotic therapy should be based on intraoperative findings. In the presence of normal looking appendix, no further antibiotic is required. Single dose combined antibiotic therapy has been demonstrated to be adequate surgical prophylaxis in non-perforated appendicitis (Mui et al., 2005). In addition, Lee et al., (2010) observed that single or double agent antibiotics were effective and of lower cost than triple therapy. The author’s recommendation in the case of an inflamed non-perforated appendix with no pus present is that a 24hr antibiotic therapy of single or double agents be given. This is because inflamed bowel is known to be associated with some micro-organism translocation. If heart rate and temperature remain within normal limits at 24 hours, antibiotics can be discontinued. A perforation may not be evident on resected specimen due to the extensive inflammation (Fenoglio-Preiser et al., 2008). In a cutaneous abscess, it is possible to clear out the pus. In the abdominal cavity, this is not possible and one must assume that infective agents remain free in the peritoneal cavity even after extensive peritoneal lavage. Five days of intravenous antibiotics is recommended in the presence of pus or an obvious perforation, preferably a triple agent therapy. The results of the microbiology analysis of any pus sample should be ascertained before the end of the five-day antibiotic therapy There is evidence that intraperitoneal abscess formation is commoner with Streptococcus milleri (Feneglio-Preser et al., 2008) and a longer duration of antibiotics, about 7days, is recommended in these situations. Thromboprophylaxis should be administered by mechanical and/or chemical means as appropriate to each patient. In particular, older children above average weight or on contraceptive medication or who smoke should have peri-operative thromboprophylaxis. Some children show signs of gastric irritation following appendicectomy more with advanced appendicitis. If features of gastric irritation are observed including new onset epigastric pain and coffee-ground appearance of the vomitus, H-2 antagonists or proton pump inhibitors should be given to cover the period of acute illness till symptoms resolve. 11. Special considerations 11.1 The normal appendix: Remove or not remove? Since the introduction of the Antegrade Colonic Enema procedure to aid the management of functional problems of the large bowel, the need to preserve the normal appendix particularly in children has been the subject of much discussion. Children without functional bowel problem or spina bifida at the time of presentation are unlikely to require the ACE procedure in the future. The appendix is also used for urinary diversion or vesicocutaneous channel in the Mitrofanoff procedure and for biliary drainage (Swain, 2005). Arguably, while this may not be required at the time of surgery, the child’s condition might www.intechopen.com 162 Appendicitis – A Collection of Essays from Around the World change in the future. The likelihood that a child would need an appendix-related reconstructive surgery in the absence of a previous health problem is less than the likelihood of having appendicitis (Morrow, 2005). On this premise, it would appear that incidental appendicectomy has more benefits to offer by avoiding a future appendicitis. Contraindications of incidental appendicectomy include impaired immunity, presence of surgical implants, presence of Crohn’s disease in the adjacent caecum, intra-operative instability, history of recent abdominal radiation and an inaccessible appendix (Stevenson, 2003). As discussed earlier, a normal-appearing appendix may be pathological. It is arguable that the presence of a Lanz incision may imply that appendicectomy had previously been undertaken which could be misleading with needless delay to the diagnosis of appendicitis where the appendix had actually not been removed. On the other hand, removing a normal appendix converts a clean operation into a dirty operation with increased risk of complications. The author recommends appropriate pre-operative evaluation and the removal of the normal-appearing appendix discovered intra-operatively. 11.2 Neurogenic appendicopathy This condition is caused by the proliferation of nerve fibres in the appendix and can only be diagnosed with certainty on histological analysis. It may be present in up to 4.2% of specimens removed for presumed appendicitis. It is commoner in girls and older children, with up to 80% of specimens showing no histological features of inflammation. The use of antiserotonin or antihistamine therapy is advocated in suspicious cases (Zaupa et al., 2011). 11.3 Inflammatory bowel disease in appendicitis Crohns disease: The appendix is involved in 25% of Crohn’s disease leading to surgical treatment. However, Crohn’s disease manifesting as appendicitis at the time of diagnosis is rare with less than 100 cases reported in literature. 7-10% of these are thought to progress to Crohn’s disease at other sites. Ulcerative colitis: The appendix is involved in up to 50% of resected ulcerative colitis specimens. Some of these manifest as skip lesions without caecal involvement or in continuity with caecal disease (Petras & Goldblum, 1996). 11.4 Acute appendicitis in the neonate Neonatal acute appendicitis is rare but associated with high morbidity and mortality of about 50-80%. Diagnosis is often late or missed and found at post-mortem (Swain, 2005). It may result from the presence of necrotising enterocolitis, cystic fibrosis, Hirschsprung’s disease or bacteraemia associated with maternal chorioamnionitis (Pressman et al., 2001). The neonatal anatomy presents special challenge due to its difference from the rest of the paediatric population. The abdomen in these children is often protuberant due to the flat diaphragm, shallow pelvis and reduced sacral curvature. Consequently, the organs that would have been within the rib cage and pelvis are intra-abdominal (Standring et al., 2005). 11.5 Chronic appendicitis An organising phase of acute appendicitis occurs with the finding of granulation tissue and a mixture of acute and chronic inflammatory changes as well as recently laid down connective tissue. However, true chronic appendicitis with lymphocyte and plasma cells present in the muscularis propria and serosa without significant acute inflammation is rare (Petras and Goldblum, 1996). www.intechopen.com Appendicitis in Children 163 11.6 Tuberculosis of the appendix Appendicular tuberculosis occurs in 0.1-3% of patients with tuberculosis but isolated tuberculosis of the appendix is rare. Appendicectomy followed by antituberculous chemotherapy is the treatment of choice. Abdominal tuberculosis in children affects the immunocompromised and those who have not received the BCG vaccine. It manifests with weight loss, malaise, abdominal distension, abdominal pain, anaemia raised white cell count and altered albumin: globulin ratio (Rangabashyam et al., 2000) 11.7 Neoplasm of the appendix Neoplasm of the appendix is found in 1.08 to 1.3% of appendicectomy specimens. The carcinoid tumour is very rare but it is the most common neoplasm of the gastrointestinal tract in children. It may be found in 0.3% of paediatric appendicectomies. Mean peak age of incidence in children is 15years though children as young as 6years old may be affected (Stevenson, 2003). Carcinoid syndrome comprises of flushing, diarrhoea and cardiac disease. It is usually associated with liver or retroperitoneal metastasis; with increased urinary 5hydroxyindoleacetic acid. Lymph node metastasis is seen in 4-5% of paediatric patients with carcinoid tumour but distant metastasis of appendicular carcinoid is very rare in children. It may also be associated with multiple endocrine neoplasia type 2. (Christianakis et al., 2008). Adenocarcinoma of the appendix is exceedingly rare. More a problem of older patients, it develops in the appendicular base with appendicitis from luminal occlusion being the commonest mode of presentation. 50% are metastasized at diagnosis. It commonly spreads to the peritoneum directly. Adenocarcinoids are also rare. The histological features are of combined carcinoid and adenocarcinomas. The treatment of neoplasm of the appendix is largely limited to appendicectomy. Extension beyond the appendix requires treatment by right hemicolectomy (Liu & McFadden, 2003). Cystadenocarcinomas are mucin-filled. Perforation results in mucin-secreting peritoneal deposits manifesting as pseudomyxoma peritonei which is treated by repeated debulking and eventually fatal. In advanced cases, the abdomen is filled with tenacious semisolid mucin. 11.8 Recurrent appendicitis Recurrent appendicitis is becoming increasingly accepted as a diagnosis. Appendicitis like any other inflammation in the human body may become arrested and not progress to fullblown process. To lend support to this, approximately a quarter of patients with histologically proven acute appendicitis report a history of prior episodes of abdominal pain of similar character as that which culminated in appendicectomy. Furthermore, sometimes histopathological analysis of acute appendicitis specimen shows both acute and chronic inflammatory characteristics. Also, about 60% of patients who respond well to treatment of advanced appendicitis report abdominal pain suggestive of recurrent appendicitis prior to interval appendicectomy (Swain, 2005). 11.9 Antibiotic-associated Clostridium difficile infection Clostridium difficile (C. diff.) is a spore-forming gram positive rod. It produces its pathogenic features by the production of toxins. Toxin A is an enterotoxin while toxin B is a cytotoxin. Diagnosis is by detecting these toxins in stool. C. diff. toxins can be detected in the stool of 2-5% of the general population and up to 50% of infants. Its clinical significance is anchored on being the causative agent of antibiotics associated diarrhoea included in this www.intechopen.com 164 Appendicitis – A Collection of Essays from Around the World chapter as a likely complication of prolonged perioperative antibiotic administration. It has been found to be responsible for approximately 30% of cases of the simple uncomplicated diarrhoea that often follows antibiotic administration. It is associated with 90% of cases where pseudomembranous colitis is present. Alteration of colonic flora especially by Ampicillin or Cephalosporins and Clindamycin favours the proliferation and virulence of C. diff. The clinical manifestation of C diff. colitis depends on which toxin is predominant in the colon. In situation of toxin A predominance, watery diarrhoea manifests; with toxin B predominance pseudomembranous colitis results. C. diff. diarrhoea onset is usually 5-10 days after starting antibiotics but this could range from day 1 to weeks after cessation of the therapy. Clinical effects may be mild and watery or bloody with or without severe crampy abdominal pain, raised levels of white blood cells and raised temperature. Treatment of C. diff colitis consists of discontinuation of implicated antibiotics which would usually lead to complete resolution of symptoms. However, if there is no response to antibiotic withdrawal or the patient is severely ill, Metronidazole or Vancomycin given orally is recommended. The risk of relapse or re-infection requiring repeat treatment may be up to 20% (Ryan & Ray, 2010). 11.10 Appendicitis in cancer patients on chemotherapy Appendicitis in the neutropaenic child on anticancer chemotherapy is a great challenge. As much as possible, surgery should be avoided and conservative management with antibiotics instituted with a plan to perform interval appendicectomy when the child is better. A CT scan is often required to differentiate this from typhlitis. Joint care should be undertaken with specialist paediatric oncology staff. Granulocyte Colony Stimulating agents are often used to improve the neutrophil count. Some authors recommend elective appendicectomy in patients diagnosed with malignancy who are about to commence chemotherapy if surgery for other reason was to be performed before chemotherapy with a view of preventing appendicitis that may occur in neutropenic patients while on chemotherapy. This practice was not found to be associated with increased complications rate but only 0.2% of patients who did not have incidental appendicectomy went on to have appendicitis during a median follow up period of 5years (Morrow, 2005). 12. 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Presented at the 12th European Congress of Paediatric Surgery, (June 2011). www.intechopen.com Appendicitis - A Collection of Essays from Around the World Edited by Dr. Anthony Lander ISBN 978-953-307-814-4 Hard cover, 226 pages Publisher InTech Published online 11, January, 2012 Published in print edition January, 2012 This book is a collection of essays and papers from around the world, written by surgeons who look after patients of all ages with abdominal pain, many of whom have appendicitis. All general surgeons maintain a fascination with this important condition because it is so common and yet so easy to miss. All surgeons have a view on the literature and any gathering of surgeons embraces a spectrum of opinion on management options. Many aspects of the disease and its presentation and management remain controversial. This book does not answer those controversies, but should prove food for thought. The reflections of these surgeons are presented in many cases with novel data. The chapters encourage us to consider new epidemiological views and explore clinical scoring systems and the literature on imaging. Appendicitis is discussed in patients of all ages and in all manner of presentations. How to reference In order to correctly reference this scholarly work, feel free to copy and paste the following: Ngozi Joy Nwokoma (2012). Appendicitis in Children, Appendicitis - A Collection of Essays from Around the World, Dr. Anthony Lander (Ed.), ISBN: 978-953-307-814-4, InTech, Available from: http://www.intechopen.com/books/appendicitis-a-collection-of-essays-from-around-the-world/appendicitis-inchildren InTech Europe InTech China University Campus STeP Ri Unit 405, Office Block, Hotel Equatorial Shanghai Slavka Krautzeka 83/A No.65, Yan An Road (West), Shanghai, 200040, China 51000 Rijeka, Croatia Phone: +385 (51) 770 447 Phone: +86-21-62489820 Fax: +385 (51) 686 166 Fax: +86-21-62489821 www.intechopen.com © 2012 The Author(s). Licensee IntechOpen. This is an open access article distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 

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DOI: 10.1501/Tarimbil_0000000982 TARIM BILIMLERI DERGISI 2000, 6 (3), 132-134 Yaş l ı Broiler Dam ı zl ı k Sürüsünden Elde Edilen Yumurtalar ı Plastik Torba İ çinde K ısa Süre Bekletmenin Kuluçka Özelliklerine Etkisi Okan ELIBOL' Geli ş Tarihi: 10.04.2000 Özet : Bu araşt ı rma, yaşl ı broiler dam ızl ı klar ından elde edilen yumurtalar ı plastik torba içinde k ısa süreli bekletmenin kuluçka özelliklerine etkisini belirlemek amac ı yla yürütülmü ştür. Çal ışman ı n yumurta materyali, 57 haftal ı k yaş da olan Ross Pm3 broiler dam ızl ı kları ndan elde edilmi ştir. Ara şt ı rmada toplam 4500 adet kuluçkal ı k yumurta kullan ı lm ışt ı r. Depolama süresi olarak 4 gün ve bekletilmeyen (0 gün) olmak üzere 2 grup olu şturulmuştur. Bunun yan ı nda depolama süresi 4 gün olan yumurtalar ı n yarıs ı plastik torba içinde di ğer yarı sı ise ayn ı koşullarda plastik torbaya konulmadan tutulmu ştur. Depolama süresi 4 gün olan plastik torbas ı z ve plastik torba içinde bekletilen gruplar ile depolanmayan(depo süresi 0 gün ) grubun ç ı k ış gücü de ğerleri s ı ras ı yla % 86.7, % 90.9 ve % 90.7 olarak tespit edilmi ştir. Gruplar ı n mukayesesi, plastik torbas ı z olarak 4 gün bekletilen grupta ç ı kış gücünün, di ğer 2 gruptan dü şük olduğ unu ortaya koy ınuştur.(P < 0.05). Sonuç olarak, ya şl ı broiler dam ızl ı klardan elde edilen yumurtalar ı n ovipozisyon sonial en k ı sa sürede geli şim makinesine yüklenmesi eğ er bu sağ lanam ıyorsa k ı sa süreli de olsa plastik torba içinde bekletilmesinin ç ı k ış gücünü art ı rd ığı tespit edilmiştir. Anahtar Kelimeler: Broiler dam ızl ı k, yumurta depolama süresi, plastik torba, ç ı k ış gücü The Effect of Short Term Storage in Plastic Bags on Hatchability of Eggs from OId Broiler Breeders Abstract : The experiment was conducted to study the effect of storing eggs in plastic bags during short-term storage(0 and 4 days). The trial involved a total of 4500 hatching eggs from broiler breeder flock aged 57th wks. The eggs kept for 4 days were divided into 2 groups, one of them was stored in plastic bags(Group 1), other was stored without plastic(Group 2). Hatchability of fertile eggs, group 1 and group 2 were 90.9% and 86.7% respectively whilst this fıgure was 90.7% for stored 0 days (group 3). Group 2 gaye signif ıcantly lower hatchability than the other two groups(P< 0.05) This data suggests that for optimum hatchability, eggs from older hens should be set as soon as possible, if it is not available, even short-term stroge periods, eggs might be stored in plastic bags Key Words:Broiler breeder, egg stroge period, plastic bag, hatchability Giri ş Genellikle broiler civciv üreten kuluçkahanelerde depolama süresi 1 haftay ı geçmemektedir. Bu sürenin ç ı k ış gücü üzerine olumsuz etkisinin olmad ığı bildirilmektedir ( Funk,1950; Oluyemi ve George1972; Mayes ve Takeballi,1984). Ancak Wilson(1991) ve Walsh(1993) kuluçkal ı k yumurtalarda maksimum ç ı k ış gücü de ğ erinin elde edilmesi için yumurtalar ı n ovipozisyon sonras ı hemen makineye yüklenmesi gerekti ğ ini belirtmi ş lerdir. Ayr ı ca depolama süresi ile sürü ya şı aras ı nda önemli seviyede interaksiyon oldu ğ unu, ya ş l ı broiler anaçlardan elde edilen yumurtalar ı n en k ı sa sürede geli ş im makinesine konulmas ı gerekti ğ ini, aksi takdirde ç ı k ış gücünde önemli seviyelerde dü ş ü ş ün olaca ğı n ı belirten ara şt ı r ı c ı lar da vard ı r(Reis ve ark. 1997, Lapao ve ark 1999). Ç ı k ış gücüne önemli etkisi olan albümin kalitesinin depolama ko ş ullar ı ve süresi(Kosin ve Konishi 1973; Hurnik ve ark. 1978) yan ı nda sürü ya şı ndan da etkilendi ğ i ve ya ş l ı sürülerin yumurtalar ı nda albümin kalitesinin daha dü ş ük oldu ğ u belirtilmi ştir ( Burley ve Vadehra 1989; Walsh 1993; Brake ve ark. 1993, Lapao ve ark 1999 ). Ya ş l ı sürü yumurtalar ı nda depolama süresinin artmas ı ile ç ı kış gücünde meydana gelen kayb ı n, albümin kalitesinin dü ş mesinden kaynaklanabilece ğ i bildirilmi ştir (Walsh 1993, Brake 1996, Brake ve ark. 1997). 1 Ankara Univ. Ziraat Fak. Zootekni Bölümü - Ankara Depolama ko ş ullar ı ndan kaynaklanan sorunlar ı en aza indirebilmek için yap ı lan uygulamalardan birisi de, yumurtalar ı n depolama süresince plastik torba içinde bekletilmesidir. Bu uygulama, yumurtada CO2 kayb ı n ı n azalmas ı ile albümin pH seviyesinin yükselmesini engelleyerek albümin kalitesindeki dü ş ü ş ün yava ş lamas ı ve yumurtada a ğı rl ı k kayb ı n ı n azalmas ı sonucu ç ı k ış gücünü olumlu yönde etkilemektedir(Davis ve Beeckler 1962; Becker ve ark. 1964; Proudfoot ve Hulan 1976). Plastik torban ı n olumlu etkisinin depolama süresi uzad ı kça daha belirgin olarak görülebilece ğ ini ifade eden ara şt ı r ı c ı lar ço ğ unluktad ı r (Davis ve Beeckler,1962; Becker,1964; Warren ve ark.1965; Proudfoot ,1966; Kirk ve ark. 1980; Elibol, 1997). Bu ara şt ı rmada üretimin do ğ as ı gere ğ i bekletme süresi uzun olmayan ya ş l ı broiler anaçlardan elde edilen yumurtalar ı plastik torba içinde k ı sa süreli bekletmenin kuluçka özelliklerine etkisini ortaya koymak amaçlanm ışt ı r. Materyal ve Yöntem Ara şt ı rma bir özel broiler entegre i ş letmesinin kuluçkahanesinde yürütülmü ştür. Çal ış mada, Petersime ELiBOL, O. "Yaşl ı broiler dam ızl ı k sürüsünden elde edilen yumurtalar ı plastik torba içinde k ı sa süre bekletmenin kuluçka özelliklerine etkisi" (57600 yumurta kapasiteli geli şim, 19200 yumurta kapasiteli ç ı k ı m makinesi) marka tam otomatik kuluçka makineleri kullan ı lm ışt ı r. Ara şt ı rman ı n yumurta materyali, ayn ı entegrasyona ait dam ızl ı k i ş letmesinde 57 haftal ı k ya şta bulunan broiler anaçlardan (Ross-Pm3) elde edilmi ştir. i ş letmede yumurtalar, kümesten kuluçkahaneye ö ğ le ve akşam olmak üzere her gün 2 parti halinde getirilmi ştir. Bütün yumurtalar kuluçkahane giri ş inde fümigasyon i ş lemine tabii tutulmu ş lard ı r. Denemede, 1. partideki (ö ğ le) yumurtalardan tesadüfi olarak seçilmi ş ve tasnif i ş lemi sonras ı 20 °C ve °k 75 nispi nem içeren depoda 4 gün muhafaza edilmi ş yumurtalar ile yükleme günü ayn ı şekilde toplanan yumurtalar, depolanmaks ız ı n kullan ı lm ışt ı r. Dört gün depolanacak yumurtalar kendi içinde rasgele e ş it 2 gruba ayr ı lm ış ve depolama süresince bir gruptaki yumurtalar plastik torba içinde muhafaza edilirken, di ğ er grup ayn ı depoda normal ko ş ullarda bekletilmi ştir. Depolama süresi 4 gün olan yumurtalar, yükleme günü elde edilen yumurtalarla(depolanmayan) birlikte önce ikinci kez fümigasyon i ş lemine ve daha sonrada 25 °C de yakla şı k 8 saat ön ı s ıtmaya tabii tutulduktan sonra ayn ı geli ş im makinesine yüklenmi ş lerdir. Çal ış mada, ç ı k ış zaman ı nda ı skarta ve ölü civcivler ile ç ı kışı olmayan yumurtalar ayr ı lm ışt ı r. Ç ı k ışı olmayan yumurtalar k ı rı larak döllü olup olmad ı klar ı tespit edilmi ş , döllü yumurtalarda ise embriyo ölüm ya şı ( erken dönem,0-5 gün;orta dönem, 6-17 gün; son dönem ölümleri, 18-21 gün ve kabu ğ u k ı rı p ölen embriyo) ile kontamine yumurtalar belirlenmi ştir. Bu veriler kullan ı larak ba şta ç ı k ış gücü olmak üzere kuluçka özelliklerine ait de ğ erler hesaplanm ışt ı r. Ara şt ı rmada toplam 4500 adet kuluçkal ı k yumurta, her biri 150 adet yumurta alabilen tepsilere dizilmi ş ve her bir tepsi bir tekerrür olarak de ğ erlendirilmi ştir. Gruplar aras ı ndaki farkl ı l ığı belirlemek için varyans analizinden, farkl ı l ığı n hangi gruptan ileri geldi ğ inin tespiti için de Duncan testinden yararlan ı lm ışt ı r (Düzgüne ş ve ark. 1983). Bulgular ve Tart ış ma Sürü ya şı 57 hafta olan broiler anaçlar ı ndan elde edilen, yükleme öncesi farkl ı süre ve ş ekillerde bekletilen yumurtalar ı n kuluçka özelliklerine ait ortalama de ğ erler Çizelge 1 de sunulmu ştur. Depolama süresi 4 gün (plastik torbas ı z) ve O gün (depolanmaks ız ı n yüklenen) olan yumurtalar aras ı nda, erken ve son dönem embriyo dlümleri, ı skarta oran ı ile ç ı k ış gücü bak ı m ı ndan farkl ı l ığı n önemli (P<0.05) oldu ğ u belirlenmi ştir. Bu sonuçlar, ya ş l ı sürülerden elde edilen yumurtalarda optimum ç ı k ış gücü için yumurtalar ı n, ovipozisyon sonras ı en kı sa sürede makineye yüklenmesini tavsiye eden Reis ve ark. (1997), Lapao ve ark: (1999yn ı n bulgular. ile uyum halindedir. Fakat depolama süresinin 7 güne kadar artmas ı n ı n ç ı k ış gücünü dü ş ürmedi ğ ini belirten Mayes ve Takeballi 133 Bunun yan ı nda (1984)'nin bulgular ı ndan farkl ı d ı r. depolama süresi 4 gün olan yumurtalar ı n plastik torba içinde bekletilmesi, erken ve son dönem embriyo dlümleri, ı skarta oran ı ve ç ı kış gücü üzerine olumlu etkide bulunmu ştur. Nitekim, 4 gün plastik torba içinde bekletilen grupta ç ı k ış gücü % 90.90 olurken plastik torbas ı z 4 gün depolanan ve depolanmayan gruplarda ç ı k ış gücü s ı ras ı yla % 86.70 ve % 90.70 olarak tespit edilmi ştir(Çizelge 1). Yap ı lan istatistik kontrolde plastik torbas ız olarak 4 gün bekletilen grubun ç ı kış gücünün di ğ er 2 gruba göre dü ş ük oldu ğ u saptanm ıştı r(P<0.05). Bu sonuçlar, depolama süresi k ı sa da olsa, ya ş l ı sürü yumurtalann ı n plastik torba içinde bekletilmesinin ç ı kış-gücü üzerinde pozitif etkiye neden olabilece ğ ini belirten., Brake (1996)'in bulgulan ile de uyum içindedir. Ovipozisyon s ı ras ı nda vizkositesi yüksek ve pH's ı 7.6 civar ı nda olan albüminin, pH değ eri depolama süresinin artmas ı yla 9.0-9.5' a kadar yükseli ı-(Kosin ve Konishi 1973; Goodrum ve ark. 1989) ve yo ğ unlu ğ u azal ı r. Bu değ i ş iklikler albümin kalitesinin dü ş mesi anlam ı na gelir. Albümin kalitesinin dü ş mesine ba ğ l ı olarak yumurtan ı n merkezinde bulunan blastodermin konumu değ i ş ir, kabu ğ a doğ ru hareket eder (Brake 1996) ve sonucunda erken dönem embriyo ölümleri artar(Romanoff 1960). Ancak bu de ğ i ş likler, genç ve ya ş l ı sürü yumurtalar ı nda ayn ı değ ildir. Genç sürülerde, ya ş l ı lara göre albümin kalitesi daha yüksektir. Ayr ı ca albümin kalitesinin bozulma ve pH s ı n ı n yükselme h ız ı daha dü ş üktür( Sauveur 1988;Brake ve ark. 1993). Bir ba ş ka ifadeyle genç sürü yumurtalar ı nda albümin kalitesi daha uzun süre muhafaza edilebilmektedir. Bunun yan ı nda Sauveur ve ark. (1967) ve Walsh(1993) albürnin kalitesinin belirli bir seviyenin üstünde ve alt ı nda olmas ı durumunda embriyo ölümlerinin artaca ğı n ı ileri sürerek optimurn ç ı kış gücü için inkubasyon ba ş lang ı c ı nda uygun pH seviyesinin 8.2-8.8 aras ı nda olabileceğ ini bildirmi ş lerdir. Genç sürülerden elde edilen yumurtalarda albümin yo ğ unlu ğ u blastoderme oksijen ve besin maddeleri aktarı m ı n ı s ı n ı rlayacak kadar yüksektir(Meuer ve Baumann 1988; Brake 1996). Bu durum genç sürü yumurtalann ı n depolanmadan makineye konulmas ı nda sorunlar yaratmakta, özellikle erken dönem embriyo ölümlerini art ı rmaktad ı r(Brake ve ark. 1993; Walsh ve ark.1995; Brake ve ark. 1997). elde edilen sürülerden karşı l ı k ya ş l ı Buna yumurtalarda gençlere göre zaten dü ş ük olan albümin kalitesi h ı zla azalmakta, pH seviyesi de yükselmektedir. Albümin kalitesindeki bu h ızl ı dü ş ü ş ün embriyo geli ş imini engelleyecek seviyeye ula ş mamas ı için de ya ş l ı sürü yumurtalar ı n ı , genç sürülerden elde edilenlerin aksine, en k ı sa sürede makineye koymak gerekrnektedir(Brake (1996; Reis ve ark.1997; Brake ve ark. 1997; Lapao ve ark.1999). Bu sa ğ lanamad ığı takdirde CO2 ç ı kışı engellenerek albümin kalitesindeki kayb ı en az seviyede tutacak yollar aranmal ı d ı r. Bu yollardan birisi de yumurtan ı n plastik torbada bekletilmesidir. Nitekim, Brake (1996) de ayn ı yolu önermekte yaln ı z yumurtalar ı plastik torba içinde bekletmenin sadece; ya ş l ı sürü yumurtalar ı nda, albürnin kalitesi dü ş ük olan -hatlarda ve uzun süre depolanacak yumurtalarda olumlu etki gösterebilece ğ ini belirtmektedir. 134 TARIM BILIMLERI DERGISI 2000, Cilt 6, Say ı 3 Çizelge 1. K ısa süreli depolama ve plastik torba kullan ım ı n ya şlı broiler dam ızl ı kları ndan elde edilen yumurtalarda kuluçka özellikleri (%) Depolama süresi Özellikler 4 gün Depolama şekli 0 gün Plastik torbas ız 5.38±0.43a 4.03± 0.43b Plastik torba 3.84±0.43b Orta dönem 0.75±0.21 0.97±0.21 0.45±0.21 Son dönem 3.85±0 56 5.78±0.56a Iskarta oran ı 4.04t0.56b w 0.53±0.35 b 1.72±0.35 a Ç ı kış gücü 90.7±0.89b 90.9±0.89b 86.7±0.89b Embriyo blüm leri Erken dönem 0.45±0.35 a-b: P<0.05 Sonuç Sonuç olarak, ya ş l ı broiler dam ızl ı klardan elde edilen yumurtalar ı n ovipozisyon sonras ı en k ı sa sürede geli ş im rnakinesine yüklenmesi, e ğ er bu sa ğ lanam ı yorsa bunlar ı n, k ı sa süreli de olsa, basit ve kolay bir uygulama olan plastik torba içinde bekletilmesi tavsiye edilebilir Kosin, I. L. and T. Konishi, 1973. Pre-incubation storage conditions and the effect on the subsequent livability of chicken embryos: Exogenous CO2, plastic bags and extended holding periods as factors. Poultry Sci. 52: 296-302. Lapao. C., L. T. Gama. and M. C. Soares, 1999. Effects of broiler breeder age and lenght of egg storage on albumen characteristics and hatchability. Poultry Sci 78:640-645. . Kaynaklar Mayes, F. J. and M. A. Takeballi, 1984.Storage of the eggs of the fowl before incubation. World Poultry Sci. Journal, 40:131140. Becker, W. A. 1964. The storage of White Leghorn hatching eggs in plastic bags. Poultry Sci.43: 1109-1112. Meuer, H. J., R. Baumann, 1988. Oxygen pressure in intra and extraembryonic blood vessels of early chick embryo.Resp. Physiol. 71:331-342. Becker,W. A., J. V. Spencer and J. L.Swartwood, 1964. The preincubation storage of turkey eggs in closed environments. Poultry Sci.43: 1526-1534. Oluyemi, J. A. and O. George, 1972. Some factors affecting hatchability of chicken eggs. Poultry Sci. 51: 1762-1763. Brake, J. T., J. Walsh, and S. V. Vick, 1993. Relationship of egg storage time,storage conditions,flock age,eggshell and albumen characteristics, incubation conditions and machine capacity to broiler hatchability-Review and model synthesis. Zootech Int. 16(1):30-41. Brake, J. T., J. Walsh, C. E. Benton., J. N. Petittle, R. Meijerhof., G. Penalva, 1997. Egg handling and stroge. Poultry Sci. 76: 144151. Brake. J. T. 1996. Optimization of egg handling and storage.World Poultry-Misset, vol 12, no 9:33-39. Burley. R. W. and D. V. Vadehra., 1989. Pages 68-71,372 in: The avian egg. John Wiley and sons New York. Davis. G. T. and A. F. Beeckler., 1962. Plastic packaging of eggs.1. Methods of packaging. Poultry Sci. 41:453-458. Düzgüneş , O., T. Kesici, F. Gürbüz, 1983. Istatistik Metotlar ı . A.Ü. Bas ı mevi,Ankara. Elibol, O. 1997. Kuluçka sonuçlar ı n ı etkileyen etmenler ve kuluçka aksakl ı klar ı n ı n giderilmesi, belirlenmesi üzerinde ara şt ı rmalar. Doktora tezi. A.Ü. Fen Bilimleri Enstitüsü(bas ı lmam ış). Funk, E. M., J. Forward and H. C. Kempster, 1950. Effect of holding temperature on hatchability of eggs. Missouri Agric. Exp.Stn. Bull 539,Columbia. Goodrum, J. W., W. M. Britton and J. B. Davis, 1989. Effect of storage conditions on albumen pH and subsequent hardcooked eggs peelability and albumen shear strength. Poultry Sci. 68:1226-1231. Hurnik, G. I, .B. S. Reinhart and J. F. Hurnik ,1978. Relationship between albumen quality and hatchability in fresh and stored eggs.Poultry Sci. 57:854-857. Kirk., S, G. C. Emmans, R. McDonald and D.Arnot, 1980. Factors affecting the hatchability of eggs from broiler breeders.British Poultry Sci. 21:37. Proudfoot , F .G. 1966. Hatchability of stored chicken eggs as affected by daily turning during storage and prewarming and vacuuming eggs enclosed in plastic with nitrogen.Canadian J.Animal Sci .46:47-50. Proudfoot ,F. G. and H. W. Hulan, 1976. Care of hatching eggs before incubation. Pages 1-17 in: Agriculture Canada Pub.1573/E,Research Station, Kentville,NS, Canada. Reis, L. H., T. Gama, M. C.Soares, 1997. Effects of short storage conditions and broiler breeder age on hatchability,hatching time and chick weights. Poultry Sci 76: 1459-1466. Romanoff, A. L., 1960. The avian embryo;structural and functional development.MacMillan Co. New York. Sauveur, B., R. Ferre, and L. Lacassagne, 1967. Conservation d'oeufs de poule sous atmosphere enrichie en gaz carbonique.Ann.Zootech 16:351. Sauveur, B. R. 1988. Reproduction des volailles et production d'Oeufs. Institut national da la recherche agronomique,Paris. Walsh, T. J. 1993. The effects of flock age, storage humidity,carbon dioxide and lenght of storage on albumen characteristics, weight loss and embriyonic development of broiler eggs. Master's thesis. North Caroline State University.NC. Walsh, T. J., R. E. Rizk and J. Brake, 1995. Effects of storage for 7 and 14 days at two temperatures in the presence or absence of carbon dioxide on albumen characteristics, weight loss and early embriyonic mortality of broiler hatching eggs. Poultry Sci. 74: 1403-1410. Warren, D. C., H. A. Roff and E. Long, 1965. Hatchability of eggs stored in plastic lined egg cases.Poultry Sci. 44:1278-1280. Wilson, H. R. 1991. Interrelationships of egg size, chick size, posthatching growth and hatchability . World's Poultry Sci.J. 47:5-20. TARIM BILIMLERI DERGISi 2000, 6 (3) 135-140 Türkiyede Üretilen Konik Hüzmeli Baz ı Meme Plakalar ı nda Delik Çap ı ve Düzgünlü ğ ünün Belirlenmesi Ergin DURSUN' Yurdaer KARAHAN 2 Ibrahim ÇILINGIR' Geli ş Tarihi :28.04.2000 Özet : Konik hüzmeli memeler gövde, süzgeç, girdap plakas ı , meme plakas ı ve başl ı k olmak üzere be ş parçadan ibarettir. Meme plakas ı ; ilaç da ğıl ı m düzgünlüğ ü, verdi, damla büyüklü ğ ü ve dağı l ı mı nı etkileyen en önemli parçad ı r. Bu çal ış mada, ülkemizde dört farkl ı üretici firma taraf ı ndan üretilen pülverizatör meme plakalar ı nda delik çaplar ı ve delik kenar düzgünlükleri belirlenmi ştir. Bundan ba ş ka incelenen plakalarla verdi ve hacimsel s ı vı dağı lı m ı ölçümleri yap ı lm ışt ır. Araşt ı rma sonuçları na göre plaka delikleri düzgün olmay ı p tam bir dairesellik göstermemektedir. Ayn ı grup plakalar ı n delik çaplar ı nda farkl ı l ı klar bulunmuştur. ölçülen ortalama delik çaplar ı ile firman ı n çap de ğerleri aras ında önemli farkl ı lı klar belirlenmi ştir. Plaka delikierinin pürüzlülü ğ ü ve delik çap ı farkl ıl ı kları , verdi ve hacimsel s ı v ı da ğı l ı mlar ı nda de ğiş imlere neden olmu ştur. Anahtar Kelimeler : Meme plakas ı , delik çap ı , delik düzgünlü ğ ü The Determination of the Orifice Diameter and Correctness of Some Cone Nozzle Orifice Plates Produced ir ı Turkey. Abstract : Cone nozzles have five parts including body, fı lter, swirl plate, orifice plate and cap. Orifice plate is the most important nozzle part effecting spray distribution, flow rate and droplet size. In this study, orifice diameters and correctness of orifice edges of the sprayer orifice plates were determined produced by four different company in Turkey. Moreover, flow rate and volumetric spray distribution measurements were performed for examined orifice plates. According to the research results, orifıces of the plates were inccorrect and not perfect circle. Office diameters of the same group plates were different. The important differences were determined between measured orifice diameters and company's diameter values. The roughness of orifices and the differences of orifice diameter resulted in variations at flow rate and volumetric spray distributions. Key Words : Orifıce plate, orifice diameter, orifıce correctness Giriş Memeler pülverizatörlerin en ucuz parçalar ı ndan biri olmalar ı na karşı n hastal ı k, zararl ı ve yabanc ı otlarla mücadelede arzulanan biyolojik kontrolün sa ğ lanmas ı nda oldukça önemli bir etkiye sahiptirler. Pülverizatörün ba ş ar ı s ı , büyük ölçüde ilac ı n son ç ı k ış noktas ı olan memelerin, görevlerini uygun bir ş ekilde yapmalar ı na ba ğ l ı olmaktad ı r. Memelerin verdilerine, damla çaplar ı na ve ilaç da ğı l ı m paternlerine ba ğ l ı olarak ilaç uygulama etkinli ğ i de ğ i şmektedir. Memelerin görevi; s ı vı ilac ı olabildi ğ ince ayn ı büyüklükteki damlalar halinde ilaçlama süresince verdilerinde de ğ i ş me olmaks ız ı n ve hedef yüzeylerde düzgün bir ilaç de ğ ilim] olacak şekilde uygulamakt ı r. Pülverizatörlerde en fazla kullan ı lan meme tiplerinden birisi konik hüzmeli memelerdir. Bu memelerde s ı vı , bir girdap plakas ı veya yivli govdeden geçerek dönü hareketi kazanmakta ve meme plakas ı deli ğ inden ç ı kan ince s ı v ı şeridi, eksenel ve te ğ etsel h ız bile ş enlerinin etkisiyle içi bo ş konik hüzme şeklini almaktad ı r. Içi dolu bir konik hüzme elde edilmesi için di ğ er bir s ıvı jetinin girdap odas ı na eksenel yönde girmesi ve meme ç ı kış deli ğ i içinde olu ş an hava çekirde ğ ini ortadan kald ı rmas ı gerekmektedir ( Matthews 1992 ). Içi bo ş konik hüzmeli 2 memeler, yüzey kaplaman ı n ve penetrasyonun gerekli oldu ğ u insektisit, fungusit ve büyüme düzenleyici hormonlar ı n uygulanmas ı nda, içi dolu konik hüzmeli memeleler ise ekim öncesi herbisit uygulamalar ı nda yayg ı n bir ş ekilde kullan ı lmaktad ı rlar (Ozkan 1995). Konik hüzmeli mernelerde ilaç da ğı t ı mı n ı etkileyen çok say ı da faktör vard ı r. Meme plakas ı delik ça' p ı , girdap plakas ı ndaki yar ı klar ı n say ı s ı , girdap odas ı 'yüksekli ğ i ve s ıvı bas ı nc ı na ba ğ l ı olarak memelerin verdileri , hüzme aç ı lar ı , damla büyüklükleri ve ilaç da ğı l ı m düzgünlükleri de ğ i ş mektedir (Matthews 1992). Bu faktörlerin d ışı nda meme imalat ı ve imalatta kullan ı lan teknoloji de oldukça önemlidir. Zeren (1974), pülverizatörlerde kullan ı lan konik ve yelpaze hüzmeli memelerde da ğı l ı m ve pülverizasyon karakteristikleri üzerinde yapt ığı çal ış mada, yerli yap ı memelerde süzgeç, meme deli ğ i ve helezon gövde yap ı lar ı n ı n yeterli düzgünlükte olmad ığı n ı belirtmi ştir. Meme deli ğ inin pürüzlü olmas ı n ı n ve deli ğ in eksantrik aç ı lmas ı n ı n, meme plakas ı n ı n meme ba ş l ığı içinde sa ğ a sola kaymas ı n ı n ve helisel kanallar ı n yeterli düzgünlükte i ş lenememesinin pülverizasyon karakteristiklerini önemli ölçüde etkiledi ğ ini vurgulam ışt ı r. Lefebre (1989), düzgün Ankara Üniv. Ziraat Fak. Tanm Makinalan Bölümü - Ankara Harran Only. Meslek Yüksekokulu Tar ı m Makirıaları Program ı - Ş anl ı urfa 136 TARIM BILIMLERI DERG İ S İ 2000, Cilt 6, say ı 3 bir ilaç da ğı l ı m ı ve tekdüze bir damla da ğı l ı m ı n ı n hassas olarak i ş lenmi ş memelerle elde edilebildi ğ ini aç ı klam ıştı r. Dursun ve Çilingir (1991), meme deli ğ inin tam bir daire şeklinde ve pürüzsüz olmas ı gerekti ğ ini, çapak, kanalc ı k ve düzgün aç ı lmam ış deli ğ in ilaç da ğı l ı m ı n ı bozdu ğ unu aç ı klam ış lard ı r. Naman ve Nordby (1965) yapt ı kları ara şt ı rmalarda meme ba ş l ığı n ı n meme gövdesine ba ğ lanmas ı s ı ras ı nda, meme plakas ı ekseninin meme eksenine göre sapmas ı n ı n ilaç dağı l ı m düzgünlü ğ ü ve damla büyüklü ğ ünü önemli derecede etkiledi ğ ini belirtmi ş lerdir. Balc ı ve Ya ğ c ı o ğ lu (1994), s ı rt pülverizatorlerinde kullan ı lan yerli tip hidrolik memelerin verdilerini ve da ğı l ı m düzgünlüklerini belirlemi ş lerdir. Verdi ölçümlerinden elde edilen sonuçlara göre ortalama verdideki sapman ı n, ele al ı nan 40 memede % 0-5,5, 5 memede % 6,3-10,4, 6 memede ise % 14-21,1 aras ı nda değ i şti ğ ini belirtmi ş lerdir. Delik çaplar ı n ı n düzgün olu şturulamamas ı n ı n dairesel da ğı l ı m ı olumsuz yönde etkiledi ğ ini ortaya koymu ş lard ı r. Bu çal ış man ı n amac ı , ülkemizde imal edilen konik hüzmeli meme plakalar ı nda hem delik kenar düzgünlükleri ve delik çap ı de ğ i ş imleri, hem de bunlar ı n meme verdisi ve hacimsel s ıv ı da ğı l ı m ı na etkilerini belirlemektir. Materyal ve Yöntem Bu ara ştı rmada, deneme materyali olarak ülkemizin değ i ş ik bölgelerinde faaliyet gösteren 4 farkl ı üretici firma taraf ı ndan imal edilen konik hüzmeli meme plakalar ı ele al ı nm ışt ı r. Her meme plakas ı bir anma ad ı ile belirtilmi ştir. Anma adlar ı s ı ras ı yla Al, A2, B, G1, G2 ve T'dir. Bu plakalar ı n üretici fı rmalarca belirtilen delik çaplar ı Al ve T plakalar ı nda 1 mm, A2 ve G2 plakalar ı nda 1,5 mm, B ve G1 plakalar ı nda 1,2 mm'dir. Bütün plakalar paslanmaz çelik malzemeden imal edilmi ş lerdir. Her meme plakas ı grubundan 5'er adet tesadüfi olarak seçilmi ş ve gerekli incelemeler bu plakalar üzerinde yap ı lm ışt ı r. Meme plakas ı delik çaplar ı n ı n ölçülmesi için öncelikle plakalar ı n görüntüleri bir taray ı c ı (scanner) yard ı m ı yla bilgisayar ortam ı na aktar ı lm ışt ı r. Delik çaplar ı n ı n hassas bir ş ekilde ölçülmesi amac ı yla görüntüler 15 kat büyütülmü ştür. Daha sonra bir görüntü i ş leme program ı yla delik çaplar ı her bir plaka için delik çevresi boyunca 5 farkl ı noktadan ölçülmü ştür. Bu ölçüm de ğ erlerinden ortalama delik çaplar ı hesaplanm ışt ı r. Ayr ı ca incelenen meme plakalann ı n hepsi için verdi ve hacimsel da ğı l ı m ölçümleri yap ı lm ışt ı r. Verdi ölçümlerinde püskürtme süresi 60 saniye olup ölçümler 6 ve 8 bar bas ı nçlarda yap ı lm ışt ı r. Püskürtülen s ı v ı bir dereceli silindirde toplanarak verdi de ğ erleri Umin olarak belirlenmi ştir. Verdi ölçümleri, her meme plakas ı için 5 tekerrürlü yap ı lm ış ve ölçülen de ğ erlerin ortalamas ı al ı nm ışt ı r. Hacimsel ilaç da ğı l ı m ı , Tar ı m Bakanl ığı Tar ı m Alet ve Makinalar ı Test Merkezi Müdürlü ğ ünde bulunan tek meme denemesine uygun bir paternatör yard ı m ı yla belirlenmi ştir. ölçümler her meme plakas ı için 3 tekerrürlü olarak yap ı lm ış ve elde edilen de ğ erlerin ortalamas ı al ı narak hacimsel da ğı l ı mlar ve bu da ğı l ı mlara ili ş kin varyasyon katsay ı ları (% CV) bulunmu ştur. Hacimsel da ğı l ı m ölçümlerinde bas ı nç 8 bar, meme yüksekli ğ i ise 50 cm olarak sabit tutulmu ştur. Bu ölçümler s ı ras ı nda özellikle meme ekseni ile meme plakas ı ekseninin ayn ı olmas ı na dikkat edilmi ştir. Bulgular ve Tart ışma Deney materyali olarak ele al ı nan meme plakalar ı deliklerinin 5 farkl ı noktadan ölçülen çap de ğ erleri, ortalama çap de ğ erleri ve bu çap de ğ erlerine ili ş kin standart sapma (S) ile varyasyon katsay ı s ı (% CV) değ erleri Çizelge l'de verilmi ştir Çizelge 1'de görülebilece ğ i gibi incelenen meme plakalann ı n herbiri için 5 farkl ı noktadan ölçülen delik çaplar ı ve bu çaplardan hesaplanan ortalama delik çaplar ı birbirinden farkl ı l ı klar göstermektedir. Al plaka grubunda ortalama delik çap ı 0,989-1,047 mm aras ı nda, A2 grubunda 1,504-1,603 mm aras ı nda, B grubunda 1,193-1,238 mm aras ı nda, G1 grubunda 1,133-1,180 mm aras ı nda, G2 grubunda 1,4441,605 mm aras ı nda ve T grubunda 0,904-0,980 mm aras ı nda bulunmu ştur. Plaka deliklerinin 5 farkl ı noktadan ölçülen çap de ğ erlerine ili ş kin CV değ erleri Al grubundaki plakalarda % 1,547-5,190, A2 grubunda % 1,696-4,478, B grubunda % 0,992-3,772, G1 grubunda % 2,128-5,286, G2 grubunda % 2,743-3,024 ve T grubunda ise % 2,1824,741 aras ı nda de ğ i şmi ştir. Bu sonuçlardan anla şı labilece ğ i gibi ; ayn ı gruptaki meme plakalar ı n ı n ortalama delik çaplar ı nda farkl ı l ı klar oldu ğ u gibi, her plakan ı n delik çevresi boyunca farkl ı noktalardan ölçülen delik çaplar ı nda da farkl ı l ı klar olmaktad ı r. Meme plakas ı delik çaplar ı ndaki düzgün ş üzlük ve buna ba ğ li olarak deliklerin daireselliklerindeki de ğ i şimler, deliklerin i ş lenme durumuyia ilgilidir. Ş ekil l'de her plaka grubundan üçer adet meme plakas ı n ı n , ,delik görünümleri verilmi ştir. Plaka deliklerinin genel görünümleri incelendi ğ inde, delik çaplar ı ndaki düzgünsüzlük çok net bir şekilde görülebilir. Bütün plakalarda delik kenarlar ı girintili ç ı kı nt ı l ı ve delik ş ekli tam bir dairesellik göstermemektedir. Deliklerin düzgünsüzlü ğ ünden ba ş ka deliklerin delinmesi s ı ras ı nda oluş an havş alar da Şekil l'de görülebilir. Bu havş alar özellikle Al, G1 ve T grubu plakalarda oldukça belirgindir. Ayrı ca gözle ve mikroskop alt ı nda yap ı lan incelemelerde ço ğ u meme plakas ı n ı n delik çevresi boyunca çapaklar oldu ğ u saptanm ışt ı r. firmalar üretici meme plakalar ı n ı n Incelenen tarafı ndan belirtilen delik çap ı değ erleri ile ölçülen ortalama delik çaplar ı Çizelge 2'de kar şı la şt ı rmal ı olarak verilmi ştir. Çizelge 2'deki % fark de ğ erlerinden (-) ile gösterilenler, ölçülen delik çap ı n ı n firman ı n bildirdi ğ i delik çap ı ndan küçük oldu ğ unu ifade etmektedir. Çizelge 2 incelendi ğ inde, üretici firmalar ı n bildirdikleri plaka delik çaplar ı ile ölçülen delik çaplar ı n ı n birbirinden farkl ı oldu ğu görülebilir. Al, A2 ve B grubu plakalarda ölçülen ortalama hariç delik çaplar ı , Al ve B gruplar ı nda birer plaka değ erlerinden daha büyük firmalar ı n bildirdi ğ i çap plakalar ı n bulunmu ştur. G1, G2 ve T gruplar ı ndaki ölçülen ortalama delik çaplar ı ise G2 grubundaki . 2 plaka hariç firma de ğ erlerinden daha küçük bulunmu ştur. En küçük farkl ı l ı klar % -0,58 ile % 3,17 aras ı nda B grubu, en büyük farkl ı l ı klar % -2,00 ile % -9,60 aras ı nda T grubu plakalar ı nda elde edilmi ştir. DURSUN, E. ve ark."Türkiye'de üretilen konik hüzmeli baz ı meme plakalar ı nda delik çap ı ve düzgünlüg' ünün belirlenmesi" Çizelge 1.Meme plakalarm ın delik çaplar ına ilişkin sonuçlar Plaka tipi Al A2 G1 G2 No di 1 2 3 4 5 1 1,106 1,015 1,038 O 987 1 067 1,521 1 535 1 535 1 459 1 570 1 264 1 219 1 254 1 238 1 228 1,196 1,219 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 T 3 4 Standart sapma Delik çap ı (mm) d2 0,970 1,015 1,033 1 040 O 975 1,468 1 467 1 584 1 543 1 488 1 223 1146 1 241 1167 1 270 1,053 1,103 d3 1,015 ,z0,945 0,999 1 022 1 055 1,532 1 625 1,625 1,582 1 496 1 264 1 219 1 241 1189 1 190 1,151 1,151 1,061 0,989 1,020 1 072 0993 1,509 1 502 1 555 1 490 1 567 1 229 1196 1 233 1 282 1 230 1,188 1,072 d5 1,083 0,980 1,011 1 030 1 017 1,490 1 557 1 716 1 603 1 612 1 181 1187 1 220 1,195 1 208 1,102 1,120 don 1,047 0,989 1,021 1 030 1,021 1, 04 1 537 1 603 1 535 1-546 1,232 1193 1 238 1 214 1 225 1,138 1,133 1,169 1,180 dı Varyasyon katsay ısı (S) . , 0,054 0,029 ,' O .015 O 030 O 039 0,025 O 059 0 071 O 060 . O 053 O 034 0 029 O 012 O 045 O 029 0,060 (% CV) 5,190 2,954 1,547 2 982 3,850 1,696 3 882 4,478 3 947 3 426 2 786 2 510 O 992 3 772 2 439 5,286 0,055 0,025 0,025 0,057 0,444 0,043 4,915 2,173 2,128 4,879 0,044 0,026 3,009 1 675 1,196 1,205 1,188 1,670 1,142 1,172 1,151 1,188 1,196 1,193 1,161 1,140 1,063 1,177 1,205 1,180 1,163 1,548 1,593 1,596 1,615 1,605 1,467 1,535 1 548 1,472 0 880 0,880 O 993 O 890 0,938 1,500 1,523 1 560 1 433 O 926 0,925 1,399 , 1,444 1 572 1 495 O 903 0,948 1,453 1,436 1 508 1 422 O 890 0,921 1,401 1,495 1 569 1 517 O 921 0,910 1,444 1,487 1 551 468 O 040 2 743 O 904 0,917 0 903 O 970 1 029 O 932 0,970 O 917 0,993 O 895 0,971 0,940 0,988 O 973 O 915 0,980 O 019 0,024 0 046 O 022 2 182 2,699 4,741 2,409 0,028 2,849 1,012 A2 Şekil 1. Meme plakası deliklerinin genel görünümleri 2,749 3,024 137 138 TARIM BIL İ MLER İ DERG İ S İ 2000, Cilt 6, Sayı 3 B G1 Şekil 1'in devam ı DURSUN, E. ve ark."Türkiye'de üretilen konik hüzmeli baz ı meme plakalar ı nda delik çap ı ve düzgünlü ğ ünün belirlenmesi" 139 Çizelge 2. Meme plakası deliklerinin ölçülen ve üretici fırmalarca bildirilen delik çaplar ı nı n karşı laştırılmas ı Plaka tipi Delik çap ı (mm) ölçülen 1 1,047 1 0,989 1 1,021 1 1,030 1 1,021 1,5 1,504 1,5 1,537 1,5 1,603 1,5 1,5 1 546 1,2 1,232 1,2 1,2 1,238 1,2 1,214 1,2 1,225 Firma değ eri Al A2 B 1,53 Fark (96) 4,70 -1,10 2,10 3,00 2,10 0,27 2,47 6,87 2,33 3,07 Basınç (Bar) 6 8 6 8 Al A2 B 8 6 8 6 8 6 8 G1 G2 T Ortalama verdi (Umin) 0,710 0,815 1,210 1,361 0,947 1 070 0,800 0,921 1,119 1,405 0,690 O 804 Standart sa ma (S) 14,5 19,0 16,3 30,4 36,1 28,3 10,9 10,1 28,4 48,8 20,7 25,8 -0,58 3,17 1,17 2,08 Varyas. kat. (% CV) 2,0 2,3 1,3 2,2 3,8 2,7 1,3 1,1 2,5 3,5 3,0 3,2 Çizelge 4. Hacimsel da ğılı mlara ait varyasyon katsay ı ları Plaka tipi Al A2 B G1 G2 T 1 29 , 4 47,0 25,7 37,2 32,4 37,7 G1 G2 2,67 1,93 Çizelge 3 Incelenen meme plakaların ı n kullan ı lmas yla ölçülen ortalama verdiler Plaka tipi Plaka tipi Varyasyon katsay ısı (% CV) 2 3 4 38,3 39,6 45 , 3 33,3 36,5 27,0 35,2 28,6 42,5 38,4 33,2 33,3 40,4 35,4 35,2 35,5 31,9 44,1 5 41,5 40,6 44,4 37,0 31,3 39,7 ilaç da ğı l ı m düzgünlü ğ ünü etkileyen önemli faktörlerden biri meme verdilerindeki de ğ iş imdir. Verdiyi etkileyen önemli faktörlerden biri ise meme plakas ı delik çap ı d ı r. Diğ er ko ş ullar sabit iken delik çap ı ndaki de ğ i şime ba ğ l ı olarak meme verdisi de ğ i şmektedir. Incelenen meme plakalar ı n ı n kullan ı lmas ı yla ölçülen ortalama verdiler ile bu verdilere ili şkin standart sapma ve varyasyon katsay ı ları Çizelge 3'te verilmi ştir. 6 bar bas ı nçta en dü ş ük CV değ eri % 1,3 ile A2 ve G1 grubu plakalar ı nda, en yüksek CV değ eri % 3,8 ile B grubu plakalar ı nda elde edilmi ştir. 8 bar bas ı nçta ise en dü ş ük CV % 1,1 ile G1, en yüksek CV 3,5 ile G2 grubu plakalar ı nda bulunmuştur. ilac ı n son çı kış noktas ı olan meme plakas ı deliklerinin düzgün olmamas ı ve delik kenarlar ı nda çapak ve havş aları n bulunmas ı , ilaç dağı l ı m düzgünlü ğ ünün bozulmas ı na neden olmaktad ı r. incelenen meme T Delik çap (mm) Firma de ğ eri ölçülen 1,2 1,138 1,133 1,2 1,169 1,2 1,180 1,2 1,163 1,2 1 605 1,5 1,5 1 444 1,487 1,5 1,5 1,551 1,468 15 0,904 1 0,917 1 1 0,973 0,915 1 0,980 1 Fark (96 ) -5,17 -5,58 -2,58 -1,67 -3,08 7,00 -3,73 -0,87 3,40 -2,13 -9 60 -8 30 -2 70 -8,50 -2,00 plakalar ı n ı n kullan ı lmas ı yla elde edilen hacimsel s ı vı da ğı l ı mlanna ait varyasyon katsay ı ları Çizelge 4'te verilmi ştir. Varyasyon katsay ı ları örtme (overlapping) yap ı lmaks ız ı n tek meme için hesaplanm ışt ı r. Çizelge 4 incelendi ğ inde, ayn ı gruptaki plakaları n % CV değerlerinde önemli farkl ı l ı klar olduğ u görülebilir. Ayn ı gruptaki plakalar ı n % CV değ erleri aras ı nda en büyük farkl ı l ı k % 20 ile A2 grubunda, en küçük farkl ı l ı k ise % 5,2 ile G1 grubunda bulunmu ştur. Ayn ı gruptaki farkl ı plakalar ı n kullan ı lması yla elde edilen hacimsel s ıvı da ğı l ı mları na ilişkin varyasyon katsay ı lar ı n ı n % 20'lere varan bir farkl ı l ı k göstermesi, ilaçlama s ı ras ı nda dü ş ük veya a şı rı dozlu alanlar ı n olu şmas ı na neden olacakt ı r. Sonuç Bu ara ştı rmadan elde edilen sonuçlar a şa ğı daki şekilde özetlenebilir; • Bütün plaka gruplar ı nda plaka delik çevresi boyunca 5 farkl ı noktadan ölçülen çap de ğ erleri ayn ı plaka için farkl ı l ı klar göstermektedir. Çap farkl ı l ı kları n' ifade eden CV değ erleri, meme plakas ı gruplar ı na ba ğ l ı olarak % 0,992 ile % 5,286 aras ı nda değ i ş mektedir. • Ayn ı gruptaki meme plakalar ı n ı n ortalama delik çaplar ı nda farkl ı l ı klar bulunmaktad ı r. • Üretici firmalar taraf ı ndan bildirilen plaka delik çaplar ı ile ölçülen ortalama delik çaplar ı aras ı nda önemli farkl ı l ı klar bulunmaktad ı r. Baz ı plakalar ı n ölçülen ortalama delik çaplar ı firmalar ı n bildirdikleri çap de ğ erlerinden daha küçük, baz ı ları daha büyüktür. • Meme plakas ı delikleri yeterli düzgünlükte olmay ı p, delik kenarlar ı nda çapak ve hav şalar bulunmaktad ı r. kullan ı lmas ı yla • incelenen meme plakaları n ı n ölçülen meme verdileri de belirli s ı n ırlar aras ı nda değ i şmi ştir. Verdi değ erlerine ili şkin CV'ler plaka gruplar ı ve bas ı nca ba ğ l ı olarak % 1,1 ile % 3,8 aras ı nda de ğ i şmektedir. • Hacimsel da ğı l ı mlara ili ş kin % CV de ğerleri her plaka grubundaki plakalar aras ı nda önemli farkl ı l ı klar göstermi ştir. % CV de ğ erlerindeki bu farkl ı l ı klar ayn ı gruptaki plakalar için en az % 5,2 ve en fazla % 20 olarak bulunmu ştur. 140 TARIM B İ L İ MLER İ DERG İ S İ 2000, Cilt 6, Say ı 3 Kaynaklar Balc ı , Y. ve A. Ya ğc ı oğ lu, 1994. S ı rt Pülverizatörlerinde Kullan ı lan Baz ı Hidrolik Memelerin Volumetrik Da ğı l ı m Karakteristikleri. Tar ı msal Mekanizasyon 15. Ulusal Kongresi, s.231-240, Antalya. Lefebre, A. H. 1989. Atomization and Sprays. Purdue University, West Lafayette, Indiana, 421 p, USA. Matthews, G. A. 1992. Pesticide Application Methods. 2.Edition, Longman, 405 p., New York. Dursun, E. ve Çilingir, 1991. Pülverizasyon Karakteristikleri Ve Buna Etkili Faktörler. A.O. Ziraat Fakültesi Yay ı nlar ı ; 1206, Derlemeler 49, 34 s., Ankara. Ozkan, H. E. 1995. Herbicide Application Equipment. Chapter 6.1n; Handbook on Weed Management Systems, Ed; A.E. Smith, Marcel Dekker Inc., USA. Naman, J. and A. Nordby, 1965. Intluence of the Eccentric Nozzle Orifıce Position on the Spray Pattern and Droplet Size. Journal of Agricultural Engineering Research, 10 (4): 342347. Zeren, Y. 1974. Mekanik Pülverizatörlerde Kullan ı lan Konik ve Yelpaze Hüzmeli Memelerde Da ğı l ı m ve Pülverizasyon Karakteristikleri Üzerinde Bir Ara şt ı rma. Ç.Ü. Ziraat Fakültesi Doktora Tezi, 85 s., Adana. 

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Turnbull et al. BMC Family Practice (2018) 19:25 DOI 10.1186/s12875-018-0716-7 RESEARCH ARTICLE Open Access What gives rise to clinician gut feeling, its influence on management decisions and its prognostic value for children with RTI in primary care: a prospective cohort study Sophie Turnbull1,10*, Patricia J. Lucas2, Niamh M. Redmond3, Hannah Christensen4, Hannah Thornton1, Christie Cabral1, Peter S. Blair5, Brendan C. Delaney6, Matthew Thompson7, Paul Little8, Tim J. Peters9 and Alastair D. Hay1 Abstract Background: The objectives were to identify 1) the clinician and child characteristics associated with; 2) clinical management decisions following from, and; 3) the prognostic value of; a clinician’s ‘gut feeling something is wrong’ for children presenting to primary care with acute cough and respiratory tract infection (RTI). Methods: Multicentre prospective cohort study where 518 primary care clinicians across 244 general practices in England assessed 8394 children aged ≥3 months and < 16 years for acute cough and RTI. The main outcome measures were: Self-reported clinician ‘gut feeling’; clinician management decisions (antibiotic prescribing, referral for acute admission); and child’s prognosis (reconsultation with evidence of illness deterioration, hospital admission in the 30 days following recruitment). Results: Clinician years since qualification, parent reported symptoms (illness severity score ≥ 7/10, severe fever < 24 h, low energy, shortness of breath) and clinical examination findings (crackles/ crepitations on chest auscultation, recession, pallor, bronchial breathing, wheeze, temperature ≥ 37.8 °C, tachypnoea and inflamed pharynx) independently contributed towards a clinician ‘gut feeling that something was wrong’. ‘Gut feeling’ was independently associated with increased antibiotic prescribing and referral for secondary care assessment. After adjustment for other associated factors, gut feeling was not associated with reconsultations or hospital admissions. Conclusions: Clinicians were more likely to report a gut feeling something is wrong, when they were more experienced or when children were more unwell. Gut feeling is independently and strongly associated with antibiotic prescribing and referral to secondary care, but not with two indicators of poor child health. Keywords: Child, Respiratory tract infections, Primary health care, Prognosis, Emotions, Decision making, Paediatric, Cough * Correspondence: Sophie.turnbull@bristol.ac.uk 1 Centre for Academic Primary Care, Population Health Sciences, Bristol Medical School, University of Bristol, Canynge Hall, 39 Whatley Road, Bristol BS8 2PS, UK 10 Centre for Academic Primary Care, School of Social and Community Medicine, University of Bristol, Bristol BS8 2PS, UK Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Turnbull et al. BMC Family Practice (2018) 19:25 Background Acute cough with respiratory tract infection (RTI) in children is the most common problem managed by health services internationally [1, 2]. While the majority of childhood RTIs are self-limiting, a small number result in serious illness and hospitalisation [3]. Clinicians report that uncertainty regarding both the diagnosis and prognosis of some children with RTIs are important drivers of antibiotic prescribing, [4–6] contributing to over-prescribing [7] and antimicrobial resistance [8]. Knowing the prognosis of children presenting to primary care with acute RTIs can be challenging as clinicians cannot always be certain where in the illness trajectory the child has presented, and whether red flag symptoms and signs are absent or are yet to develop. In cases where there is uncertainty, clinical intuition or ‘gut feeling’ is thought to play a part in management decisions. The current literature offers two definitions of clinician gut feeling. Both propose it arises when the clinician feels uncertain or has a low ‘feeling of rightness’, [9, 10] and both have been shown to influence management decisions [11]. In some studies, a comparison has been made between a sense of reassurance versus a feeling that ‘something is wrong’ which defines a broad set of cases where clinicians are worried [11]. In others, authors define a gut feeling as a sense of dissonance between intuitive and analytic reasoning; a gut feeling something is wrong despite a lack of clinical markers [9]. The latter definition draws on theories of clinical reasoning distinguishing between “intuitive” processing, which is rapid and unconscious and “analytic” processing that involves slow deliberative reasoning [12–14]. In primary care, gut feeling has been described as an incorporation of clinician knowledge, experience and information about the patient [9]. In the case of childhood RTI, clinicians report using immediately apparent symptoms and signs (the child’s energy, pallor and breathing) to distinguish severe from non-severe cases, using pattern recognition and drawing on past experience [5]. A recent study in children with any acute illness, found that: general appearance, breathing pattern, weight loss, history of convulsions and parental concern that the illness was different from any previously experienced predicted the feeling something was wrong when clinical impression was of non-serious illness [15]. This evidence goes some way to characterising the factors influencing clinical gut feeling, but does not identify the specific clinical features associated with particular illnesses, or provide objective assessments of illness severity. Despite qualitative studies indicating the importance of clinician intuition for primary care doctors and in nursing, [16] there is a paucity of quantitative evidence regarding the prognostic value of gut feelings and Page 2 of 12 whether primary care clinicians utilise them in their decision making. A systematic review suggested that gut feeling in itself should be viewed as a ‘diagnostic red flag’ and that it had greater diagnostic value than the majority of illness specific symptoms and signs [17]. Another study indicated that in situations where there was uncertainty after clinical assessment, gut feeling was highly predictive of serious infective illness [15]. We used a large prospective cohort study of children presenting to primary care with acute cough and RTI to address three objectives regarding ‘gut feeling that something is wrong’ (from here on ‘gut feeling’): (i) to describe the clinician and child characteristics that drive clinician to have a gut feeling that something is wrong; (ii) to investigate if gut feeling influences management decisions; and (iii) to evaluate the prognostic value of gut feeling in relation to primary care reconsultations with evidence of illness deterioration, and hospital admissions. Methods Design and setting The ‘TARGET’ study [18] was a multicentre, prospective cohort study that recruited children presenting to general practices with acute cough and RTI, between July 2011 and May 2013. Practices were recruited and trained by four University hubs (Bristol, London, Oxford and Southampton). The primary aim of the study was to develop a clinical rule that could help clinicians improve their use of antibiotics by using baseline clinical characteristics to predict the children that would be hospitalised in the next 30 days [3]. Here we present findings from secondary analysis of this dataset, which are reported following the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) recommendations [19]. Participants Clinicians (General Practitioners: GPs and ‘prescribing’ Nurse Practitioners: NPs), were eligible to recruit to the study if they were working in participating primary care practices and reported that they prescribed antibiotics in ≤30% children with RTIs (to reduce the possibility of confounding by indication). Clinicians were asked to specify a priori their preferred recruitment strategy (for example, consecutive or first eligible on the day). Children were eligible if aged ≥3 months to < 16 years and presenting with an acute (≤28 days) cough and other symptoms of an RTI. Children with an infected exacerbation of asthma and those severely unwell (for example, requiring same day hospital assessment or admission) were included. Children were excluded if: presenting with a non-infective exacerbation of asthma; immunocompromised; previously recruited or recently Turnbull et al. BMC Family Practice (2018) 19:25 participated in other research; or temporarily registered at the GP practice. Data collection Clinician characteristics Clinician characteristics included the clinician type (GP or NP), number of years qualified and evidence of further medical/ health qualifications (Diploma in Child Health (DCH) or Membership of the Royal College of General Practitioners (MRCGP)). Child characteristics At the baseline consultation the recruiting clinician used a structured case report form (CRF) to record sociodemographic information, parent-reported symptoms (including severity of symptoms in the past 24 h) and physical examination findings. A parent and clinician reported illness severity score was collected, scored 0-10 (see Fig. 1. Case report form). Information about the child’s past medical history was collected from the primary care medical record. The 2010 Indices of Multiple Deprivation (IMD) provided a measure of neighbourhood deprivation linked to the child’s home postcode [20]. Page 3 of 12 they prescribe antibiotics ‘just in case’ children are subsequently admitted to hospital with their RTI, and we sought to use an inclusive definition. Second, our previous [21] and more recent [22] research has shown the symptoms of acute cough and RTI take 25 days for most (90%) of children to resolve. A hospitalisation outcome with a ≤ 7 day cut off was also used as a sensitivity analysis to ensure that there was not a temporal association between the prognostic value of gut feeling and the hospitalisation outcome. We calculated the Kappa statistic to assess inter-rater reliability of the two reviewers that independently assessed the primary care medical record for these two health outcomes. Statistical analysis Treatment of variables At the end of their clinical evaluation, the clinician was also asked to respond ‘yes’ or ‘no’ to the statement “my gut feeling is ‘something is wrong’” with the child. A definition was provided in the clinician instruction booklet that gut feeling in this context “represents clinicians gut feeling that the child’s illness may be more serious than is superficially apparent”. This question was located at the end of the CRF, following the socio-demographic, symptoms and physical examination findings, but prior to the reporting of management decisions. Parent-reported symptom severity (mild, moderate, severe) in the 24 h prior to consultation was transformed into a binary outcome with a threshold dependent on overall prevalence: to either ‘severe/not severe’ (where > 5% of children reported as being severe or ‘moderate or severe/not moderate or severe’ (where < 5% of children reported being severe). This pragmatic cut-off was chosen prior to analysis to avoid severity variables with very low prevalence. We used accepted clinical cut offs (temperature > 37.8 °C, [23] capillary refill time (CRT) ≥3 s, [24, 25] oxygen saturation ≤ 95% [26]) or 25th percentile or 75th percentile thresholds to dichotomise continuous data, and we used age-adjusted thresholds for heart and respiratory rates [27]. Given the large number of variables being tested, continuous outcomes were initially categorised to help interpret and contextualise the final model, a sensitivity analysis was then conducted to assess the distribution of these continuous variables if included in the model. Management decisions Determinants of gut feeling Clinicians were asked to record if the child was referred for a secondary care assessment and if they prescribed antibiotics. We first used univariable multilevel random effects logistic regression (accounting for between-clinician differences) to assess associations between clinician profile and children’s baseline characteristics, with gut feeling. Modelling was conducted using a multivariable multilevel random effects logistic regression model (accounting for clustering at the clinician level) and variable selection using the backward stepwise method with a 5% model entry threshold. For the ‘determinant’ analyses, the outcome variable was the clinician’s gut feeling and (given the large number of variables investigated) model retention was set at the 1% level. A more liberal threshold was employed for the univariable stage than for the multivariable stage because the former was used as a selection filter for the latter and we did not want to miss any potential associations of interest, whereas for the final selection of variables it was necessary to be more Gut feeling Prognostic value Two indicators of poor child health were collected from the primary care medical record by trained notes reviewers at least 3 months after study entry (to allow sufficient time for the hospital discharge information to reach the medical record): primary care re-consultations for the same RTI with evidence of symptom deterioration; and hospital admissions for RTI in the 30 days following recruitment (using hospital discharge summaries). The 30 days threshold for hospitalisation was selected for two reasons. First, the rationale for the primary aim of the TARGET study [3] (from which the ‘gut feeling’ paper derives its data) was that clinicians report Turnbull et al. BMC Family Practice (2018) 19:25 Page 4 of 12 Fig. 1 Case report form conservative given both the number of variables considered and the large sample size (which could potentially result in small effects being detected statistically). Global clinician illness severity score was not included in this analysis as it was suspected that there was overlap in what this and gut feeling variables represented to the clinician. Considering prior literature indicating prognostic value of gut feeling in the absence of clinical signs of severe illness, we undertook a sensitivity analysis to explore the contribution of high illness severity score to the model. Evidence that clustering by clinician was important in the multivariable model was explored using the Likelihood-ratio test (LRT). The accuracy of the resulting model was represented by the Area Under the Receiver Operating Characteristic (AUROC) curve. Turnbull et al. BMC Family Practice (2018) 19:25 Multiple imputation was employed as a sensitivity analysis to check whether the model was robust [28]. Management decisions and prognostic value Univariable and multivariable multilevel logistic regression modelling was employed, using backward stepwise selection (accounting for between-clinician differences), as well as variables associated with each of those outcomes retaining variables with a p-value of < 0.05. Two models were used to investigate the associations between clinician gut feeling and the decision to: 1) prescribe antibiotics, and 2) refer the child to hospital for acute admission. Two further models were used to investigate the associations between gut feeling and: 1) reconsultation for the same RTI with evidence of deterioration, and; 2) hospitalisation for a RTI in the 30 days following recruitment. A further multivariable model was used in the sensitivity analysis exploring the association between gut feeling and the hospitalisation outcome with a ≤ 7 day cut off. To ensure the prognostic value of gut feeling did not differ with definition of gut feeling, a univariable analysis was conducted to explore the association between hospitalisation and the more focused definition described by a previous study: gut feeling when clinical impression was of non-serious illness [15]. Due to the exploratory nature of these analyses, and to ensure we did not miss any potential influences, the more liberal threshold was employed for the multivariable analysis. LRT was used to explore the influence of clustering by clinician in the multivariate models. Data were analysed using STATA version 13.1. Results Clinicians and children Of the 842 clinicians agreeing to participate, 519 (62%) located at 247 general practices recruited at least one child (median 5, inter-quartile range (IQR) 2-15 children) into the study. Of the clinicians who recruited at least one child who was retained in the study, data were available about clinician type for 502 (97%) clinicians; of these 77 (15%) were NPs and 425 (85%) were GPs. Data on years since qualification were available for 498/518 (96%) of the clinicians: median of 20 years (range 1-44, IQR 13-26 years). Of the 519 clinicians, 85 (16%) reported having at least one additional qualification (DCH or MRCGP). A total of 8613 children were recruited to the study, of whom 219 (3%) children were excluded: 181 did not meet the eligibility criteria, 32 did not have baseline data and six were withdrawn. Of the 8394 children recruited and retained, clinicians recorded their gut feeling for 8377 (99.8%) - all analyses that follow use these 8377 children. Participant flow through the study is available in Fig. 2. Oxygen saturation was removed from all analysis as these Page 5 of 12 data were missing for 50% of children due to a lack of available paediatric oxygen saturation monitors. All other reported variables had < 2% missing data. Children’s median age was 3 years (range 3 months-15 years, IQR, 16 years) and 1390 (17%) were under 1 year. There were slightly more boys (52%) than girls, and of the 8333 (99.5%) for whom ethnicity data were available the majority (6540, 78%) were white (similar to UK Census 2011 data) [29]. Families’ median deprivation (IMD) score was 16.7 (IQR 8.8-29.5), similar to the English median (17.2, IQR 9.8-30.2) [20]. Median clinician reported illness severity was 3 (range 0-9, IQR 2-4). Clinicians reported a gut feeling something was wrong in 1706 (20%) cases. 3110 (37%) were prescribed an antibiotic, with 2341 (28%) given an immediate script and 769 (9%) given a delayed script. Seventy-four children were immediately referred for secondary care assessment at the recruiting consultation, of whom 13 were subsequently admitted. A further 65 children were admitted to hospital with a RTI in the subsequent 30 days. For the 78 hospitalised children the median number of days to hospitalisation was 2 days (IQR 1-12 days). The hospital discharge diagnoses were: lower respiratory tract infection (19%); bronchiolitis (18%); viral wheeze (15%); upper respiratory tract infection (13%); croup (8%); infected exacerbation of asthma (8%); tonsillitis (6%); viral illness (5%); febrile illness (3%); and pneumonia (1%). Of the 8193 children for whom data were available relating to primary care reconsultations in the 30 days following baseline, 1847 reconsulted for an RTI. Of these 354 (4%) reconsulted for the same illness with evidence of symptom deterioration, with a median of 5 days (IQR 2-11 days) to this reconsultation. The percentage interrater agreement (kappa) for hospitalisation and reconsultation for the same illness with evidence of deterioration were 90% (Kappa 0.80) and 84% (Kappa 0.67) respectively. There were 242/8377 (3%) children in whom the clinician reported a low illness severity score (≤2) whilst also reporting they a gut feeling something was wrong. Determinants of gut feeling Univariable analyses Fifty-one (84%) of the 61 variables measured were associated with a gut feeling (with p-values < 0.05 (Additional file 1: Web Appendix, Table S1). Two of the three clinician characteristics were associated: those with more years since qualification and NPs reported having a gut feeling in a higher proportion of the children than GPs (27% vs. 18%), while additional qualifications were not associated. Only one of the five sociodemographic variables collected were associated: being male (55% vs 51%). There was no evidence of a difference in the children’s ethnicity, age, the mother’s age at the time of the child’s birth, or home IMD score in the two groups. Turnbull et al. BMC Family Practice (2018) 19:25 Page 6 of 12 Fig. 2 Flow of participants through the study All the four variables collected regarding the child’s medical history were associated with gut feeling. The child was more likely to: have a chronic condition (21% vs 18%), a previous or current asthma diagnosis (12% vs 8%), and to have consulted for an RTI on two or more occasions in the 12 months prior to recruitment (36% vs 35%), than the children for whom the clinician did not have a gut feeling. There was evidence that 44/49 (90%) of the symptoms and signs that were recorded at baseline were associated with gut feeling. The five symptoms and signs that did not reach the 5% threshold were low illness duration prior to recruitment, barking or croupy cough, blocked or runny nose, diarrhoea and stridor (Additional file 1: Web Appendix, Table S1). Multivariable analysis There was strong evidence that increasing years since clinician qualification, five carer-reported symptoms Turnbull et al. BMC Family Practice (2018) 19:25 Page 7 of 12 and eight clinically-reported signs were independently associated with gut feeling (Table 1). The strongest was crackles and crepitations. In the multivariable model there was no evidence of association with clinician type (NP versus GP), additional clinician qualifications or any of the socio-demographic variables. There was strong evidence that clustering by clinician was important in the model (LRT of rho = 0: p < 0.001) when compared with the multivariable model without clustering. The AUROC curve was 0.82, 95% confidence interval (CI) 0.80-0.83. Imputing the missing data for the final model made little difference to the odds ratios, confidence intervals and p-values. The sensitivity analysis exploring the impact of high clinician illness score on the model resulted in few changes: high clinician illness severity score was retained, while severe fever in the last 24 h and parent reporting that that illness was worse recently dropped out of the model. There was little change to the remaining estimates, except for crackles and crepitations, where the OR fell from 14.56 (95% CI 11.71- 18.12) to 9.57 (95% CI 7.64-11.99) and the AUROC increased from 0.81 (95% CI 0.80-0.83) to 0.82 (95% CI 0.83-0.85). Management decisions and gut feeling There was strong evidence in both the univariable and multivariable analysis that when clinicians report a gut feeling, they were more likely to prescribe antibiotics and refer for same day further secondary care assessment (Table 2, including description of the covariates retained in the multivariate model at the < 1% level). These findings were independent of clinician’s impression of illness severity. Prognostic value of gut feeling There was no evidence to suggest that the children for whom the clinician had a gut feeling were more likely to re-consult for the same illness with evidence of illness deterioration in the 30 days following recruitment in the univariable or multivariable analysis. They were over twice as likely to be admitted to hospital for a RTI Table 1 Determinants: Multivariable associations between parent-reported symptoms, clinician-reported observations, clinician profile and ‘gut feeling’ Significant predictors OR 95% CI p-value 10.3 Ref Ref < 0.001 5 d.f.* 423/2213 19.1 0.55 0.30-1.02 80.5 235/1206 19.5 1.44 0.71-2.90 76.9 367/1586 23.1 1.28 0.57-2.87 No gut feeling Yes gut feeling n/N % n/N % 1-9 years 646/720 89.7 74/720 10-14 years 1790/2213 80.9 15-19 years 971/1206 20-24 years 1219/1586 Years qualified (Categorical) 25-29 years 875/1159 75.5 284/1159 24.5 1.67 0.76-3.68 30+ years 1170/1493 78.4 323/1493 21.6 1.08 0.47-2.46 High parent illness severity score (≥7/10) 1459/6650 21.9 681/1702 40.0 1.67 1.39-2.01 < 0.001 Fever in the last 24 h (severe) 363/6647 5.5 177/1700 10.4 1.66 1.23-2.26 0.001 Low energy during illness 3404/6669 51.0 1100/1704 65.6 1.65 1.36-1.99 < 0.001 Parent reported symptoms Shortness of breath during illness 2115/6670 31.7 857/1704 50.3 1.50 1.25-1.81 < 0.001 Illness worse recently 4182/6663 62.8 1339/1704 78.6 1.44 1.18-1.76 < 0.001 695/6661 10.4 901/1704 52.9 14.56 11.71-18.12 < 0.001 Recession 164/6663 2.5 239/1704 14.0 3.31 2.29-4.79 < 0.001 Pallor 6185/6663 7.2 345/1705 20.2 3.10 2.30-4.18 < 0.001 Clinical signs Crackles and crepitations Bronchial breath 136/6658 2.0 142/1703 8.3 2.99 1.90-4.70 < 0.001 Wheeze (recorded in consultation) 675/6663 10.1 560/1703 32.9 2.78 2.23-3.46 < 0.001 High temperature (≥37.8 °C recorded in consultation) 629/6653 9.5 415/1702 24.4 2.32 1.84-2.91 < 0.001 High respiratory rate (age related cut offs) 825/6634 12.4 414/1698 24.4 1.64 1.30-2.07 < 0.001 Inflamed pharynx 1873/6647 28.2 521/1702 30.6 1.51 1.23-1.86 < 0.001 N = 8217/8377 (98.1% of the sample) 514 clusters (clinicians) * Degrees of freedom Turnbull et al. BMC Family Practice (2018) 19:25 Page 8 of 12 Table 2 Management decisions: relationship between ‘gut feeling’ and subsequent clinical management Treatment No gut feeling Yes gut feeling Univariable analysis controlling for clustering Multivariable analysis controlling for clustering and significant covariates n/N % n/N % OR 95% CI P-value OR 95% CI P-value 25.6 1403/1706 82.2 20.80 17.42-24.83 < 0.001 5.85 4.67-7.32 < 0.001± 3.4 19.27 9.83-37.79 < 0.001 12.64 6.31-25.32 < 0.001¥ Prescribed antibiotics at baseline consultation Any antibiotics prescribed 1707/6671 Referred for acute admission (during the recruiting consultation) Referral for acute admission 16/6671 0.2 58/1706 ± Co-variates of any antibiotic prescription (retained at the < 1% level): illness has got worse recently, child’s age (< 2 years), barking cough, fever, diarrhoea, low energy and productive cough during the illness, moderate/severe wheeze and severe fever in the last 24 h, recession, crackles and crepitations, wheeze (as reported by the clinician), bronchial breath, inflamed pharynx, high temperature, high clinician and parent illness severity scores, low illness duration and gut feeling that something is wrong ¥ Co-variates of referral for acute admission (retained at the < 1% level): Recession, gut feeling that something is wrong and capillary refill time (CRT) in the 30 days following recruitment. However, after adjusting for other variables associated with hospital admission, this relationship was attenuated. This was also true for the sensitivity analysis examining the 7day hospitalisation outcome. The presence of clinician reported wheeze and/or recession in the multivariate model reduced the association with gut feeling below the < 5% threshold (Table 3, including description of covariates retained in the multivariate model). There was evidence of clinician clustering effect for reconsultation (LRT of rho = 0: p < 0.001) but not hospitalisation (30 days LRT of rho = 0: p = 0.30, 7 days LRT of rho = 0: p = 0.43), indicating that there was a relationship between the clinician who had seen the child and the likelihood of child reconsulting, but not being hospitalised. There was also no evidence of an association between hospitalisation and the more focused definition of gut feeling (described in previous studies) was explored at univariate level(OR 1.26, 95% CI 0.384.13, p = 0.71). Discussion Summary of findings In a large, rigorously conducted, prospective cohort study, gut feeling was more commonly reported by more experienced clinicians and in children with parent-reported symptoms and physical examination signs suggesting more severe illnesses. Gut feeling was strongly associated with management decisions – both antibiotic prescribing and same-day referral for admission. Despite this, we did not find strong evidence that gut feeling was associated with indicators of poorer prognosis in the following 30 days, such as reconsulting in primary care with evidence of illness deterioration or being hospitalised for a RTI, at least not after controlling for potential confounders. Strengths and limitations To our knowledge, ours is the largest, prospective study to describe the determinants, management decisions and prognostic value of gut feeling for respiratory tract infections in children in routine, office-based primary Table 3 Prognosis: relationship between ‘gut feeling’ and children’s health outcomes Health outcome No gut feeling Yes gut feeling Univariable analysis controlling for clustering Multivariable analysis controlling for clustering and significant covariates n/N % n/N % OR 95% CI P-value OR 95% CI P-value Re-consultation for the same illness with evidence of illness deterioration (≥1) in the 30 days following recruitment 271/6523 4.2 83/1670 5.0 1.30 0.99 -1.71 0.06 1.08 0.80-1.46 0.60¥ Admitted to hospital in the 30 days following recruitment 48/6671 0.7 30/1706 1.8 2.47 1.55-3.95 < 0.001 1.11 0.65-1.89 0.71≠ 24/6647 0.4 25/1681 1.5 4.20 2.34-7.54 < 0.001 1.62 0.83-3.14 0.16α Health outcome Sensitivity analysis Admitted to hospital in the ≤7 days post recruitment ¥ Co-variates of re-consultation for the same RTI with evidence of deterioration (retained at the < 5% level): ≥2 consultations for RTI in primary care in the year prior to baseline, low illness duration prior to baseline, previous asthma diagnosis, current asthma diagnosis, child’s age at baseline (< 2 years), white ethnicity, parent reported barking cough, wheeze and taking fewer fluids during the illness, moderate/severe vomiting and fewer fluids in the 24 h prior to baseline, severe eating less in the 24 h prior to baseline, wheeze and recession (as reported by the clinician) ≠ Co-variates of hospitalisation (retained at the < 5% level): child’s age (< 2 years), low illness duration prior to baseline, current asthma diagnosis, moderate/severe vomiting, recession, wheeze (as reported by the clinician), high temperature (age related cut offs) α Co-variates of hospitalisation ≤7 days post recruitment (retained at the < 5% level): child’s age (< 2 years), low illness (< 3 days) duration prior to baseline, current asthma diagnoses, moderate/severe vomiting, recession, wheeze (as reported by the clinician), high temperature (age related cut offs) Turnbull et al. BMC Family Practice (2018) 19:25 care [2]. We included a broad range of clinician, demographic, child and clinical characteristics and the study focussed on a clinical group in whom clinicians report diagnostic and prognostic uncertainty [6]. Determinants of gut feeling were measured according to routine clinical practice using standardised reporting forms on the day of recruitment, blind to the prognostic outcomes, which were reliably measured from the primary care record. We used rigorous techniques to adjust for confounding. We are aware of five main limitations. First, while we offered some instructions to support completion of the question “My gut feeling is ‘something is wrong’”, including a definition, there was no attempt to interpret it and clinicians were simply asked to report its presence/absence. While the low question refusal suggests clinicians understood the question, it is likely the presence of gut feeling means different things to different clinicians. Definitions of gut feeling overlap, and may not be clear to clinicians. Given our relatively high (20%) prevalence of gut feeling, the strong associations found with symptoms and signs (such as crepitations/crackles) and with high clinician reported illness severity score, it is likely our clinicians were using a broad definition, such as “the presence of a significant illness”. Second, although focussing on an extremely common primary care presentation, [2] findings may not generalise to other illnesses or different age groups. Third, we adjusted for a wide range of potential confounders but it is possible that other unmeasured factors could be important. We are aware of another study that found ‘parent concern that the illness was different to other illnesses’, weight loss and history of convulsions have been shown to be associated with gut feeling and these were not collected in our study [15]. Fourth, while our conservative model entry and exit thresholds have minimised scope for Type I error, the relative infrequency of the hospitalisation outcome, and the fact this was a secondary analysis of data collected for another purpose, [18] could have led to Type II error and us incorrectly concluding that gut feeling does not have prognostic value. The large study size mitigates against this for re-consultation. It is plausible some cases of re-consultation or hospitalisation were avoided where antibiotics were given and this may mask associations with gut feeling (so-called confounding by indication). However, there is limited evidence of the mitigating effects of antibiotics on prognostic outcome for RTI. Two studies, one with adults [30] and the other with children (in submission [31]) with RTIs found antibiotics did not reduce hospitalisation, and immediate antibiotics did not reduce reconsultations. Finally, the outcomes used to explore the prognostic value of gut feeling are indicators of poor health, but we acknowledge that other factors beyond the child’s illness severity can influence these, such as parent behaviour and available services in the area. Page 9 of 12 Comparison with existing literature Determinants of gut feeling Previous studies have described gut feeling in primary care clinicians as being derived from a combination of patient characteristics and clinicians’ knowledge and past experience [32, 33]. Like others we found that pallor or appearance, energy and breathing patterns were associated with gut feeling [5, 15]. Our results contrast with a previous study, which did not find evidence to support their hypothesis that high temperature contributed towards gut feeling, but we found high temperature and carer-reported severe fever both contributed [15]. The literature is contradictory regarding the role of clinician experience. For instance, both we and a previous qualitative study found more experienced family primary physicians were more likely to report having a gut feeling something was wrong, [17] whereas another quantitative study found that more senior doctors were less likely to report it [15]. These differences may be due to differences in participating children or definition of gut feeling; van den Bruel’s study included children with any non-serious acute illness, whereas we studied children with RTI regardless of clinician-reported illness severity. In their study they conceptualised gut feeling as “intuitive feeling that something was wrong even if the clinician was unsure why” and included the 3.1% children where the clinician reported a gut feeling in addition to the illness was not severe. Our definition was broader and included 20% of the children. However, in our sample a clinician reported a gut feeling combined with a low illness severity score approximates this definition, and we found the same proportion (3%). The concept that gut feeling is more commonly reported in more experienced clinicians is supported by the learning perspective of intuition: where intuition is the consequence of unconscious drawing on an implicit knowledge base and recognition of patterns of clinical markers that is developed though experience and may be associated with previous negative or uncertain outcomes. As demonstrated by our findings, more experienced does not mean more accurate. The validity of the gut feeling as a guide to decision-making relies not only on the quantity but also the quality of the feedback. If there is good feedback during the learning process, this results in good or accurate gut feeling and associated decisionmaking. Conversely, poor or absent feedback results in poor intuition [34]. Management decisions and gut feeling Our findings agree with previous studies [32] that found gut feeling can play a substantial role in treatment decisions. Turnbull et al. BMC Family Practice (2018) 19:25 Prognostic value of gut feeling A recent systematic review suggested that gut feeling was of greater diagnostic value than the majority of symptoms and signs [17]. Two recent prospective studies reported that gut feeling in primary care was also associated with serious infectious illness in children in Belgium [15] and serious disease in adults and children in Denmark [35]. In contrast, we did not find evidence that gut feeling was associated with subsequent reconsultations with evidence of deterioration. When considered in isolation, we found gut feeling was associated with hospitalisation, however, when other clinical signs (wheeze and/or recession) were taken into account, gut feeling was no longer associated. Indeed, our recent paper identified seven other characteristics that were independently associated with hospitalisation – namely age less than 2 years, current asthma, illness duration of 3 days or less, parent-reported moderate or severe vomiting in the previous 24 h, severe fever in the previous 24 h or a body temperature of 37·8 °C or more at presentation, clinician-reported intercostal or subcostal recession, and wheeze on auscultation [3]. Use of these may reduce prognostic uncertainty and reduce reliance on gut feeling. Clinical, teaching and research implications Our study suggests that for children presenting to primary care with acute cough and RTI, the gut feeling that something is wrong, which appears to drive clinical decision-making in this group, are not associated with either re-consultation or hospital admission, at least not after potential confounding effects were taken into account [30, 31]. Given the high levels of uncertainty reported by clinicians looking after this group of patients, [5, 36] it is not surprising they wish to use all the tools at their disposal, including gut feeling. However, our results suggest some clinicians may over-value gut feeling in their clinical decision making in this group of patients and that they are using gut feeling as a proxy or unconscious marker of other important symptoms and signs. Gut feeling may have value in the absence of known, objectively assessed markers of risk, however, attending to objective assessment of these risks is preferable when this information is available. Future research may wish to focus on a more specific definition of gut feeling, including perhaps the sense of dissonance arising when conscious and unconscious assessment processes are in conflict. Conclusions Clinicians were more likely to report a gut feeling something is wrong, when they were more experienced and when children were more unwell. Gut feeling is independently and strongly associated with antibiotic Page 10 of 12 prescribing and referral to secondary care, but not with two indicators of poor child health. Additional file Additional file 1: Web Appendix Table S1. Determinants: univariable associations between sociodemographic, parent-reported symptoms, clinician-reported observations, clinician profile and gut feeling. This table presents the univariable associations between child and clinician variables and gut feeling. (DOCX 21 kb) Abbreviations AUROC: Area under the receiver operating Characteristic; CRF: Case report form; CRT: Capillary refill time; DCH: Diploma in child health; GPs: General practitioners; IMD: Indices of multiple deprivation; IQR: Inter-quartile range; LRT: Likelihood-ratio test; MRCGP: Membership of the royal college of general Practitioners; NPs: ‘Prescribing’ nurse practitioners; RTI: Respiratory tract infection; STROBE: Strengthening the reporting of observational studies in epidemiology Acknowledgements The National Institute for Health Research funds the Programme Grant for Applied Research TARGET Programme grant at the University of Bristol and NHS Bristol Clinical Commissioning Group. This paper summarises independent research funded by the National Institute for Health Research (NIHR) under its Programme Grants for Applied Research Programme (Grant Reference Number RP-PG-0608-10018). HC is supported by the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Evaluation of Interventions at the University of Bristol, in partnership with Public Health England (PHE). ADH is funded by NIHR Research Professorship (NIHR-RP-02-12-012). NMR’s time is supported by the NIHR Collaboration for Leadership in Applied Health Research and Care West (CLAHRC West) at University Hospitals Bristol NHS Foundation Trust. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, and the Department of Health of Public Health England. The authors are extremely grateful to the children, parents/carers and families who have participated in the study, all GP practices including recruiting clinicians, administrative and research contacts and all other staff whose participation made this study possible. We thank all our colleagues from the TARGET Programme, the TARGET Programme Management Group and the TARGET Programme Steering Committee (Sandra Eldridge, Nick Francis, Joe Kai, Victoria Senior, Anna Thursby-Pelham, Mireille Williams) for their time, expertise and support. We are grateful to the following individuals who have helped with the study; James Austin, Denis Baird, Tony Beard, Stephen Beckett, Issy Bray, Peter Brindle, Kate Brooks, Sue Broomfield, Joanna Cordell, Judy Cordell, Tania Crabb, Hazel Crabb-Wyke, Mike Crawford, Julie Cunningham, Christina Currie, Rachel Davies, Elizabeth Derodra, Elena Domenech, Stevo Durbaba, Lucy Feather, Caroline Footer, Emily Gale, Anna Gilbertson, Victoria Hardy, Rose Hawkins, Abigail Hay, Lisa Hird, Sandra Hollinghurst, Julie Hooper, Jonathan Hubb, Catherine Jameson, Grania Jenkins, Amy Jepps, Mari-Rose Kennedy, Michael Lawton, Mel Lewcock, Lyn Liddiard, Sandra Mulligan, Sharen O’Keefe, Lucy O’Reilly, Marilyn Peters, Aled Picton, Ilaria Pinna, Fiona Redmond, Isabel Richards, Kim Roden, Sharon Salt, Douglas Shedden, Ella Simmonds, Sue Smith, Carol Stanton, Kate Taylor, Elizabeth Thomas, Nicki Thorne, Sara Tonge, Abby Waterhouse, Eleanor Woodward. The TARGET study team acknowledges the support of the National Institute for Health Research, through the Comprehensive Clinical Research Network. To Robert Ross from the Department of Psychology at the Royal Holloway who advised on the dual process theory of clinical reasoning. Also to George Leckie, from the multi-level modelling centre at Bristol University who advised on statistical methods. Funding This paper reports independent research funded by the National Institute for Health Research (NIHR) under its Programme Grant for Applied Research (Grant Reference Number RP-PG-0608-10018). The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. The funder did not have a role in the design of the Turnbull et al. BMC Family Practice (2018) 19:25 study, data collection, analysis, interpretation of the data or in writing the manuscript. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Authors’ contributions PLu and ST were responsible for conceiving the research question. ADH, PLu, PLi, PSB, BD, MT, and TJP conceived the original idea for the TARGET cohort study and were responsible securing funding. ADH, CC, PLu, PLi, PSB, HC, BD, NMR, MT, ST and TJP were responsible for the study design, data collection and interpretation. NMR, HC and ST were responsible for study management and co-ordination. ST, HT, NR, PSB and TJP were responsible for study analysis; ST led the analysis for this paper. ST drafted the paper. All authors have read and approved the final manuscript. ST (corresponding and guarantor author) affirms that the manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained. Ethics approval and consent to participate The study was approved by the South West Central Bristol Research Ethics Committee, UK (reference number: 10/H0102/54) and research governance approvals were obtained for all areas prior to the start of recruitment. The study was sponsored by the University of Bristol, which ensured the study met all regulatory approvals. All participants’ parent or legal carers gave written, informed consent on behalf of the child. All children aged 11 and over gave informed assent. Consent for publication Not applicable. 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. Author details 1 Centre for Academic Primary Care, Population Health Sciences, Bristol Medical School, University of Bristol, Canynge Hall, 39 Whatley Road, Bristol BS8 2PS, UK. 2School for Policy Studies, University of Bristol, 8 Priory Road, Bristol BS8 1TZ, UK. 3National Institute for Health Research Collaborations for Leadership in Applied Health Research and Care West (NIHR CLAHRC West), University Hospitals Bristol NHS Foundation Trust, 9th Floor, Whitefriars, Lewins Mead, Bristol BS1 2NT, UK. 4Population Health Sciences, Bristol Medical School, University of Bristol, Oakfield House, Oakfield Grove, Bristol BS8 2BN, UK. 5Population Health Sciences, Bristol Medical School, University of Bristol, Level D, St Michael’s Hospital, Southwell St, Bristol BS2 8EG, UK. 6 Department of Surgery and Cancer, Imperial College London, St Mary’s Hospital, W2 1NY, London, UK. 7Department of Family Medicine, University of Washington, Seattle, WA 98195-4696, USA. 8Primary Care and Population Sciences Unit, University of Southampton, Aldermoor Health Centre, Aldermoor Close, Southampton SO16 5ST, UK. 9School of Clinical Sciences, University of Bristol, 69 St Michael’s Hill, Bristol BS2 8DZ, UK. 10Centre for Academic Primary Care, School of Social and Community Medicine, University of Bristol, Bristol BS8 2PS, UK. Received: 5 April 2017 Accepted: 24 January 2018 References 1. Hay AD, Heron J, Ness A. The prevalence of symptoms and consultations in pre-school children in the Avon longitudinal study of parents and children (ALSPAC): a prospective cohort study. Fam Pract. 2005;22(4):367–74. 2. Okkes IM, Oskam SK, Lamberts H. The probability of specific diagnoses for patients presenting with common symptoms to Dutch family physicians. J Fam Pract. 2002;51(1):31–6. Page 11 of 12 3. Hay AD, Redmond NM, Turnbull S, Christensen H, Thornton H, Little P, Thompson M, Delaney B, Lovering AM, Muir P, et al. Development and internal validation of a clinical rule to improve antibiotic use in children presenting to primary care with acute respiratory tract infection and cough: a prognostic cohort study. Lancet Respir Med. 2016;(11):902–10. 4. Lucas PJ, Cabral C, Hay AD, Horwood J. 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Exosomal MicroRNA-151a-3p Improves the Sensitivity to Radiotherapy via the Interaction between p53 and Histone Deacetylase 5 Seung Min Lee Asan Medical Center Bo Hyun yoon Asan Medical Center Myoung-Hee Kang Asan Medical Center Dong Ha Kim Asan Medical Center Yong-Hee cho Asan Medical Center Min choi Asan Medical Center Jin Woo Lee Asan Medical Center Tae Keun Kim Asan Medical Center Je-Won ryu Asan Medical Center Kyunggon Kim Asan Medical Center Young Hoon Sung Asan Medical Center Chan-Gi Pack Asan Medical Center Jin Kyung rho Asan Medical Center Sang-wook Lee Asan Medical Center Chang Hoon Ha (  chhoonha@amc.seoul.kr ) Asan Medical Center https://orcid.org/0000-0001-7606-4340 Page 1/30 Research Keywords: Exosome, microRNA, HDAC5, p53, radiotherapy, hepatocellular carcinoma Posted Date: August 31st, 2021 DOI: https://doi.org/10.21203/rs.3.rs-847360/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 2/30 Abstract Background: Tumor-derived exosomal microRNAs are key elements of the cell-cell communications response to lots of stimuli. However, various functions of the exosome in tumor suppression by radiotherapy (RT) are not clearly understood. Our study showed a previously unknown interaction of p53 and histone deacetylase 5 (HDAC5) by radiation exposure in hepatocellular carcinoma (HCC). Methods: Using serial ultracentrifugation methods, radiation and non-radiation exosomes were purified to investigate the radioresistance of miRNA151a-3p. Radiation doses were treated in 2gy and 4gy using radiation equipment X-RAD 320 to observe the expression of HDAC5 and p53 in hepatic cancer cells. Exosomal miRNA bioinformatics analysis was conducted to find a variation in the miRNA configuration inside Exosome after radiation exposure. Results: HDAC5 and p53 interacted by exposure to radiation, which increased exosome release and altered microRNAs' composition within exosomes. Also, we have described the intercommunication occurring between irradiated and untreated cells via exosomal microRNAs that affect tumor proliferation. In particular, the expression of exosomal microR151a-3p was markedly reduced by radiation treatment. We confirmed that inhibition of exosomal microR151a-3p promotes suppression of non-irradiated cancer cells, thereby increasing RT sensitivity. Conclusion: our present findings demonstrated HDAC5 is a key component of the p53-mediated release of exosomes resulting in tumor suppression through exosomal microRNA-151a-3p in response to radiation. Finally, we highlight the important role of exosomal microRNA 151a-3p as a biomarker in enhancing RT sensitivity. Background The Exosomes are extracellular vesicles of 30-150 nm in size that are released outside of the cell, which structurally constitutes a phospholipid bilayer membrane, so there is no possibility of being broken down by various enzymes in the body. Cancer cells secrete 10 times more exosomes than normal cells, and include growth factors, chemokines, miRNAs, etc. inside exosomes to regulate the microenvironment around cancer, and promote the growth and communication of cancer cells [1, 2]. Recent studies on miRNA contained within exosome have been actively conducted [3, 4]. After release, tumor-derived exosomal microRNAs (miRNAs) regulate such functions as retaining tumor growth, invasion, metastasis and therapyresistance in tumor cells through cell-cell communication within the tumor derived exosome. Therefore, exosomal miRNAs have an essential function to mediate tumor progression and can likely be applied as a promising biomarker in the diagnosis and prediction of cancer [5] [6]. p53 is a tumor suppressor that is very important in cancer and regulates cell-cycle control, aging, apoptosis, and genomic stability in response to various external stresses [7, 8]. Recent studies have shown that p53 modification is temporarily regulated by histone deacetylase 5 (HDAC5) [9]. It is believed that various posttranslational modifications of p53 play an important role in determining p53 promoter specificity toward its various target gene sets. During post-translational modifications, Acetylation of p53 at lysine 120 of the Page 3/30 DNA binding domain has been associated with p53-mediated apoptosis [10]. Further, it is known that the secretion of exosomes in cells undergoing p53 response by stress is increased by effector TSAP6 downstream of p53. So, exosomes can be regulated by the p53 response [11, 12]. Histone acetylation/deacetylation plays a critical role in the regulation of gene expression [13] [14]. In particular, HDAC5, one of class II HDACs, has been implicated in modulating cellular signalings by interactions with various transcriptional cofactors and confers responsiveness to gene expressions. Previous studies have shown that tumor suppressor p53 is activated by DNA damage that causes genotoxicity, and as a temporary regulator, HDAC5 interacts with p53 and HDAC5 in the early stages of stress, thereby deacetylating p53. The pro-apoptotic target gene, modulated by p53, is transactivated by k120 deacetylation of p53 by HDAC5, which determines the fate of the cell as a regulator of p53 modification [9, 15]. However, little is known about the role of HDAC5 in response to radiation therapy in tumor. Radiotherapy (RT) is one of the most commonly used therapeutic methods that can suppress the proliferation of cancer cells. However, there may be some side effects that reduce sensitivity to RT and become radiation resistant. In recent years, the goal of RT is to improve the quality of life of patients by reducing the side effects of radiation treatment. In particular, in hepatocellular carcinoma (HCC), RT has become a key treatment that prevents the progression of cancer and helps surgical treatment. RT is the most effective treatment for local tumor metastasis in patients who potentially need liver resection or transplantation [16, 17]. Here, we showed our findings regarding tumor-derived exosomes, including the miRNA delivering mechanism. We then described the interactions that occur between irradiated and untreated cells via exosomal miRNAs that affect tumor proliferation. HDAC5 and p53 interacted by exposure to radiation, the effect of which increased exosome release and altered the composition of miRNAs within exosomes. In particular, the expression of Exosomal miRNA 151a-3p was decreased, which is the result of irradiation. As a result of the experiment, it was confirmed that inhibition of exosomal miRNA 151a-3p promotes apoptosis of non-irradiated tumor cells, thereby enhancing the RT effect. Finally, we highlight the potential role of miRNA-151a-3p as biomarkers in tumor therapeutics and sensitivity to RT. Methods Cell culture, X-Radiation treatment HepG2, SK-Hep1 was purchased from the American Type Culture Collection (Rockville, MD) (ATCC HTB177). Hep3B was purchased by Korea Cellular Bank (seoul, Korea). It was cultured in DMEM (Corning) with 10% fetal bovine serum, 4.5 g/L glucose, L-glutamine, sodium pyruvate, and 100 U/mL of penicillin and streptomycin, and maintained at 37°C in a humidified chamber containing 5% CO2. Radiation was exposed with 2Gray, 4Gray a using X-RAD320 (1Gray/min at 320KV, 12.5mA, 50cm SSD (HVL≈ 4mm Cu)) equipment. Page 4/30 Exosome purification Radiation and non-radiation exosomes purified using ultracentrifugation methods. 1 × 106 cells were seeded in 100 mm dishes and allowed to recover for overnight. Then, the cells were washed twice with prewarmed PBS, and the culture medium was replaced with exosome-free medium supplemented with 5% exosome-depleted FBS (SBI). The Radiation exosome is treated 4Gray Radiation using X-RAD 320 (1Gray/min at 320KV, 12.5mA, 50cm SSD (HVL≈ 4mm Cu)) equipment. The medium was gathered and subjected to gradient centrifugation. Briefly, the medium was first centrifuged at 1000g for 10 min, at 3000g for 30 min, then centrifuged in a Beckman Coulter Optima™ L-XP Ultracentrifuge System at 100,000 gavg at 4℃ for 90 minutes with a SW 28 Swinging-Bucket Rotor (k-factor: 71) to pellet exosomes. The supernatant was carefully removed, and crude exosome-containing pellets were resuspended in 1 mL of ice-cold PBS and pooled. A second round of ultracentrifugation [100,000 gavg at 4℃ for 90 minutes with a Type SW 28 Swinging-Bucket Rotor (k-factor: 71)] was carried out, and the resulting exosome pellet resuspended in 500 μl of PBS. Western blot analysis The cells were collected, washed with PBS, and lysed in lysis buffer containing protease inhibitors (GenDEPOT, USA). After determining the protein concentration with a Bradford Protein Assay Reagent (BioRad), equal amounts of protein were separated on 10% SDS-PAGE, electrically transferred to nitrocellulose membrane, and blocked with 5% skim milk and 5% BSA. The membranes were incubated with anti-HDAC5 (1:1000; Cell Signaling Technology, USA), anti-p53 (1:2000; Santacruz, USA), anti-p21 (1:2000; Cell Signaling Technology), anti-Puma (1:2000; Cell Signaling Technology), anti-STEAP3 (1:1000; Proteintech, USA), anti-Maspin (1:1000; Cell Signaling Technology), anti-CD63 (1:1000; Santacruz), anti- CD81 (1:1000; Santacruz), anti-HSP90 (1:1000; BD bioscience), anti-BCL2 (1:1000; Cell Signaling Technology), anti-Ecadherin (1:1000; Agilent Dako), anti-Twist1 (1:1000; abcam), N-cadherin (1:1000; Cell Signaling Technology), Vimentin (1:1000; Calbiochem) or anti-beta-actin (1:1000; Santacruz) primary antibody overnight shaking at 4 °C. After washing twice, the membranes were then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse secondary antibody (Jackson ImmunoResearch Inc. USA) at room temperature for 1 hour. Finally, the membranes were incubated with WESTSAVE-UP western blotting substrate (Youngin frontier, Korea), and images were visualized using ChemiDoc imaging system (Bio-Rad, USA) and recorded. Duolink proximity ligation assay (PLA) The DuoLink® In Situ Red Starter Kit Mouse/Rabbit (DUO92101, Sigma-Aldrich, Darmstadt, Germany) was used to detect interacting target proteins. Cells were seeded in eight-well chamber removable slides (ibidi GmbH Am Klopferspitz, Germany) and cultured 24hours. Then, HepG2 cells were exposed to 4 g of radiation, and after 12 hours, the slides were washed with cold 1xPBS and fixed in 4% paraformaldehyde for 30 minutes. Then slides were blocked with Duolink Blocking Solution in a pre-heated humidified Page 5/30 chamber for 30 min at 37°C. The primary antibody to detect HDAC5 and p53 was added to the slides and incubated overnight at 4°C. Then slides were washed with 1×Wash Buffer A and subsequently incubated with the two PLA probes (1:5 diluted in antibody diluents) for 1 h, then the Ligation-Ligase solution for 30 min, and the Amplification-Polymerase solution for 100 min in a pre-heated humidified chamber at 37°C. Before imaging, slides were washed with 1×Wash Buffer B and mounted with a cover slip using Duolink In Situ Mounting Medium with DAPI. Fluorescence images were acquired using a zeiss LSM 780 confocal microscope. 17 Transmission electron microscopy (TEM) In order to photograph exosomes, 0.1M of exosome pellets were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate solution (pH 7.0) for 1 hour and then fixed in 2% osmium tetroxide for another hour. (4°C) After dehydration using the graded acetone series, embedding was performed through Spurr's medium (Electron Microscopy Sciences). The resulting section sample was cut at 60 nm with an ultramicrotome (RMC MTXL, USA), followed by double staining using 2% uranyl acetate for 20 minutes and citrate for 10 minutes. Prepared sections were photographed at 80 kV using Hitachi H-7600 TEM (Hitachi, Japan) equipment. Exosome size distribution and concentration measurement. To determine exosome size distribution and concentration, nanoparticle tracking-based analyses were performed using a NanoSight (NS500) apparatus (Malvern Instruments Ltd.). Samples were diluted to provide counts within the linear range of the instrument. The videos of 1-minute duration were recorded for each sample, with a frame rate of 30 frames per second. Particle movement was analyzed by NTA software (NTA 2.3; NanoSight Ltd.) according to the manufacturer's protocol. The NTA software was optimized to first identify and then track each particle on a frame-by-frame basis. 18, 19 Small RNA sequencing RNA isolation HepG2-derived Exosomal small RNA was extracted using a miRNeasy Mini Kit (Qiagen Korea Ltd.) according to the manufacturer’s instructions. RNA quality was assessed by Agilent 2100 bioanalyzer using the RNA 6000 Pico Chip (Agilent Technologies, Amstelveen, The Netherlands), and RNA quantification was performed using a NanoDrop 2000 Spectrophotometer system (Thermo Fisher Scientific, Waltham, MA, USA). Library preparation and sequencing Page 6/30 For control and radiation induced HepG2-drived exosomal small RNAs, the construction of the library was performed using NEBNext Multiplex Small RNA Library Prep kit (New England BioLabs, Inc., USA) according to the manufacturer’s instructions. Briefly, for library construction, total RNA from each samples were used 1ug to ligate the adaptors and then cDNA was synthesized using reverse-transcriptase with adaptor-specific primers. PCR was performed for library amplification and libraries were carried out clean-up using QIAquick PCR Purification Kit (Quaigen, Inc, German) and AMPure XP beads (Beckmancoulter, Inc., USA). The yield and size distribution of the small RNA libraries were assessed by the Agilent 2100 Bioanalyzer instrument for the High-sensitivity DNA Assay (Agilent Technologies, Inc., USA). High-throughput sequences were produced by NextSeq500 system as way of single-end 75 sequencing (Illumina, SanDiego, CA., USA). Data analysis Sequence reads were mapped by bowtie2 software tool in order to obtain bam file. Mature miRNA sequence is used as a reference for mapping. Read counts mapped on mature miRNA sequence were extracted from the alignment file using bedtools v2.25.0 (Quinlan AR, 2010) and Bioconductor 20 that uses R statistical programming language (R development Core Team, 2016). Read counts were used in order to determine the expression level of miRNAs. Quantile normalization method was used for comparison between samples. For miRNA target study, miRWalk 2.0 21 was performed. Functional gene classification was performed by DIANA 22. Exosomal small RNA sequencing arrays were prepared, hybridized, and scanned at the local authorized Illumina array service provider (Ebiogen, Seoul, South Korea). TCGA(The Cancer Genome Atlas) and GEO(Gene Expression Omnibus) Database analysis The two gene expression profiling data sets (GSE74618, GSE147889) we analyzed were downloaded from the NCBI GEO database.23 The GSE74618 database consists of 218 human HCC tumours samples, 10 adjacent cirrhotic non-tumoral tissue samples, and 10 healthy liver samples. We used adjacent cirrhotic non-tumoral tissue samples and healthy liver samples as controls and compared them with human HCC tumours samples. The GSE1477889 database consists of 97 samples of HCC tumours and 97 samples of the same patient's surrounding chronic hepatitis tissue. Surrounding chronic hepatitis tissue was used as a control and compared with HCC tumour samples. Two data sets were analyzed as volcano plots and mean difference plots, respectively, using the GEO2R analysis tool. TCGA (The Cancer Genome Atlas) data analysis was performed using oncomiR (WashU Pan-Cancer miRNome Atlas) online bioinformatics tool.24 Among the TCGA data, the analysis was conducted using the LIHC (Liver Hepatocellular Carcinoma) database (n=366), and the days' survival rate graph of liver cancer patients was exhibited. Page 7/30 Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses KEGG is a database that contains genetic information on the human genome, biological cell signalling pathways, and various diseases and chemicals. The KEGG pathway analysis of exosomal miRNA was analyzed using the DIANA-miRPath v3.0 analysis tool. DIANA-miRPath v3.0 is a KEGG-based miRNA pathway prediction tool that actively interacts with the major miRNA analysis tools, DIANA-microT CDS and TargetScan v6.2 database, to analyze the target pathway.22 miRNA Transfection Cells were seeded in 6-well plates at 1 × 105 cells/mL/well before the transfection and radiation. On the following day, After HepG2 cells were exposed 4gy of radiation using X-RAD 320 and transfection was performed when the cells had reached approximately 80% confluence. miRCURY LNA miR-151a-3p mimic (miRCURY LNA miRNA Mimic, MIMAT0000757) were purchased from QiAGEN (Qiagen Korea Ltd.). And scrambled miRNA control were purchased from BIONEER (Seoul, Korea). The final concentrations of miRNA were 50-100nM Transfections were conducted with Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. Quantitative real time reverse transcription polymerase chain reaction(qRT-PCR) Total RNA was extracted using a miRNeasy Mini Kit (Qiagen Korea Ltd.) in the manufacturers' protocols. For the quantification of matured cellular and exosomal miRNAs, the extracted total RNA was polyadenylated with a poly(A) tailing kit (Ambion, Austin, TX) prior to reverse transcription. And was reverse transcribed to cDNA using PrimeScript™ Reverse Transcriptase (Takara Bio Inc.). The cDNA samples were used for quantitative RT-PCR analysis in triplicate to determine the expression levels of miR-151a-3p using a TB Green® Premix Ex Taq™ (Takara Bio Inc.) and CFX96 Detection System real-time PCR instrument. Proliferation assay The cell proliferation assay was performed using the Chromo-CK Cell Viability Assay KIT (monobio seoul, korea) according to the manufacturer’s instructions. Transfected cells were harvested at the designated times after seeding. Briefly, the reagent (10 μl/well) was added to 100 μl of medium containing cells in each well of a 96-well plate and incubated for 1 hours at 37℃ under humidified 5% CO2 in air. For colorimetric analysis, absorbance at 450 nm was recorded using an ELx800 Absorbance Microplate Reader (SpectraMax 340PC Microplate Reader, Molecular Devices, USA). Each experiment was repeated at least 3 times. Page 8/30 Migration invasion assay Tumor cell migration and invasion were analyzed in 24-well plates with 8-μm pore size polycarbonate membranes (BD, NJ, USA). For invasion assays, the membranes were coated with diluted Matrigel (BD, NJ, USA) to form matrix barriers. HepG2 were transfected with 151a-3p or negative control. For the migration and invasion assays, the cells (5×10^4 for migration, 1×10^5 for invasion) were resuspended in 200μl of serum-free DMEM at 24 hours post-transfection and added to the upper compartments of the chambers, and the lower compartments were filled with 600μl of DMEM with 10% FBS (different substrates were added accordingly). After incubation at 37°C for 10 hours (migration) or 24 hours (invasion), the cells remaining on the upper surfaces of the membrane were removed. The cells on the lower surfaces of the membrane were fixed, stained with crystal violet and counted under a light microscope. Each experiment was repeated at least 3 times. Analysis of cell cycle The Transfected cells were collected and resuspended after radiation exposure. Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining assays were conducted to detect the percentage of apoptotic (FITC-stained) and necrotic (PI-stained) cells in a given population. Analyses were performed by a FACS canto flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. The procedures were repeated in triplicate. 25 Analysis of cell apoptosis The Transfected cells were collected and resuspended with cold PBS after radiation exposure. And fixed in cold 70% ethanol added dropwise to the cell pellet while vortexing. Stored at -20℃ for overnight. Cells were rehydrated with PBS for 10 min at RT and then cells were stained with propidium iodide (PI) staining solution contained with 50 μg/ml PI (BD Biosciences, Franklin Lakes, NJ, USA), 100 μg/ml RNase A, DNase and protease-free (Thermo Scientific) in 1X binding buffer. Analyses were performed by a FACS canto flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. The procedures were repeated in triplicate. Subcutaneous tumor xenograft models BALB/c nude mice (5-week old male) were purchased from the Charles River Laboratories, Inc (Wilmington, Massachusetts). The fodder was feed to the normal rodent laboratory animal feed. 5x10^6 HepG2 cells in total volume 100ul were injected into the right flank of a 5-week-old nude mouse in a 1:1 ratio with Matrigel (corning 356234). After the average of the transplanted solid cancer size increased by more than 100 mm3, the invivo-jetPEI/target miRNA complex was treated. invivo-jetPEI was used as an efficient carrier for delivering miRNA and was mixed with 5% glucose solution at a ratio of miRNA 10ug / invivo-jetPEI 1.2ul and treated in a total dose of 100 ul. The invivo-jetPEI/target miRNA complex was administered 3 times Page 9/30 every 3 days by intratumoral injection. Tumor size was measured using a standard ABS digimatic caliper (CD-15AX) every 3 days after transplantation. Tumor volume was measured using the following formula: ∏ x 4/3 x larger diameter x smaller diameter square. At the time when the volume of the largest tumor of the implanted nude mice in the experimental group exceeded 1000 mm3, the tumor was separated after sacrifice, the weight of the tumor was measured, and expression of the target protein in cancer tissue was analyzed. Statistical analysis Each experiment in our study was repeated three times. Mathematical data are expressed as mean ± SD. Unless indicated, the differences between two groups were analyzed using a Student's t-test (two-tailed). Unless otherwise noted, significant numerical differences between the two groups were analyzed using Student's t-test (two-tailed). The overall survival curve was graphed using the Kaplan-Meier method and compared through the log-rank test. Differences were evaluated statistically significant at P< 0.05. All statistical analysis was performed using SPSS13.0 software (SPSS, Chicago, IL, USA). Results Modulation of p53 and HDAC5 by RT An experiment was conducted to verify that protein p53 and HDAC5 were modulated by radiation in HCC. Radiation doses were treated in 2gy and 4gy using radiation equipment X-RAD 320 to observe the expression of HDAC5 and p53 when radiation was treated in three types of hepatic cancer cells. The interaction of HDAC5 and p53 by DNA damage has been previously described [9]. However, the interaction of HDAC5 and p53 in HCC by radiation is not yet known. HepG2 and SK-Hep1 are p53 wild types (p53+/+) and Hep3B are p53 deletion types (p53-/-). The amount of expression of each protein was measured through the Western Blot in order of 6 hours, 12 hours, 18 hours, and 24 hours (Fig. 1A-F). In HepG2, the expressions of HDAC5 and p53 were significantly increased from 6 hours compared to the control group by radiation 2gy and 4gy. Later, at 2 gy of radiation, HDAC5 showed a time-dependent decrease again. However, HDAC5 and p53 were significantly increased in radiation 4gy regardless of time. This shows that the expression of HDAC5 and p53 and its downstream is regulated by irradiation in HepG2 (Fig. 1. A,B). SKHep1 increased the expression of HDAC5 24 hours after 2gy irradiation. However, in 4 gy, it was reduced compared to the control group. p53 and its downstream showed a significant increase by irradiation, but HDAC5 did not show a significant expression pattern (Fig. 1. C, D). Finally, Hep3B showed an independent increase in HDAC5 expression without p53 after irradiation. As a result, it was confirmed that among p53 and HDAC5 increased by irradiation, HDAC5 independently increased protein expression (Fig. 1E, F). Subsequent experiments were conducted on HepG2, which showed the most significant change by RT among the three liver cancer cells. To check the relationship between HDAC5 and p53, a HepG2 cell line that p53-/- was created using the CRISPER CAS9 system. The target site of Human p53: TGTAACAGTTCCTGCATGGG was investigated by the NGS method. The insertion/deletion frequency was Page 10/30 99.98% (T insertion:19.9%, CCTG deletion:18.8%, AT insertion:19.5%), and the elimination of the p53 gene was confirmed to be close to perfection (supplementary Fig. 1). The radiation was exposed for p53+/+, p53-/-cells and measured the protein expression of HDAC5 and p53, p21, Puma with western blot. The expression of p53, p21 did not almost appear because p53 was deleted. Regardless of p53 identified an increase in the expression of HDAC5. As a result, it was confirmed that the expression of HDAC5 was increased by irradiation regardless of the expression of p53 in cells, which showed the most significant expression by irradiation, and the relationship between HDAC5 and p53 was identified (Fig. 1. G, H). Using FACS equipment, it was confirmed that cell cycle arrest and apoptosis are induced in p53+/+, p53-/-cells by radiation treatment. In p53+/+cell, powerful cell cycle arrest in the G2/M phase was observed from 6 hours after irradiation and gradually switched to G1 phase cell cycle arrest as time passed. A similar cell cycle alteration was observed in p53-/-cell, but it was verified that the effect was smaller than that of p53+/+cell. Also, p53-/-cell had significantly less apoptosis 48 hours after irradiation than p53+/+cell. It showed stronger cell cycle arrest and apoptosis at p53+/+cell compared to p53-/-cell (supplementary Fig. 2). Intercommunication between p53 and HDAC5 by RT resulting in increasing the expression of TSAP6, exosome modulating protein To identify the more accurate interaction between p53 and HDAC5, this was investigated using the duolink PLA assay. When the cells were not exposed to radiation, no red dots were found to interact, and 4 Gy of radiation revealed multiple red dots, the interaction between p53 and HDAC5. It has been demonstrated that radiation exposure induces a significant increase in the interaction of p53 and HDAC5 in cells (Fig. 2. A). Using western blot validated whether TSAP6 (Tumor Suppressor Activation Pathway 6), an exosomemodulated protein in the direct downstream of p53 and controlled by p53 is increased by RT [11, 12]. The expression of TSAP6 was verified with a western blot at 12 hours and 24 hours after exposure to 4Gy of radiation. Our results showed that the interaction between HDAC5 and p53 was enhanced by radiation, and the downstream TSAP6 of p53 was also significantly increased (Fig. 2. B, C). The characteristics of exosomes derived from p53+/+, p53-/-cells were identified. Exosomes secreted by cells were observed using an electron microscope. (Fig. 2. D) Western blot analysis showed that microvesicles released from p53+/+, p53-/-cells expressed exosome markers CD63, CD81, and HSP90. All exosome markers have been demonstrated [18]. We also identified the discriminating expression of protein maspin (mammary serine protease inhibitor), which is released by p53 dependently through exosome by radiation exposure [11]. Inside p53+/+cell derived exosomes, it was found that the expression of maspin was increased by radiation. However, p53-/-cell derived exosomes demonstrated no expression of maspin by radiation exposure. (Fig. 2. E, F) The expression of maspin inside the exosome induced by radiation exposure is regulated by the expression of p53. p53 directly binds to the p53 gene-consensus binding site present in the maspin promoter, through activating the maspin promoter and inducing maspin expression [19]. Exosome secretion in a p53-dependent manner by RT Page 11/30 After exposure to radiation, experiments were conducted to demonstrate that the secretion of exosome changes over time. The number of cultured p53+/+cells and p53-/-cells was equal to 5x106. Experiments were conducted at 6, 12, 24, and 48 hour intervals after exposure to 4 Gy of radiation, and exosomes released at each hour were extracted by ultracentrifugation. The qualitative and quantitative comparison and analysis of the isolated exosomes were performed using the Nanosight NT300 equipment (supplementary Fig. 3. A, B). As a result of the analysis, the Exosome showed a heterogeneous exosomal population with a diameter of 30–300 nm or more. These results suggest that the isolated exosomes had high purity and satisfy the size, density, structure, and characteristics of vesicles secreted by cells [18]. p53+/+cells manifested a significant increase in the secretion of overall vesicles when exposed to radiation of 4 Gy. As a result of comparing the total vesicles by each secreted time, it was found that the volume of secreted vesicles increased more than twice when exposed to radiation. In particular, 24 hours after exposure to radiation, the total quantity of vesicle secretion was found to be more than three-fold that of the control, and the most vesicle secretion exhibited at this time (Fig. 3A). Conversely, p53-/-cells exhibited a decrease in the volume of vesicle secretion after exposure to radiation. From 6 hours to 48 hours, the quantity of vesicle released in all experimental groups was significantly decreased compared to the control (Fig. 3B). The secreted extracellular vesicles were analyzed qualitatively by separating them into 30-150nm, 150-300nm, and > 300nm by size. The p53+/+cells secreted Exosome size part (30-150nm) was the most significant increase of 12 hours after radiation exposure. As time passed after exposure to radiation, the variation of vesicles was characterized by a change in the direction of increasing their size. It was verified that exosome-sized vesicles were noticeably reduced in vesicles 48 hours after exposure to radiation (Fig. 3C). In the same way, p53-/-cells derived exosome was analyzed by dividing each size. The exosome-sized vesicles released after irradiation were also rapidly reduced (Fig. 3D). The exosomes of p53+/+ and p53-/cells released after irradiation were analyzed qualitatively and quantitatively. (Fig. 3E, F) This experiment proved that the secretion of extracellular vesicles was increased by radiation exposure. Among them, 30150nm-sized exosomes proved to be increased or decreased by p53 dependency. This result suggests that the expression of p53 induced by RT increases exosome secretion through the TSAP6 pathway. Alteration of exosomal miRNA’s expressions by RT in HCC After the release in a cell, exosomes are taken up by neighboring or distant cells, and the miRNAs contained within modulating such processes as interfering with tumor immunity and the microenvironment, possibly facilitating tumor growth, invasion, metastasis, angiogenesis, and drug resistance. Therefore, exosomal miRNAs have a significant function in regulating tumor progression. Exosomal miRNA bioinformatics analysis was conducted to find a variation in the miRNA configuration inside Exosome after radiation exposure. The variation of miRNA in exosome secreted from the radiation exposed cell and miRNA in exosome secreted from without radiation exposure was analyzed through Heatmap utilizing MeV software (Small RNA sequencing, miRNA-Seq Only Analysis Program developed by Dana-Farber Cancer Institute in the United States) (supplementary Fig. 4. A). Also, to find out which intracellular function each of the miRNAs inside the exosome changed after irradiation, it was classified and analyzed into various cell functional categories using Gene Ontology terms (supplementary Fig. 4. B). Handling the miRNA's fold change values analyzed, the graph was composed in descending order (Fig. 4. A). It has been confirmed that 33 miRNAs have significantly reduced due to irradiation among 156 miRNAs that have inside exosome Page 12/30 (Fig. 4. B). Among the miRNAs with reduced expression, we analyzed using the hepatocellular carcinoma GEO data set to identify the miRNAs associated with the progression of hepatocellular carcinoma. The GEO data set was downloaded from NCBI Gene Expression Omnibus, and analyzed by volcano plot and meandifference plot using the GEO2R analysis tool (supplementary Fig. 5. A, B). As a result of analysis utilizing the HCC miRNA GEO data set (Spain, GSE74618), it was verified that 4 specific miRNAs (miR-151a-3p, miR106b-5p, miR-183-5p, miR-452-5p) were significantly increased in tumor tissues compared to normal (Fig. 4.C) [20]. The HCC miRNA GEO data set (Spain, GSE74618) was re-validated using another HCC miRNA GEO data set (Japan, GSE147889) to confirm the accuracy of the analysis results (fig S4.B). To ascertain the relationship between the four miRNAs (miRNA151a-3p, miRNA 106b-5p, miRNA183-5p, miRNA 452-5p) with increased expression in HCC and the survival rate of liver cancer patients, we analyzed using OncomiR Cancer miRNome Atlas. As a result of OncomiR analysis, it was found that the higher the expression of miRNA151a-3p, miRNA 106b-5p, miRNA183-5p, and miRNA 452-5p in the tumour tissues of liver cancer patients, the significantly decreased the survival rate of liver cancer patients (Fig. 4.D) [21]. We performed the KEGG pathway analysis to find out which molecular biological processes the four miRNAs, which were directly related to the prognosis of liver cancer patients, affect cancer (Table 1). Among the analysis results performed, the cell cycle, a mechanism directly related to radiation therapy, and the p53 signalling pathway, the central target of our study, was ascertained to be significant signaling mechanisms. In addition, targetscan (http://www.targetscan.org/vert_72/) analysis tool was used to analyze the target protein of each miRNA. As a result of the target analysis of miRNA, it was discovered that miRNA151a-3p can bind to the p53 mRNA target region with a high probability (Fig. 4E). Likewise, in previous studies, it has been reported that miRNA 151a-3p was bioinformatically analyzed as a target miRNA of p53 and binds to the p53 mRNA target region, thereby inhibiting the expression of p53 in cells [22, 23]. We considered miRNA 151a-3p interesting and continued the study, and it was confirmed that miRNA 151a-3p is deeply related to tumorigenesis of various cancers (Table. 2). Furthermore, three cancer types were identified, where patient survival was significantly associated with miRNA 151a-3p (Table. 3). As expected, miRNA 151a-3p had a great correlation with the prognosis of liver cancer patients. So, it has been confirmed that miRNA 151a-3p is a respectable relation to tumorigenesis and survival of Liver Hepatocellular carcinoma (LIHC). We actually verified that miRNA 151a-3p was reduced by radiation exposure both intracellularly and in exosomes. The expression of miRNA 151a-3p in cell and exosome after radiation exposure was analyzed using qRT-PCR. The quantification of miRNA 151a-3p was quantified using miR-Let7a [24]. Cellular miRNA 151a-3p decreased by 1/2 after radiation exposure, whereas exosomal miRNA 151a-3p decreased by more than 1/4 after radiation exposure. (Fig. 4. F, G) As a result, miRNA 151a-3p had a significant relationship with the poor survival of HCC. It also after irradiation in hepG2, a HCC cell line, demonstrated a significant decrease in the expression of miRNA 151a-3p inside the cellular and exosome. Table 1 Results of KEGG pathway analysis of major miRNAs within exosomes that were reduced after radiation exposure. Page 13/30 KEGG pathway p-value Genes Pathways in cancer 6.61E-08 103 Viral carcinogenesis 1.62E-06 56 Cell cycle 1.88E-06 43 Adherens junction 1.88E-06 26 Colorectal cancer 2.49E-06 25 Hepatitis B 2.01E-05 45 Thyroid cancer 2.61E-05 15 TGF-beta signaling pathway 0.000154 27 Bladder cancer 0.000154 19 p53 signaling pathway 0.000226 27 Wnt signaling pathway 0.000238 43 Endometrial cancer 0.000245 19 Renal cell carcinoma 0.000284 24 PI3K-Akt signaling pathway 0.000379 87 RNA transport 0.000401 48 Protein processing in endoplasmic reticulum 0.000401 50 AMPK signaling pathway 0.000401 40 Pancreatic cancer 0.001437 24 Insulin signaling pathway 0.002105 41 mTOR signaling pathway 0.002607 22 Focal adhesion 0.002607 55 Small cell lung cancer 0.002778 27 Non-small cell lung cancer 0.006262 18 Endocytosis 0.006754 50 Sphingolipid signaling pathway 0.008416 32 mRNA surveillance pathway 0.014393 27 Hepatitis C 0.015025 37 Phosphatidylinositol signaling system 0.024036 20 MAPK signaling pathway 0.024051 59 Page 14/30 Table 2 15 cancer types where tumorigenesis is significantly associated with hsa-miR-151a-3p expression. Table 3 cancer types where survival is significantly associated with hsa-miR-151a-3p. Cancer Abbreviation Log Rank Pvalue Log Rank FDR Zscore Upregulated in: Deceased Expression Living Expression T-Test Pvalue T-Test FDR LGG 4.71e02 3.48e02 1.990 Deceased 10.37 10.15 4.53e02 1.98e01 LIHC 5.47e04 1.71e02 3.471 Deceased 11.68 11.34 3.93e03 7.93e02 SARC 8.84e04 5.62e01 3.320 Deceased 11.00 10.65 2.15e02 5.56e01 LGG: brain lower grade glioma; LIHC: liver hepatocellular carcinoma; SARC: sarcoma Radiotherapy reduced expression of exosomal miRNA-151a3p resulting in tumor suppression We investigated the radioresistance of miRNA151a-3p. A protein related to cell apoptosis and cell cycle arrest that can be caused by radiation was verified 24 hours after the transfection of negative control and miRNA151a-3p in the cell through the western blot. Cancer repression gene p53 significantly reduced. A Decrease of P21 which inhibits cyclin-dependent phosphorylation to stop cell cycle has been identified. The BCL-2 controlling the Mitochondrial Outer Membrane Permeabilization Pore, which creates a channel that increases the permeability of the mitochondrial outer membrane, has been increased. And Puma associated with apoptosis has been reduced (Fig. 5. A, B). Based on the results of Fig. 5. A-B, the cell cycle arrest analysis experiment was conducted using FACS to verify the function of miRNA151a-3p in cell cycle arrest induced by radiation exposure. 24 hours after miRNA151a-3p transfection, 4Gy radiation was exposed to the cell. After six hours, the cell cycle of each experimental group was analyzed (Fig. 5. C). The G2/M phase arrest was increased in the N.C group and the 4Gy positive group. In the miR-151a-3p group, the G2/M phase arrest was reduced relatively (Fig. 5. D). Also, it has been confirmed that miRNA151a-3p has a function to inhibit cell apoptosis induced by radiation exposure. As a result of the experiment, the miRNA151a-3p group significantly reduced total cell apoptosis compared to the N.C. and 4Gy positive groups (Fig. 5. E-F). Page 15/30 Cancer Abbreviation T-Test P-value T-Test FDR Upregulated in: Tumor Expression Normal Expression BLCA 9.93e05 6.17e04 Tumor 11.82 10.21 BRCA 5.98e05 1.61e04 Tumor 10.98 10.68 COAD 7.53e03 1.78e02 Tumor 10.61 9.57 ESCA 7.78e03 3.89e02 Tumor 10.55 10.14 HNSC 7.69e05 2.63e04 Tumor 11.35 10.96 KICH 1.06e03 3.05e03 Tumor 10.99 10.49 KIRC 8.36e25 2.30e23 Tumor 10.68 9.95 KIRP 1.54e05 7.80e05 Tumor 10.60 10.13 LIHC 1.12e10 2.40e09 Tumor 11.67 10.90 LUAD 1.52e04 7.47e04 Tumor 11.68 11.20 LUSC 1.97e05 7.41e05 Tumor 11.54 11.06 PRAD 9.20e06 4.29e05 Tumor 10.58 10.26 READ 8.54e03 4.23e02 Tumor 11.28 8.51 STAD 3.86e11 2.92e09 Tumor 11.25 10.11 UCEC 9.47e08 7.54e07 Tumor 11.41 10.22 BLCA: bladder urothelial carcinoma; BRCA: breast invasive carcinoma; COAD: colon adenocarcinoma; ESCA: esophageal carcinoma; HNSC: head and neck squamous cell carcinoma; KICH: kidney chromophobe; KIRC: kidney renal clear cell carcinoma; KIRP: kidney renal papillary cell carcinoma; LIHC: liver hepatocellular carcinoma; LUAD: lung adenocarcinoma; LUSC: lung squamous cell carcinoma; PRAD: prostate adenocarcinoma; READ: rectal adenocarcinoma; STAD: stomach adenocarcinoma; UCEC: uterine corpus endometrial carcinoma The expression of EMT marker protein was verified 24 hours after the transfection of negative control and miRNA151a-3p in the cell through the western blot. E-cadherin, a representative epithelial phenotype marker, was reduced in the miRNA151a-3p group. There was a significant increase in the mesenchymal phenotype Page 16/30 marker vimentin and N-cadherin moving inversely to E-cadherin, and a noticeable increase in the expression of Twist1 associated with p53 metastasis (Fig. 6A, B) [25, 26]. For functional evaluation that induces the progression of cancer of miR-151a-3p, proliferation assay, Transwell migration, and invasion assay were implemented. A significant increase in proliferation was identified in the miRNA151a-3p 100nM group, depending on the concentration of miRNA151a-3p (Fig. 6C). Similarly, the cell movement and the increase in metastasis were identified depending on the concentration of miRNA151a-3p in cell migration, Invasion assay using transwell (Fig. 6D). The migrated and invasive cells were dyed and expressed in a graph after counting (Fig. 6E). So, evidence of the above data has demonstrated that miRNA151a-3p can induce EMTlike phenotype and increase cell proliferation. In order to confirm the tumor progression ability of miRNA151a-3p in vivo, a nude mouse subcutaneous xenograft model experiment was conducted. HepG2 cells were injected 5x10^6 subcutaneously in the flank of nude mice. Thereafter, miRNA-NC and miRNA-151a-3p / in vivo jet-PEI complexes were injected three times at 3 days intervals using the established in vivo jet-PEI transfection reagent to increase the expression of miRNA 151a-3p in cells [27]. In the in vivo experiment, the size of the tumor was significantly increased in miR-151a-3p compared to miRNA-NC, and the growth rate of the tumor was also faster (Fig. 7A-C). In addition, in western blot analysis in tumour tissue, miRNA-151a-3p significantly inhibited the expression of p53, P21, and Puma α/β (Fig. 7D, E). Comprehensive results of the experiment showed that overexpression of miR-151a-3p decreased the expression of the target protein in the tumour, and as a result, promoted tumour progression in the xenograft model. Discussion In-depth studies have shown that exosomes, which are widely present in various body fluids, contain many types of proteins, miRNAs, and various small molecules [3]. After secretion, exosomes are absorbed by adjacent or distant cells, and the miRNAs in them regulate processes such as promoting tumor growth, invasion, angiogenesis, and resistance to radiotherapy (RT). Therefore, miRNAs are promising as biomarkers in clinical targeted therapies in regulating cancer progression. [28]. Exosomes can be considered as modulators that target and bind underlying genetic molecules in the pathways regulating RT. RT is one of the important treatments for preventing tumor progression, and through various mechanisms, it affects target cancer tissue and surrounding cancer cells. Among them, p53 plays a key role in the suppression of cancer through RT [29],[30]. Our current study demonstrated a previously unknown interaction of p53 and HDAC5 in HCC by radiation exposure. Moreover, the expression of both proteins was confirmed in a time-dependent manner by radiation exposure. We confirmed that HDAC5 expression was increased by RT in p53-/-HepG2 cells, indicating that HDAC5 is an up-regulator of p53. These findings show that HDAC5 plays an important role in p53-mediated exosome release by radiation. As previously studied, TSAP6 is known as a protein that regulates the secretion of exosomes [12]. TSAP6, exosome secretion modulating protein through P53, has increased expression in a radiation exposure environment. Changes of exosomes after radiation exposure were qualitatively and quantitatively analyzed Page 17/30 by Nanoparticle Tracking Analysis (NTA). The presence or absence of the p53 gene dramatically changed exosomes' secretion depending on the time of radiation exposure. In the previous figure, p53+/+ TSAP6 was most expressed after 12 hours of radiation exposure, and secretion of exosome was also most secreted after 12 hours of radiation exposure. On the contrary, the expression of TSAP6 by radiation exposure in the p53-/-cells did not change, and the secretion of exosomes after radiation exposure decreased. This suggests that TSAP6 is clearly involved in exosome secretion and that TSAP6 is regulated by changes in p53 resulting from radiation exposure. We photographed exosomes separated for exosome characterization using electron microscopy and analyzed western blot of exosome marker protein. Among the exosome marker proteins, Mammary Serine Protease Inhibitor (maspin) is a protein regulated by the p53 gene and functions to suppress cancer, and many studies are underway. Previous studies have confirmed that maspin inside the exosome is increased by radiation exposure in the lung cancer cell line [11]. We found that the expression of maspin increased significantly within the exosome secreted from p53+/+cells after radiation exposure. Besides, we confirmed that the expression of maspin did not increase in the p53-/-cells. Maspin is mounted on the exosome and released outside the cell, indicating that it functions to suppress tumor progression in surrounding cancer cells. Maspin is expected to suppress the cancer progression of the recipient cell. The exosome is loaded with various substances (ex, protein, mRNA, miRNA., etc.) and plays an important role in communication between cells. Among them, miRNA is a potential biomarker, and many studies are underway [31] [32]. As a short RNA strand of the 18-22base pair, it binds to specific mRNAs with the same sequence and inhibits the gene's protein expression. After radiation exposure, changes in the miRNA composition balance inside the exosomes were observed. The change of miRNA inside exosome changed by radiation was thought to be the starting point for communication between cancer cells and changes in the cancer microenvironment. Therefore, we generally performed a miRNA microarray to determine the miRNAs' overall expression in exosomes released in culture and exosomes released from irradiated cells. As a result of the analysis, 156 exosomal miRNAs were expressed out of a total of 2588 miRNAs. We classified exosomal-miRNAs for each cell function using GO term to determine which cellular function the changed exosomal-miRNAs are involved in after irradiation. As a result of classifying exosomal-miRNAs with a normalized expression (log2) of 1.5 or more, the number of exosomal-miRNAs involved in cell proliferation and cell migration decreased the number of exosomal-miRNAs involved in DNA repair and apoptosis increased. Among them, we conducted an analysis focusing on 33 miRNAs whose expression almost disappeared after irradiation. To find out the relationship between the disappeared 33 miRNAs and HCC, each miRNA expression was analyzed using the miRNA microarray dataset cohort in the cancer tissues of HCC patients. Among the 33 miRNAs that disappeared after irradiation, miR-151a-3p, miR-106b-5p, miR183-5p, and miR452-5p involved cell proliferation and invasion were significantly higher in expression in HCC cancer tissues. So, we focused on miRNAs whose expression was reduced in exosomes after irradiation. As a result of analyzing using the GEO dataset (GSE74618) to find out how exosomal-miRNAs with decreased expression after irradiation were exhibited in cancer tissues of human liver cancer patients, 4 miRNA candidates with specifically increased expression were found. Four miRNA candidates showed a statistically significant Page 18/30 increase in expression in liver cancer tissues. Subsequently, it was analyzed using the TCGA database (oncomiR) to analyze the association between miRNA candidates and liver cancer patients' survival rate. All miRNA candidates showed a direct correlation with the poor prognosis of liver cancer patients. These results are expected to contribute as a part of the mechanism for inhibiting cancer growth caused by radiation therapy. MiRNA 151a-3p, one of the four miRNAs that had a significant relationship with the survival rate of liver cancer patients, was found to bind p53 with a high probability due to target protein analysis through the targetscan prediction tool. Subsequently, it was confirmed by qRT-PCR that the expression of miRNA151a3p was reduced in cells and exosomes due to radiation exposure. p53 is one of the key genes that maintain cell homeostasis by increasing expression by DNA damage such as radiation, leading to phenotypes such as cell cycle and cell death. As expected, miRNA151a-3p significantly reduced the expression of p53. Also, by inhibiting the expression of p53 downstream molecules P21 and Puma, which play an important role in the cell cycle and apoptosis in the p53 signaling pathway, it actually prevented cell cycle arrest and suppressed apoptosis after radiation exposure. Eventually, we confirmed that miRNA 151a-3p induces radiation resistance by inhibiting p53 signaling. Subsequent experiments were conducted to determine the effect of miR151a-3p on cells. When miRNA151a-3p was transfected, Epithelial-mesenchymal transition (EMT)-like phenotype was verified by expression of related markers, increase of cell growth, migration, and invasion was discovered. The expression of Twist1, which indirectly inhibits cell cycle arrest and apoptosis functions regulated by p53, was increased [25, 33]. In our experimental results, in the miRNA151a-3p overexpression group, cell cycle arrest, and apoptosis were significantly reduced compared to the miRNA NC group. So, we proved that miRNA151a-3p induces anti-cell cycle arrest and anti-apoptosis effects on cells exposed to radiation. Conclusions Here, we confirmed the microRNA delivery mechanism through tumor-derived exosomes in RT. We have described the intercommunication occurring between irradiated and untreated cells via exosomal miRNAs that affect tumor proliferation. HDAC5 and p53 interacted by exposure to radiation, which increased exosome release and altered the composition of miRNAs within exosomes. In particular, the expression of exosomal miR151a-3p was markedly reduced as a result of the investigation. We confirmed that inhibition of exosomes miR151a-3p promotes apoptosis of non-irradiated cancer cells, thereby increasing radiotherapy sensitivity. Understanding the molecular mechanisms of exosome secretion and microRNA expression will help design novel therapeutics that improve sensitivity to RT in HCC. In conclusion, our present findings establish HDAC5 as a key component of the p53-mediated release of exosome in response to radiation treatment resulting in tumor apoptosis through exosomal miR-151a-3p resulting in improvement of RT. Abbreviations Page 19/30 RT, radiotherapy; HDAC5, histone deacetylase 5; HCC, hepatocellular carcinoma; TSAP6, Tumor Suppressor Activation Pathway 6; miRNA, microRNA; EMT, Epithelial-mesenchymal transition. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and material Not applicable Competing interests The authors declare that they have no competing interests. Funding This research was supported by National Research Foundation of Korea (NRF) grants (NRF2021R1A6A1A03040260, NRF-2020R1I1A2072110, NRF-2017M2A2A7A02020212). This work was also supported by grants (2018IP0600-1, 2019IP0848-1, 2020IL0006-1, 2021IP0048-1) from the Asan Institute for Life Sciences, Asan Medical Center, Seoul, Korea. Authors' contributions SML analyzed the data and developed the study. BHY., MHK, DHK, YHC, MC, JWL, TKK, JWR, KGK, YHS, CGP, and JKR collected and performed samples in the field. SWL, and CHH planed the study and wrote the manuscript with input from all of the authors. The authors read and approved the final manuscript. there are no conflicts where this is the case. Acknowledgements We thank the Confocal Microscope core facility and the Comparative Pathology Core at the ConveRgence mEDIcine research cenTer (CREDIT), Asan Medical Center for support and instrumentation. Page 20/30 References 1. Mao L, Li X, Gong S, et al. 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The impact of acetylation and deacetylation on the p53 pathway. Protein Cell. 2011;2(6):456–62. 8. Purvis JE, Karhohs KW, Mock C, et al. p53 dynamics control cell fate. Science. 2012;336(6087):1440– 4. 9. Sen N, Kumari R, Singh MI, et al. HDAC5, a key component in temporal regulation of p53-mediated transactivation in response to genotoxic stress. Mol Cell. 2013;52(3):406–20. 10. Li T, Kon N, Jiang L, et al. Tumor suppression in the absence of p53-mediated cell-cycle arrest, apoptosis, and senescence. Cell. 2012;149(6):1269–83. 11. Yu X, Harris SL, Levine AJ. The regulation of exosome secretion: a novel function of the p53 protein. Cancer Res. 2006;66(9):4795–801. 12. Lespagnol A, Duflaut D, Beekman C, et al. Exosome secretion, including the DNA damage-induced p53dependent secretory pathway, is severely compromised in TSAP6/Steap3-null mice. Cell Death Differ. 2008;15(11):1723–33. 13. McKinsey TA, Olson EN. Toward transcriptional therapies for the failing heart: chemical screens to modulate genes. J Clin Invest. 2005;115(3):538–46. 14. Backs J, Olson EN. Control of cardiac growth by histone acetylation/deacetylation. Circ Res. 2006;98(1):15–24. 15. Zhang J, Shen L, Sun LQ. The regulation of radiosensitivity by p53 and its acetylation. Cancer Lett. 2015;363(2):108–18. 16. Citrin DE. Recent Developments in Radiotherapy. N Engl J Med. 2017;377(22):2200–1. 17. Chen CP. Role of Radiotherapy in the Treatment of Hepatocellular Carcinoma. J Clin Transl Hepatol. 2019;7(2):183–90. Page 21/30 18. Ringuette Goulet C, Bernard G, Tremblay S, et al. Exosomes Induce Fibroblast Differentiation into Cancer-Associated Fibroblasts through TGFbeta Signaling. Mol Cancer Res. 2018;16(7):1196–204. 19. Zou Z, Gao C, Nagaich AK, et al. p53 regulates the expression of the tumor suppressor gene maspin. J Biol Chem. 2000;275(9):6051–4. 20. Martinez-Quetglas I, Pinyol R, Dauch D, et al. IGF2 Is Up-regulated by Epigenetic Mechanisms in Hepatocellular Carcinomas and Is an Actionable Oncogene Product in Experimental Models. Gastroenterology. 2016;151(6):1192–205. 21. Wong NW, Chen Y, Chen S, et al. OncomiR: an online resource for exploring pan-cancer microRNA dysregulation. Bioinformatics. 2018;34(4):713–5. 22. Bisio A, De Sanctis V, Del Vescovo V, et al. Identification of new p53 target microRNAs by bioinformatics and functional analysis. BMC Cancer. 2013;13:552. 23. Liu H, Cheng Y, Xu Y, et al The inhibition of tumor protein p53 by microRNA-151a-3p induced cell proliferation, migration and invasion in nasopharyngeal carcinoma. Biosci Rep 2019;39(10). 24. Kim DH, Park S, Kim H, et al. Tumor-derived exosomal miR-619-5p promotes tumor angiogenesis and metastasis through the inhibition of RCAN1.4. Cancer Lett. 2020;475:2–13. 25. Qin Q, Xu Y, He T, et al. Normal and disease-related biological functions of Twist1 and underlying molecular mechanisms. Cell Res. 2012;22(1):90–106. 26. Powell E, Piwnica-Worms D, Piwnica-Worms H. Contribution of p53 to metastasis. Cancer Discov. 2014;4(4):405–14. 27. Koo KH, Kwon H. MicroRNA miR-4779 suppresses tumor growth by inducing apoptosis and cell cycle arrest through direct targeting of PAK2 and CCND3. Cell Death Dis. 2018;9(2):77. 28. Chen R, Xu X, Tao Y, et al. Exosomes in hepatocellular carcinoma: a new horizon. Cell Commun Signal. 2019;17(1):1. 29. Torre LA, Bray F, Siegel RL, et al. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65(2):87–108. 30. Butt Z, Parikh ND, Beaumont JL, et al. Development and validation of a symptom index for advanced hepatobiliary and pancreatic cancers: the National Comprehensive Cancer Network Functional Assessment of Cancer Therapy (NCCN-FACT) Hepatobiliary-Pancreatic Symptom Index (NFHSI). Cancer. 2012;118(23):5997–6004. 31. Zhao L, Liu W, Xiao J, et al. The role of exosomes and "exosomal shuttle microRNA" in tumorigenesis and drug resistance. Cancer Lett. 2015;356(2 Pt B):339–46. 32. Li S, Yao J, Xie M, et al. Exosomal miRNAs in hepatocellular carcinoma development and clinical responses. J Hematol Oncol. 2018;11(1):54. 33. Feng MY, Wang K, Song HT, et al. Metastasis-induction and apoptosis-protection by TWIST in gastric cancer cells. Clin Exp Metastasis. 2009;26(8):1013–23. Figures Page 22/30 Figure 1 Modulation of protein p53 and HDAC5 expression by radiation exposure in hepatocellular carcinoma. Radiation was exposed to 2Gy and 4Gy, and the protein expression of p53, P21, and HDAC5 were confirmed by Western blot analysis in a time-dependent manner up to 24 hours per six hours after exposure. (A-F). Using the CRISPER CAS9 system, a cell line with p53-/- in HepG2 was produced and the expression of HDAC5, p53, P21 and PUMA was confirmed through Western blot analysis after radiation exposure. The expression of each protein was quantified by b-actin. (G, H) Page 23/30 Figure 2 Interaction of HDAC5 and p53 by radiation exposure in HepG2 cells, and characterization of the exosome secretion pathway and the released exosomes that are altered by this union. The interaction between HDAC5 and p53 was confirmed after radiation exposure using Duolink proximity ligation assay in HepG2 (A). The expression of TSAP6 (STEAP3) by radiation exposure was confirmed by Western blot analysis. The expression of TSAP6 (STEAP3) was quantified by b-actin (B,C). Electron microscopy was used to image resuspended exosome particles isolated from HepG2 (D). The expression of exosome marker protein Page 24/30 HSP90, CD63, CD81 and Maspin with each sample 10ug after radiation exposure was verified with western blot analysis. (E,F). Figure 3 Time-dependent measurement and identification of the difference in exosome size and release amount determined by the p53 gene in liver cancer cells. Time-dependent measurements were made to determine whether the secretion of extracellular vesicles (EV) changes after radiation exposure using Nanosight's Nanoparticle Tracking Analysis (NTA) (A-B). Based on the analyzed nanosight data, three parts (30-150nm, Page 25/30 150-300nm, >300nm) were displayed by vesicle size (C,D). The secretion of EVs for the presence or absence of Tp53 genes after radiation exposure was analyzed qualitatively and quantitatively (E,F). Figure 4 The expression analysis of exosomal miRNAs altered by irradiation in liver cancer cells and the clinical meaning exploration and identification of candidates using bioinformatics analysis. Before and after radiation exposure, exosomal miRNA was arranged into a fold change value and analyzed as a graph and ven diagram (A,B). Applying the GEO data set, the expression difference between normal and cancer of each candidate miRNA was analyzed. (C). To investigate the clinical significance of each candidate, it was analyzed using OncomiR Cancer miRNome Atlas and patient survival was expressed by Kaplan-Meier analysis (D). The target protein prediction analysis of the major candidate miR-151a-3p was performed by the Targetscan analysis tool (E). The modulation of a miR-151a-3p the radiation exposure was analyzed by qRT-PCR in the cell and the exosome. (F,G). Page 26/30 Figure 5 Cell dysfunction according to radiation exposure is regulated by miR151a-3p. Western blot analysis was performed to confirm that p53 and its sub-signals P21, PUMA, and BCL-2, which are closely related to cancer suppression, are modulated by miR151a-3p. (A,B) Cell cycle arrest assay using FACS was performed by PI staining to confirm that cell cycle arrest induced by radiation exposure was modulated by miR151a3p. The Cell cycle was analyzed by dividing into G1, S, and G2 / M phases. (C,D) To confirm that cell Page 27/30 apoptosis induced by radiation exposure is modulated by miR151a-3p, it was analyzed using FACS by Annexin V (FITC), PI staining. (E,F) Figure 6 The ability of miRNA151a-3p to tumorigenesis. That the representative Epithelial-mesenchymal transition (EMT) marker of E-cadherin, Twist1, Vimentin, N-cadherin is regulated by the miR151a-3p was confirmed by Western blot analysis. (A,B) The cell proliferation according to the concentration of the miR-151a-3p was Page 28/30 analyzed by MTT assay. (C) Cell migration and invasion ability according to the concentration of miR-151a3p were analyzed using transwell assay. (D,E) Figure 7 Increased tumor progression in a nude mouse subcutaneous tumor transplant model overexpressing miRNA 151a-3p Acceleration of tumor growth by overexpression of miRNA 151a-3p in a subcutaneous xenograft model.(n = 5) (A-C) Representative images at day 18 post-transplantation with a subcutaneous xenograft nude mouse model injected with miR 151a-3p/miR-NC. (A) After the mice were sacrificed on the 18th day, the tumor was separated and the size was compared, and the weight of the tumors of each group was measured and plotted. (B) In the subcutaneous xenograft model, miR-151a-3p, miR-NC, and invivo jetPEI complex were injected three times every three days from the third day after tumor transplantation, and the size of the tumors of each group was measured every three days. (C) The expression of the related target protein and downstream in tumour tissues were analyzed by Western blot. (D,E) A schematic image Page 29/30 of Radiation-induced exosomal miRNA-151a-3p restrain hepatocellular carcinoma progression: By radiation, HDAC5 and p53 in HCC cells increase the secretion of exosomes as a result of the interaction. The exosomal miRNA-151a-3p inside the secreted exosome functions to infiltrate the surrounding recipient cells and promote the progression of cancer. (F) Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryFigures.docx Page 30/30 

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Genuino et al. BMC Health Services Research https://doi.org/10.1186/s12913-019-4715-8 (2019) 19:874 RESEARCH ARTICLE Open Access Cost-utility analysis of adjuvant trastuzumab therapy for HER2-positive early-stage breast cancer in the Philippines Anne Julienne Genuino1,2, Usa Chaikledkaew1,3* , Anna Melissa Guerrero2, Thanyanan Reungwetwattana4 and Ammarin Thakkinstian1,5 Abstract Background: Breast cancer is the leading malignancy among Filipino women, with about 23.50% of cases characterized by human epidermal growth factor receptor-2 (HER2) overexpression. Trastuzumab, in addition to standard chemotherapy, is currently recommended as primary treatment for HER2-positive early-stage breast cancer (EBC) in the adjuvant settings, and has been listed in the Philippine National Formulary (PNF) since 2008, but with no current evidence yet on its value for money, to date. Hence, despite several policy enablers, its accessibility remains to be limited in the Philippines. We performed an economic evaluation to assess the cost-effectiveness and budget impact of adjuvant trastuzumab therapy for HER2-positive EBC in the Philippines, using healthcare system and societal perspectives, in aid of guiding coverage decisions. Methods: A Markov model-based cost-utility and budget impact analyses were conducted to estimate the total costs incurred and outcomes gained in using 1 year of adjuvant trastuzumab added to standard chemotherapy versus standard chemotherapy alone, over a lifetime horizon. We discounted both costs and outcomes at 3.5% per annum. Parameters were estimated using country survival data, systematic review and meta-analysis of the relative treatment effect, local and international cost data, and published utility data. Univariate and probabilistic sensitivity analyses were used to account for parameter uncertainty. Results: Trastuzumab therapy was dominated with an incremental cost-effectiveness ratio (ICER) at PHP 453,505 per QALY gained from a healthcare system perspective or PHP 458,686 per QALY gained from a societal perspective, with 10% cost-effectiveness probability at the country cost-effectiveness threshold of PHP 120,000 per QALY gained. National implementation will cost an additional amount of PHP 13,909 million in year one alone, plus about PHP 2000 to 3000 million annually for the succeeding fiscal years. Conclusion: At its current cost, 1 year of adjuvant trastuzumab therapy compared to standard chemotherapy alone for HER2-positive EBC does not represent value for money in the Philippines. Its current cost will have to significantly lower down by one-half to achieve cost-effectiveness. Keywords: Adjuvant trastuzumab, Cost-utility analysis, HER2-positive, Breast cancer, Cost-effectiveness, Philippines * Correspondence: usa.chi@mahidol.ac.th 1 Mahidol University Health Technology Assessment (MUHTA) Graduate Program, Bangkok, Thailand 3 Social and Administrative Pharmacy Excellence Research (SAPER) Unit, Department of Pharmacy, Faculty of Pharmacy, Mahidol University, 447 Sri-Ayudhaya Rd., Phayathai, Ratchathewi, Bangkok 10400, Thailand Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Genuino et al. BMC Health Services Research (2019) 19:874 Background Breast cancer is currently the globally leading malignancy among women with about 1.7 million diagnosed cases and 521,907 deaths as estimated by the World Health Organization (WHO) in 2012 [1]. In the Philippines, it is recognized as the most prevalent cancer among women and in both sexes, as well as the most common cause of cancer deaths among women [2]. About 15 to 20% of breast cancers overexpresses human epidermal growth factor receptor-2 (HER2) [3] – a clinically important subtype of breast cancer that is associated with an aggressive disease phenotype and shortened survival outcomes [4], resulting in poorer prognosis compared to other subtypes. Two large studies of breast cancer data in the United States [5] [6] reported, however, that women of Asian descent were more likely to have HER2-positive tumours than Caucasian women suggesting possible racial differences. In the Philippines, the Department of Health (DOH) Breast Cancer Control Program reported a HER2-positivity rate of 23.17%, with an estimated 80% of them at the early stage [7, 8]. The discovery of revolutionary therapies such as trastuzumab, the first monoclonal antibody to specifically target HER2, has changed the course of treatment and improved the prognosis of affected breast cancer patients. Several key pivotal trials have demonstrated its relative treatment efficacy versus standard chemotherapy alone, in improving the disease-free and overall survival of HER2-positive early-stage breast cancer (EBC) patients [9–16]. These same trials though have reported an associated increased risk for cardiotoxic effects such as congestive health failure (CHF) and left ventricular ejection fraction (LVEF) decline [9–16]. Both global [17, 18] and national clinical guidelines [19] currently recommend the administration of trastuzumab with chemotherapy regimens as primary treatment for HER2-positive EBC in adjuvant settings. It has been listed in the Philippine National Formulary (PNF) since 2008, but with no current evidence yet on its value for money to date. Hence, despite several policy enablers, its accessibility remains to be limited as the current government insurance case rate for breast cancer does not cover the treatment for HER2-positive type; public hospitals cannot afford to procure and make it available in their facilities due to its high cost; and, while it has been recently included in the list of subsidized medicines under the DOH Breast Cancer Medicine Access Program (DOH BCMAP) under a negotiated reduced price for national hospitals, the access sites and medicine stocks are limited. Assessing cost-effectiveness is critical in establishing equitable trade-off decisions between the sustainable access to such effective health technology that can Page 2 of 12 improve survival and the limited health budget, especially for resource-constrained countries such as the Philippines. While many published economic evaluation (EE) studies have been previously conducted to assess its cost-effectiveness, all were conducted in settings that are not comparable to a lower-middle income country (LMIC) like the Philippines, as they were all from upper-middle and high-income countries [20–35]. Therefore, we conducted this economic evaluation to assess the cost-effectiveness and budget impact of adjuvant trastuzumab therapy compared to chemotherapy regimen alone for patients with HER2positive EBC in the Philippines, to guide coverage decisions. Methods We conducted a cost-utility analysis (CUA) using decision analytic Markov model to calculate and compare the costs and utilities of using 1 year of adjuvant trastuzumab combined with standard chemotherapy (i.e., doxorubicin 60 mg/m2 on day 1 plus cyclophosphamide 600 mg/m2 on day 1 every 3 weeks for 4 cycles followed by docetaxel 100 mg/m2 on day 1 plus trastuzumab 8 mg/kg initial dose then 6 mg/kg on day 1 every 3 weeks for 4 cycles then trastuzumab 6 mg/kg on Day 1 every 3 weeks for 14 cycles) versus chemotherapy alone (i.e., same chemotherapy minus trastuzumab) for Filipino women with HER2-positive EBC. The model cohorts were Filipino women with HER2positive EBC who enter the model at the age of 50 years old which is the mean age of patients enrolled in the Department of Health Breast Cancer Medicines Access Program (DOH BCMAP) [36]. We modelled over a lifetime horizon from both publicly-funded healthcare system and societal perspectives, with a discounting rate of 3.5% per year applied to both costs and outcomes. We measured the incremental costeffectiveness ratio (ICER) as Philippine Peso (PHP) per Life Year (LY) or Quality-adjusted life year (QALY) gained. We applied the current Philippine costeffectiveness threshold value of PHP 120,000 per QALY gained that was set by the Formulary Executive Council in the Philippines based on the value of one times Gross Domestic Product (GDP) per capita in the Philippines. In the case that trastuzumab was not costeffective, a threshold analysis was performed to calculate the cost-effective price of trastuzumab therapy. Further, we estimated the likely budget impact of its national coverage for five fiscal years. Model overview Our model structure as illustrated in Fig. 1 consists of five health states - disease-free survival (DFS), congestive heart failure (CHF), local recurrence, distant metastasis, Genuino et al. BMC Health Services Research (2019) 19:874 Page 3 of 12 Congestive Heart Failure Disease-Free Survival Local Recurrence Death Distant Metastasis Fig. 1 Schematic diagram of the Markov model and death. Upon administration of either of the competing interventions, the cohort enters the model at the DFS state. We applied a cycle length of 1 year which was run for 49 cycles to represent lifetime horizon. The model assumed that patients who progressed to CHF could only experience it once as cardiotoxicity from trastuzumab is only an asymptomatic decline in LVEF and is mostly reversible with interruption of trastuzumab or cardiac medication [37]. From CHF state, they have the option to move to DFS, local recurrence, or distant metastasis state. It was also assumed that the cohort does not have any baseline co-morbidities and the use of hormonal therapies was not considered. Based on the methodological domains and assumptions, a spread sheet model was developed using Microsoft Office Excel 2013 (Microsoft Corp., Redmond, WA) to generate the total lifetime costs incurred and LYs or QALYs gained through cohort simulation. distant metastasis, and mortality for the intervention model cohort when at DFS state. As for the longest follow-up data available to date showing constant treatment effect over 11 years [9], it was therefore assumed that the efficacy of trastuzumab lasts for 11 years. The cardiotoxic effect was incorporated in the model as increased risk for CHF for the intervention model cohort. Utility data The utility values associated with the model health states were sourced from a quality of life study among 30 patients each with early and advanced breast cancers at Hanoi Oncology Hospital and Da Nang Oncology Hospital Vietnam [41], which were derived using the EuroQol five-dimension with three-level (EQ-5D-3 L) questionnaires. As the only utility weight study derived from an Asian population in a fellow LMIC setting to date, it was deemed that Vietnam utilities are comparable and applicable to the Philippine setting. Model input parameters Clinical data Cost data The primary data source for the baseline TPs between the health states was obtained from published literatures [31, 38], which were estimated from the fouryear follow-up of joint analysis of data from NCCTG N9831 and NSABP B-31 trials on the efficacy and safety trastuzumab plus standard anthracycline-taxanebased CT [39]. We generated the relative treatment effect of trastuzumab by conducting a systematic review and metaanalysis of published trials [9–16] on the efficacy and safety of adjuvant trastuzumab [40]. The benefit of trastuzumab was incorporated in the model as a reduction applied in the risk of developing local recurrence, Direct medical costs (DMC) were comprised of workup, treatment and monitoring costs related to laboratory and diagnostic tests, procedures, admissions and outpatient visits, and pharmacologic therapy. A preconstructed costing sheet guided by clinical guidelines [18, 42, 43] was consulted among local experts. We then valuated the final costing items using the government standard case rate values [44] and data from a local costing study [45] for the medical services, procedures, and diagnostics; and, the government drug price reference index [46] and procurement data [47] for all drug costs. The cost per vial of trastuzumab was based on the negotiated price under the DOH BCMAP for Genuino et al. BMC Health Services Research (2019) 19:874 the 150 mg-IV-vial preparation amounting to PHP 619, 667 for all trastuzumab cycles. The dose calculation was based on a 65 kg-body weight. All costs were expressed in PHP 2017 values and inflation adjustments were applied as necessary by using the Consumer Price Indexes (CPI) [48]. Direct non-medical costs (DNMC) were estimated using the costing values for food and transportation expenses from the Thai Standard Costing Lists for Health Economic Evaluation [49], in the absence of these standard costing values in the Philippines. These costs (in 2009 Thai Baht values) were converted to 2017 PHP values by using the 2009 conversion factors 1 USD = 33.129 Thai Baht [50] and 1 USD = PHP 46.421 [51], then adjusted for inflation using CPI [48]. The utilization of such costs was assumed to incur for every health facility visit involved relevant for the particular health state. The list of all input parameters used in the model is summarized in Table 1. Sensitivity analysis Both one-way and probabilistic sensitivity analyses (PSA) using second order Monte Carlo simulation replicated for 50,000 times were performed to handle parametric uncertainties. We assigned beta distribution for TPs and utility parameters; log-normal distribution for the relative treatment effect; and, gamma distribution for cost parameters. The PSA results were illustrated as incremental cost-effectiveness planes and cost-effectiveness acceptability curves. A discount rate of 0 to 6%, and a treatment efficacy duration of 5 years to 49 years were also applied in the one-way sensitivity analysis. Results Cost-effectiveness analysis Based on a probabilistic approach, from the healthcare system perspective, trastuzumab therapy compared to chemotherapy alone was estimated to incur an additional cost of PHP 452,128 with an expected health gain of additional 1.20 LY or 1.00 QALY per patient, resulting in an ICER of PHP 377,009 per LY gained, or PHP 453,505 per QALY gained. From a societal perspective, adopting adjuvant trastuzumab therapy was estimated to cost an additional PHP 457,131 for a similar additional health benefit, resulting to an ICER of PHP 381,405per LY gained, or PHP 458,686 per QALY gained. The ICERs from both perspectives have consistently shown that adding 1 year of adjuvant trastuzumab to chemotherapy for HER2-positive EBC is not costeffective in the Philippines as they exceeded the costeffectiveness threshold by about 3.8 times more. The lifetime costs, LYs and QALYs gained from using trastuzumab therapy versus chemotherapy alone, and the Page 4 of 12 resulting ICERs in the probabilistic analysis from the two perspectives are shown in Table 2. Threshold sensitivity analysis Results have shown that the cost-effective price of adjuvant trastuzumab therapy per patient is PHP 596,239 from the healthcare system perspective, or PHP 590,314 from a societal perspective. This implies that the current cost of adjuvant trastuzumab therapy overall, including the cost of standard chemotherapy drugs and other supportive medications, chemotherapy administration cost and cardiac function tests (i.e., PHP 1,076,607), needs to be decreased further by about one-half in order to achieve an ICER that will be at least equal to the costeffectiveness threshold. Probabilistic sensitivity analysis The results the Monte Carlo simulation with 50,000 replications were plotted in a cost-effectiveness plane, presented in Fig. 2. Majority of the ICER plots appear at the upper-right hand quadrant of the plane extending to form an ellipsoid shape which implies the positive correlation of the incremental cost and the incremental outcomes. The black line represents the resulting mean ICER while the green line represents the cost-effectiveness threshold where estimates below this line are considered cost-effective. Figure 3 presents the cost-effectiveness acceptability curve for the healthcare system. Transforming the results of the CE plane to a cost-effectiveness acceptability curve as illustrated in Fig. 3 demonstrates that at the cost-effectiveness threshold of PHP 120,000 per QALY gained (green line), the probability of cost-effectiveness of adjuvant trastuzumab therapy is 10%, while that for the chemotherapy alone regimen is 90%, under both perspectives. The current cost-effectiveness threshold has to increase by about four times more (i.e., PHP 500,000 per QALY gained) in order for adjuvant trastuzumab to reach at least 48% probability of cost-effectiveness from the publicly-funded healthcare system perspective, or 51% probability of cost-effectiveness from the societal perspective. The trastuzumab curve only started to rise steeply as the threshold increases at PHP 650,000 per QALY gained. Deterministic sensitivity analysis The results of the one-way sensitivity analyses are presented in Fig. 4. Among all input parameters varied under both perspectives, the ICER results were most sensitive to variations in the hazard ratio for DFS, duration of efficacy of trastuzumab, discounting rate for outcomes, cost of trastuzumab therapy, and TP from DFS to metastasis state. On the other hand, the model estimates were negligibly sensitive with respect to Genuino et al. BMC Health Services Research (2019) 19:874 Page 5 of 12 Table 1 Input parameters used in the model and their sampling distribution for the probabilistic sensitivity analysis Parameter Mean (SE) Distribution Source Clinical Parameters Baseline Transitional Probabilities DFS ➔ CHF 0.0053 (0.0024) Beta DFS ➔ Recurrence 0.0294 (0.0029) Beta DFS ➔ Metastasis 0.0785 (0.0140) Beta DFS ➔ Death 0.0020 (0.0001) Beta CHF ➔ Recurrence 0.0294 (0.0029) Beta CHF ➔ Metastasis 0.0785 (0.0140) Beta CHF ➔ Death 0.1500 (0.0153) Beta Dokainish et al., 2017 [52] Recurrence ➔ Metastasis 0.0785 (0.0140) Beta Buendia et al., 2013 [31] Recurrence ➔ Death 0.2950 (0.2066) Beta Metastasis ➔ Death 0. 0.2950 (0.2066) Beta Pooled hazard ratio for DFS 0.65 (0.0825) LogNormal Pooled hazard ratio for OS 0.67 (0.0493) LogNormal Pooled risk ratio for CHF 3.97 (0.2240) LogNormal 5 – year prevalence of Breast Cancer in the Philippines 64,046 prevalent cases – WHO GLOBOCAN, 2012 [8] Incidence of Breast Cancer in the Philippines 21,057 new cases – Estimated based on the 2015 new cases (Philippine Cancer Facts and Estimates, 2015 [2]) adjusted to 2017 values by applying the incidence rate calculated using the total population for 2015 [53] and 2017 [54] Percentage of HER2-positivity of breast cancer in the Philippines 23.17% – DOH Breast Cancer and Control Program, 2013 [7] Estimated percentage of early-stage HER2-positive breast cancer cases in the Philippines 80% – DOH Breast Cancer and Control Program, 2013 [7] DMC of adjuvant trastuzumab therapy for the intervention PHP cohort (per patient per treatment course) – Drugs, CT 1076607 administration, cardiac function assessment (54929) Gamma DOH Philippines -Pharmaceutical Division, 2018 [47] Philippine Health Insurance Corporation, 2013 [44] DMC of adjuvant CT for the control cohort (per patient per treatment course) – Drugs, CT administration, cardiac function assessment PHP 194900 (9944) Gamma DNMC of adjuvant treatment for intervention cohort PHP 9432 Gamma (481) DNMC adjuvant treatment for control cohort PHP 3494 Gamma (178) DMC at DFS state - imaging, labs, visits/consultation PHP 9493 Gamma (484) Buendia et al., 2013 [31] Relative treatment efficacy of adjuvant trastuzumab therapy Genuino et al., 2019 [40] Epidemiological Data Cost Parameters Riewpaiboon, 2014 [49] Wong, 2018 [45] Genuino et al. BMC Health Services Research (2019) 19:874 Page 6 of 12 Table 1 Input parameters used in the model and their sampling distribution for the probabilistic sensitivity analysis (Continued) Parameter Mean (SE) Distribution Source DNMC at DFS state PHP 1747 Gamma (80) Riewpaiboon, 2014 [49] PHP 3400 Gamma (1602) Philippine Health Insurance Corporation, 2013 [44] DNMC at CHF state PHP 1049 Gamma [48] Riewpaiboon, 2014 [49] DMC at Local Recurrence state (first year) – work-up, radiotherapy, CT drugs, CT administration PHP 567156 (3061) DNMC at Local Recurrence state (first year) PHP 8166 Gamma (1488) Riewpaiboon, 2014 [49] DMC at Local Recurrence state (after first year) CT drugs, CT administration PHP 182437 (15513) Gamma DOH Philippines, 2018 [46] Philippine Health Insurance Corporation, 2013 [44] DNMC at Local Recurrence state (after first year) PHP 17817 (178) Gamma Riewpaiboon, 2014 [49] DMC at Local Recurrence state (after first year) – CT drugs and administration PHP 516904 (26373) Gamma DOH Philippines, 2018 [46] Philippine Health Insurance Corporation, 2013 [44] DNMC at Local Recurrence state (after first year) PHP 5939 Gamma (273) Riewpaiboon, 2014 [49] DMC at Distant Metastasis state (first year) - work-up, radiotherapy, CT drugs, CT administration, palliative care drugs PHP 956172 (48784) Gamma DOH Philippines, 2018 [46] Philippine Health Insurance Corporation, 2013 [44] DNMC at Distant Metastasis state (first year) PHP 10131 (465) Gamma Riewpaiboon, 2014 [49] DMC at Distant Metastasis state (after first year) – CT drugs and administration, palliative care drugs PHP 1666816 (59531) Gamma DOH Philippines, 2018 [46] Philippine Health Insurance Corporation, 2013 [44] DNMC at Distant Metastasis state (after first year) PHP 5939 Gamma (273) Riewpaiboon, 2014 [49] DFS 0.8320 (0.0084) Beta Ahn et al., 2014 [41] CHF 0.6700 (272.71) Beta Recurrence 0.8280 (0.0262) Beta Metastasis 0.7620 (0.0262) Beta DMC at CHF state -hospital admission, echocardiography, drugs, cardiac monitoring Gamma DOH Philippines, 2018 [46] Philippine Health Insurance Corporation, 2013 [44] Utility Parameters Table 2 Cost-effectiveness results of probabilistic sensitivity analysis Healthcare system perspective Trastuzumab-CT regimen CT only regimen Difference Total Lifetime Cost (PHP) 4,462,407 4,010,279 452,128 Total LYs gained 11.07 9.87 1.20 Total QALYs gained 8.99 7.99 1.00 ICER (PHP per LY gained) 377,009 ICER (PHP per QALY gained) 453,505 Genuino et al. BMC Health Services Research (2019) 19:874 Page 7 of 12 Fig. 2 Cost-effectiveness plane from a publicly-funded healthcare system perspective variations in the DMC and DNMC for all health states, and all utilities at the different health states. The yellow bars show the effect on the ICER of applying the lower limit of the specific parameter, while the green bars show the effect on the ICER of applying the upper limit of the specific parameter. We likewise explored the impact on the costeffectiveness of adjuvant trastuzumab therapy of simultaneous changes in the values of the HR of DFS and the duration of efficacy. The two-way sensitivity analysis as illustrated in Fig. 5 shows that over the plausible range of the said parameters, standard chemotherapy remained the cost-effective approach. Budget impact analysis The estimated number of prevalent cases of HER2positive EBC in the Philippines is 11,872 which was calculated based on the estimated proportion of earlystage cases (i.e., 80% [7]) among all HER2-positive patients which is about 23.17% (i.e., HER2-positivity rate in the Philippines [7]) of the total breast cancer prevalent cases in the country [8]. The same calculation was performed for the generation of the estimated new cases of 3903 by calculating the proportions of early-stage HER2-positive cases from the total breast cancer new cases in the Philippines [2] adjusted to 2017 value. Fig. 3 Cost-effectiveness acceptability curve – publicly-funded healthcare system perspective Genuino et al. BMC Health Services Research (2019) 19:874 Page 8 of 12 Fig. 4 One-way sensitivity analysis from a publicly-funded healthcare system perspective The total budget for implementing adjuvant trastuzumab therapy to cover all estimated prevalent and new cases of HER2-positive EBC patients in the Philippines will demand high health budget as it will incur PHP 16, 983 million per cohort in year one alone, plus more than PHP 5200 million annually per cohort on the next four fiscal years. On the contrary, implementing chemotherapy alone to cover the same number of prevalent and new cases will incur a total cost of PHP 3075 million in year one, and more than PHP 2300 million annually on the next four fiscal years. The incremental budget, therefore, is about PHP 13,909 million in year one alone, plus about PHP 2000 to 3000 million annually on the next four fiscal years, as presented in Fig. 6. Discussion To our knowledge, this is the first published study in Philippines and among LMICs to evaluate the health and economic impact of adjuvant trastuzumab therapy for HER2-positive EBC. The study is intended to guide coverage decisions on such effective but high cost therapy under a low-resource setting. It is believed that this economic evaluation faithfully considered and represented all the best available evidence appropriate to a developing country context by conducting a systematic review and meta-analysis to estimate the relative treatment effect. In this model, we project that shifting to 1 year of adjuvant trastuzumab therapy in addition to chemotherapy Fig. 5 Two-way sensitivity analysis from a publicly-funded healthcare system perspective Genuino et al. BMC Health Services Research (2019) 19:874 Page 9 of 12 Fig. 6 Five-year governmental budget impact for HER2-positive EBC, at its current cost, shall incur additional cost to the government of PHP 453,505 or USD 9084, or the society with additional PHP 458,686or USD 9188 (2017 Exchange Rate: 1 USD = PHP 49.9230 [51],) for every unit of QALY gained. Exceeding the cost-effectiveness threshold by 3.8 times more, our findings suggest that such therapy is not cost-effective in the Philippines with 10% of being cost-effective at the threshold compared to chemotherapy alone at 90%. Its value for money will improve if its current therapy cost will reduce by one-half. Apart from cost-effectiveness, the decision makers will also have to consider the affordability of trastuzumab coverage. The total acquisition cost for trastuzumab drug alone for a national coverage implementation will cost about PHP 7.36 billion – an amount that significantly exceeds the usual annual budget for the procurement of various breast cancer medicines covered under the DOH BCMAP (i.e., ranging from PHP 39 to 92 million from year 2009 to 2017) and can consume the chunk of the DOH budget allocation for the procurement of all drugs under the national health programs (i.e., PHP 9.87 billion for 2018) [55, 56]. Our generalizations are comparable with majority of previous EEs from upper-middle-income countries (UMICs) [31–33] which similarly concluded that trastuzumab in their settings is not cost-effective. In Iran [33], the ICER is about USD 174,901 per QALY gained which is 17 times more than its cost-effectiveness threshold. The ICERs range from about USD 63,036 to 153,554 in Latin American countries [31, 32], similarly exceeding their cost-effectiveness threshold by 6 to 15 times more. On the contrary, the CUA studies from high-income countries (HICs) [20–24, 26–30, 34, 35] concluded that trastuzumab is cost-effective in their settings with their ICERs ranging from USD 11,334 to 78,929 per QALY gained. Trastuzumab though was found to be costeffective in the analyses from two UMICs specifically in China by Chen et al., 2009 [25] and in Thailand by Kongsakon et al., 2018 [38], with ICERs at USD 9976 per QALY gained and USD 6527 per QALY gained, respectively. Such relatively lower ICER and opposing conclusion compared to the earlier mentioned findings from majority of analysis from UMICs [31–33] may be explained by several factors. Chen et al., 2009 applied a relatively lower hazard ratio (i.e., 0.54) versus those used by the studies in Iran (i.e., 0.64) and Latin America (i.e., 0.59). Second, it applied a 5-year efficacy duration with decreasing efficacy in a stepwise function, compared to the studies in Iran and Latin America which applied a 5year duration of efficacy with zero applied benefit onwards. Kongsakon et al., 2018 [38] did not incorporate cardiac events in their modelling analysis which may have resulted to an underestimated ICER. Considering ICER values alone, our ICERs (~ USD 9084 per QALY gained) were in fact lower than the ICER range from UMICs (USD 63,036 to 174,901 per QALY gained) which concluded for non-costeffectiveness [31–33], the ICER range from HICs (USD 11,334 to 78,929 per QALY gained) which concluded for cost-effectiveness [20–24, 26–30, 34, 35]. Two factors in this analysis may have contributed to our relatively low ICER: First is the longer duration of trastuzumab efficacy applied in this modelling (i.e., 11 years) versus most of the previous studies (i.e., 5 years) which was based on the currently available follow-up data during the time of those EEs; and, second is the Genuino et al. BMC Health Services Research (2019) 19:874 lower cost of trastuzumab therapy in our analysis (USD 12,412) versus other studies (USD 35,349 to 137, 677). Our low ICER, however, did not automatically translate to favourable cost-effectiveness findings because of the relatively low Philippine cost-effectiveness threshold at USD 2404 per QALY gained only, compared to the higher thresholds of UMICs (~USD 5000 to 15,000 per QALY gained) and HICs (USD 37,920 to 354,555). Our results were specifically sensitive to the following parameters i.e., the relative treatment efficacy which underscores the significance of estimating the effectiveness from real-world data where a less favourable treatment effect in the actual clinical practice setting may be possibly observed compared to follow-up studies under controlled environment; the duration of clinical efficacy which supports the significance of longer follow-up data on trastuzumab to define its real duration of benefit, and therefore its value for money; and, the cost of trastuzumab therapy which emphasizes the need to consider schemes to bring down its incurred cost to the government in order to achieve cost-effectiveness. Nevertheless, the model and its overall findings proved to be relatively robust, showing minimal sensitivity with variations in the majority of the parameters. There were several limitations in this analysis. First, we referred to the Vietnamese utility data for breast cancer in the absence of such data in the Philippines. While there may be differences between Filipino and Vietnamese patients on the valuation of health states because of numerous factors (e.g., cultural differences affecting disease perception, social support, health system structure), this approach was deemed acceptable because of a comparable Asian context and economic status. Second, we used the Thai cost values for the DNMCs in the societal perspective analysis due to data unavailability. As the only available and accessible standard costing menu from a similar Asian setting, using the Thai cost values was likewise deemed acceptable. The recommended study perspective anyway in the Philippine methods guide for economic evaluation is publicly-funded healthcare system which, in our analysis, used purely local costing data. Lastly, the estimation of baseline TPs were obtained from published data, not local data. Hence, it is recommended in future analysis to re-run the modelling using local TPs, health utilities and DNMC values. Exploring the role of subgroups (i.e., hormone-receptor and nodal status) on the value for money of trastuzumab is likewise proposed. Conclusion In conclusion, this CUA suggests that 1 year of adjuvant trastuzumab therapy, at its current cost, in addition to the standard chemotherapy for HER2-positive EBC, Page 10 of 12 might not be cost-effective and unaffordable in the Philippines. The government should consider schemes to lower down the cost to improve its value for money such as price negotiation, facilitation of the entry of cheaper biosimilar products, or targeted coverage (for the worst prognosis subtype or high risk subgroups), ultimately towards sustainable access to trastuzumab. Our findings are designed to aid policy decisions of a resource-constrained setting on the coverage of such effective but high cost therapy. As this research is the first published study among LMICs to evaluate the health and economic impact of adjuvant trastuzumab therapy for HER2-positive EBC, our findings may guide decision-makers from comparable low resource settings with low capacity to conduct economic evaluations who are also facing a similar policy and research question. Abbreviations CHF: Congestive heart failure; CPI: Consumer price index; CT: Chemotherapy; CUA: Cost-utility analysis; DMC: Direct medical cost; DNMC: Direct nonmedical cost; DOH BCMAP: Department of Heath Breast Cancer Medicine Access Program; DOH: Department of Heath; EBC: Early stage breast cancer; EE: Economic evaluation; HER2: Human epidermal growth factor receptor 2; HIC: High income country; HR: Hazard ratio; ICER: Incremental costeffectiveness ratio; LMIC: Lower-middle income country; LVEF decline: Left ventricular ejection fraction decline; LY: Life year; OS: Overall survival; PCS: Philippine Cancer Society; PHL CE threshold: Philippine costeffectiveness threshold; PHP: Philippine Peso; PNF: Philippine National Formulary; PSA: Probabilistic sensitivity analysis; QALY: Quality adjusted life year; RR: Risk ratio; TP: Transition probability; UMIC: Upper-middle income country; WHO: World Health Organization Acknowledgements The authors are grateful to the Pharmaceutical Division and the National Breast Cancer Control Program of the DOH Philippines, and to Dr. Rachel Rosario and Mr. Romeo Marcaida of the PCS. Authors’ contributions AJG performed the research, analysed data and drafted manuscript. UC designed the research, validated and interpreted data, as well as drafted manuscript. AMJ designed the research and drafted manuscript. TR and AT interpreted clinical data and drafted manuscript. All authors have agreed and approved the author’s contribution and final manuscript. Funding Our study was not supported by a pharmaceutical company. This work is a part of training in Health Technology Assessment (HTA)'s Master degree, which scholarship is provided by Mahidol University and the International Decision Support Initiative (iDSI). This work was produced as part of the International Decision Support Initiative (www.idsihealth.org), which supports countries to get the best value for money from health spending. iDSI receives funding support from the Bill & Melinda Gates Foundation, the UK Department for International Development, and the Rockefeller Foundation. The funding agencies of this study have no role in study design, data collection, data analysis, findings, interpretations, conclusions or writing of this manuscript. Availability of data and materials The patient data that support the findings of this study are available from the DOH and PCS but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of the DOH and PCS. Genuino et al. BMC Health Services Research (2019) 19:874 Ethics approval and consent to participate The Faculty of Dentistry/ Faculty of Pharmacy, Mahidol University, Institutional Review Board reviewed the protocol and granted the ethics approval to access the available aggregated patient data from the PCS, owned by the DOH Philippines. The DOH Philippines also released a certification to allow the use of the aggregated survival data for the sole purpose of this study. Consent for publication Not applicable Competing interests The authors declare that they have no competing interests. Author details 1 Mahidol University Health Technology Assessment (MUHTA) Graduate Program, Bangkok, Thailand. 2Pharmaceutical Division, Department of Health Philippines, Manila, Philippines. 3Social and Administrative Pharmacy Excellence Research (SAPER) Unit, Department of Pharmacy, Faculty of Pharmacy, Mahidol University, 447 Sri-Ayudhaya Rd., Phayathai, Ratchathewi, Bangkok 10400, Thailand. 4Division of Medical Oncology, Department of Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand. 5Section for Clinical Epidemiology and Biostatistics, Faculty of Medicine Ramathibodi Hospital, Bangkok, Thailand. Received: 9 May 2019 Accepted: 6 November 2019 References 1. Cancer WHO-IAfRo. 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Postoperative pneumonia in geriatric patients with a hip fracture: incidence, risk factors and a predictive nomogram Xin Zhang Changhai hospital affiliated to the Second Military Medical University Xu-zhou Duan Changhai hospital affiliated to the Second Military Medical University Da-ke Tong Changhai hospital affiliated to the Second Military Medical University Fang Ji (  jifangdirector@sina.com ) Changhai Hospital Kai-hang Xu Changhai hospital affiliated to the Second Military Medical University Rong-zhi He Changhai hospital affiliated to the Second Military Medical University Research Keywords: hip fracture, geriatric, postoperative pneumonia, nomogram Posted Date: July 16th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-35763/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/25 Abstract Background : To evaluate the incidence and risk factors of postoperative pneumonia (POP) in geriatric patients with a hip fracture after surgery, to design a predictive nomogram and to validate the accuracy of the nomogram. Methods : We retrospectively studied 1285 surgical-treated geriatric patients with a hip fracture from April 2010 to April 2018. Surgical treatment was performed on the patients of this study. The procedure methods were classified as: total hip arthroplasty, hemiarthroplasty, percutaneous fixation, intramedullary nail fixation and plate/screw fixation. The primary interest of end point of this study is the development of POP during the postoperative period. The postoperative period in this study was defined as the time from 24 hours after surgery to discharge. The diagnostic criteria for pneumonia were set according to the guidelines built by the Infectious Diseases Society of America and the American Thoracic Society (Guidelines for the Management of Adults with Hospital-acquired, Ventilator-associated, and Healthcareassociated Pneumonia, 2005). Potential variables for developing POP were identified using logistic regression analyses initially and were further selected via the method of LASSO. Then the independent risk factors were identified by multivariable regression analyses. A predictive nomogram was built based on the multiple regression model, and the calibration abilities of the nomogram was measured by Harrel C-index, calibration plot and Hosmer-Lemeshow test respectively. Decision curve analysis was carried out to assess the net benefit due to threshold probability and an on-line questionnaire survey was conducted among the clinicians to assess the applicability of the nomogram coherently. Results: Of the 1285 patients, 70 (5.4%) developed POP. COPD, number of comorbidities, ASA classification > 2, preoperative dependent functional status and cognitive impairment were identified as independent risk factors of POP. The nomogram built based on the results showed good accordance between the predicted probabilities and the observed frequency. The decision curve analysis confirmed the clinical utility of the nomogram when the threshold probabilities were between 5% and 65% due to the net benefit, while the results of on-line questionnaire among 200 clinicians showed that 91.5% of the participants had a mental shreshold of intervention between 5-50%. Conclusion: 1. COPD, number of comorbidities, ASA classification > 2, preoperative dependent functional status and cognitive impairment were independent risk factors for POP. 2. The nomogram built in this study has a good accordance between the predictive risk and the observational incidence. The results of decision curve and questionnaire among clinicians show well applicability of the nomogram. Background As a worldwide public health concern, hip fracture has ranked one of the top 10 causes of disability [1] and contributed to high mortalities and morbidities in the elderly population, which leads to increasing socioeconomic cost [2, 3]. The one-year mortality rate could be as high as 36% despite progressive surgical management and accelerated rehabilitation [4]. Many life-threatening complications have been Page 2/25 investigated in current literature, such as cardiac infarction, stroke, deep vein thrombosis and subsequent pulmonary embolism [5, 6], while little has been studied regarding pneumonia, a troubling complication following hip fracture surgery [7]. Little is known for the occurrence of postoperative pneumonia (POP) during the hospital stay. The purpose of this study is to determine the incidence and independent risk factors of POP after hip fracture surgeries in geriatric patients, to construct a predictive nomogram for the development of POP and to evaluate the accuracy and clinical applicability of the nomogram. Patients And Methods Patient Population In this study, we retrospectively studied 1285 patients with hip fractures and treated by surgical management between April 2010 and April 2018 in a single center. The inclusion criteria of patients are as follows: 1) age ≥ 65, 2) postoperative diagnosis of unilateral femoral neck fracture, or intertrochanteric fracture; 3) receiving total hip arthroplasty, or hemiarthroplasty, or percutaneous fixation, intramedullary nail fixation, or plate/screw fixation. The exclusion criteria included: 1) pathological fractures or history of diagnostic malignant tumor, 2) multiple injuries, 3) open fractures. Patients were characterized at baseline according to sex, age, body mass index (BMI), comorbidities, current smoking status, fracture pattern, risk classification of anesthesia according to American Association of Anesthesiologists (ASA), type of anesthesia, surgery option, time to surgery, operation time, blood loss, transfusion, preoperative hemoglobin and serum albumin concentration, intensive care, and length of hospital stay, etc. (Table 1).. Age was stratified into 65–74, 70–74, 75–79, 80–84, and ≥ 85 years. Hemoglobin was stratified into <120g/L in male and <110g/L in female (anemia) versus ≥120g/L in male and ≥110g/L in female (normal). Serum albumin was stratified into < 35g/L (hypoalbuminemia) versus ≥ 35g/L (normal). Transfusion was stratified into > 2 units of RBC or not. Time to surgery, which was calculated in days, was defined as the time elapsed from hospital admission to surgery [8]. Surgery procedure was stratified as described above. Patients were characterized according to whether or not they developed pneumonia in the postoperative period. The postoperative period in this study was defined as the time from 24 hours after surgery to discharge. The diagnostic criteria for pneumonia were set according to the guidelines built by the Infectious Diseases Society of America and the American Thoracic Society (Guidelines for the Management of Adults with Hospital-acquired, Ventilator-associated, and Healthcare-associated Pneumonia, 2005) [9]. In the guidelines, pneumonia was defined as the presence of “new lung infiltrate plus clinical evidence that the infiltrate is of an infectious origin, which includes the new onset of fever, purulent sputum, leukocytosis, and decline in oxygenation.” Accordingly, in our study, the identification of development of pneumonia should accord with the following events that could be confirmed in the patients’ medical/nursing records and radiological database: 1) a radiographic (X-ray or CT scan) infiltrate that is new or progressive, 2) observable clinical symptoms, which included a new onset of fever with a axillary temperature over 37.0℃, and/or purulent sputum with a positive sputum culture, 3) Page 3/25 leukocytosis. Two experienced doctors (the co-first authors of this study) independently identified the diagnosis of POP for all patients following the diagnostic strategies. Any controversies in diagnosis between the two doctors were settled by discussion, and a final diagnosis for POP on each patient was made. Statistical Analysis The primary end point of interest in this study was the development of POP. Multivariable regression analysis was conducted by logistic regression model, and this model was the basis of the POP prediction model. The potential predictors of POP analyzed in this study included age, sex, body mass index, smoking status, hypertension, diabetes, COPD, congestive heart failure, preoperative functional status, fracture pattern, ASA classification, serum albumin, hemoglobin, time to surgery, anaesthesia type, operation type, operation time, blood loss, transfusion, and ICU stay. An initial univariate logistic regression model was fitted to evaluate the association between the clinical factors and pneumonia. To identify independent risk factors, variables achieving a significance level of p < 0.05 were initially selected out. The method of LASSO was used to select potential strong factors and then independent predictors were identified by multiple regression analysis. The independent statistically significant predictors in the final model were determined with the backward method based on the AIC criteria. A predictive nomogram was established based on results of the multiple regression. A predictive discrimination was evaluated using Harrel Concordance index (C-index), which measured the probability that, given a pair of randomly selected patients, the model correctly predicted which patient will experience an event first. The C-index of the model can range between 0.5, which represents random chance, and 1.0, which represents a perfectly discriminating model (Harrell et al, 1982). The relatively correctional C-index of the nomogram was calculated by bootstraps with 1000 resamples. In calibration, the predicted probability of postoperative pneumonia generated by 10-fold cross validation was compared with the observed cumulative incidence estimates of pneumonia probability. The consistency of the curves was evaluated by Hosmer-Lemeshow (H-L) test. All statistical analyses were conducted using R software version 3.5.0 (http://www.rproject.org/), with rms package (Harrell, 2012).In addition, the decision curve analysis was carried out using the code found at https://www.mskcc.org/departments/epidemiology biostatistics/ healthoutcomes/decision-curve-analysis–01 according to its tutorials, and an on-line questionnaire (Appendix. 1) was conducted on Tencent Questionnaire Platform from February 11, 2019 to February 15, 2019 combined with the decision curve to study the threshold for intervention among clinical practitioners, which was used to evaluate the clinical applicability of the nomogram comprehensively. Results Baseline characteristics There were 1409 patients who met the inclusion criteria for this study, of which 124 were excluded due to the exclusion criteria. Finally, 1285 patients were included in this study. The clinical and pathologic Page 4/25 characteristics of patients were listed in Table 1. There were 389 (30.3%) male and 896 (69.7%) female patients in total with a median age of 80 (IQR, 75–84 years) at the time of diagnosis. 770 patients (59.9%) had comorbidities preoperatively. 334 patients (26.0%) had two or more than two kinds of comorbidities. Hypertension (43.5%) and diabetes (16.4%) were the most common comorbidities. COPD (3.8%) and congestive heart failure (2.8%) also constituted a small amount of comorbidities. Eventually, 70 (5.4%) patients developed POP, of which 30 (42.9%) were male and 40 (57.1%) were female. Comparison of the clinicopathological features between patients with and without POP Patients were characterized according to whether or not they developed pneumonia in the postoperative period. The postoperative period in this study was defined as the time from 24 hours after surgery to discharge. So the observation period of this study was limited to the duration of hospitalization. Compared to patients without POP, those with POP had more older age individuals (82 vs. 79, p = 0.001), a higher portion of male sex (42.9% vs. 29.5%, p = 0.026), a higher portion of patients who had an ASA classification higher than grade 2 (52.6% vs. 25.0%, p = 0.017), more individuals who had at least two kinds of comorbidities (72.9% vs. 23.3%, p<0.001), more dependent functional status (47.1% vs. 21.3%, p＜ 0.001) and more preoperative hypoproteinemia (55.7% vs. 36.1%, p = 0.002), a higher rate of diabetes (28.6% vs. 15.7%, p = 0.008), more COPD (28.6% vs. 2.39%, p < 0.001), more congestive heart failure (30.0% vs. 1.23%, p < 0.001), postoperative ICU stay (36.8% vs. 15.7%, p＜0.001) and more postoperative delirium (24.3% vs. 13.8%, p = 0.025). (Table 1).. Multiple regression analysis The results of the univariate regression analysis were shown in Table 2. In the univariate logistic regression, male sex (p = 0.022), age (p = 0.002), preoperative cognitive impairment (p<0.001), number of comorbidities ≥ 2 (p<0.001), diabetes (p = 0.009), COPD (p<0.001), ASA classification > 2 (p<0.001), preoperative hypoproteinemia (p = 0.001), preoperative dependent functional status (p<0.001) and postoperative ICU stay (p = 0.006) were found to be significant predictors of POP. These factors were further filtrated by the method of LASSO. Comparatively weaker factors were eliminated so that the strength of influence of the factors was enhanced. Finally, in multiple regression analysis, COPD, number of comorbidities, ASA classification > 2, preoperative dependent functional status and cognitive impairment were identified as independent risk factors of POP. (Table 3).. Development of a predictive nomogram and utility of the nomogram Page 5/25 To provide the physicians with a quantitative method to predict the risk of postoperative pneumonia, we constructed a predictive nomogram (Figure 1) according to the strength of influence of each independent risk factors based on the multivariable logistic regression (Table 3).. To use the nomogram, a vertical line was drawn up to the top point row to assign points for each variable. Then, the total number of points was calculated, and a vertical line was drawn downwards from the total point row to obtain the probability of POP. The risk of POP increased with the increasing total points, due to the nomogram. The total points of the nomogram ranges from 0 to 35 points, corresponding to the predicted risk of POP from less than 10% to greater than 85%. (Figure 1).. For example, when the total points was calculated to be 15, the corresponding risk of postoperative pneumonia was 20%. Validation of the nomogram The nomogram was validated by the method of Harrel C-index and a calibration plot using the R software 3.0. H-L test was performed to evaluate the statistical significance of the consistency between the curves in the calibration plot. The nomogram displayed a C-index of 0.84 (95% CI 0.78–0.90) which indicated good accuracy of the predictive model. The calibration plot showed good concordance between predicted probability and actual probability. (Figure. 2).. The H-L test showed that the curves in the calibration plot had statistical significance. (p = 0.88) Based on the threshold probability, the decision analysis curve was leveraged to evaluate the clinical application of a prediction model. In the decision analysis curve, the nomogram is compared with the null model for its added value, which turned out that the nomogram was applicable when thresholds were in the range of 0.05 to 0.65 due to the net benefit (Figure. 3).. The results of on-line questionnaire among 200 clinicians showed that 89.5% of the participants have obtained Identification of Physician’s Qualification for more than 5 years. 52.5% had senior professional qualifications. 88.5% have been working as an orthopedist for more than 5 years. 62% had an experience of surgical treatment for geriatric hip fractures for more than 100 cases. 97% of the participants declared that they had encountered POP among geriatric hip fracture patients in their career. 91.5% had a threshold of intervention when predictive risk of POP ranged from 5% to 50%, which slickly conformed to the net-benefit threshold interval produced by the decision curve, indicating well clinical applicability of the nomogram. Discussion The increasing number of hip fractures worldwide predicts greater impact to health status in elderly population. In the United States, it has been estimated that the total number of hip fractures might double by the year 2040 compared to the 1980s [2]. Greater contributions should be made by Asian countries for Page 6/25 the increasing number of osteoporotic hip fractures in future decades [10]. The risk of mortality and morbidity within one year of fracture remains high according to the existing literatures [4, 11~14]. The improvements in surgical and rehabilitation managements these years have improved clinical outcomes of hip fractures, while complications following hip fracture surgery remain frequent and troubling and are often complex, which might be caused by various factors, such as general conditions, comorbidities, traumatic impairment, surgery, anesthesia, and immobilization, etc. Several studies have focused on the fatal adverse events during the postoperative periods, such as acute myocardial infarction, stroke, deep vein thrombosis, etc. [16 ~ 20], which were known as the most dangerous events. Pneumonia is also known to be a devastating complication after hip fracture surgery, which can also be life-threatening when correlative respiratory and circulatory failure was induced subsequently, and is especially risky to elderly patients who are more easily affected by the impactions of trauma and immobilization after fractures and surgeries. The reported incidence of pneumonia in elderly patients with a hip fracture was 1.2% preoperatively [22] and approximately 4% ~ 5% postoperatively according to recent literatures [14, 15, 21]. Although the overall incidence was not alarmingly great, it was found that patients who developed pneumonia would have a higher readmission rate, a higher rate of sepsis, and a higher mortality rate [15]. In our study, 70 (5.4%) of 1285 patients developed postoperative pneumonia, which involved the time from 24 hours after surgery to discharge. The latest guidelines built by the Infectious Diseases Society of America and the American Thoracic Society in 2016 [23] has differed Ventilator Associated Pneumonia (VAP) from the traditional concept of Hospital Associated Pneumonia (HAP). However, in developing countries, such as China, situation remains different. Community mechanical ventilator equipment is far from popularized yet, so ventilators still could only be seen in the intensive care unit in hospital in most cases. The development of POP will significantly increase the length of stay, while clinical data would be hard to collect after discharge. Therefore, in this study, we defined POP as a new-onset pneumonia from 24 hours after surgery to discharge, so that all the clinical and radiological evidences could be exactly obtained. Little was known regarding the occurrence of pneumonia in postoperative periods. In a recent retrospective study of 29, 377 patients, male sex, older age, low body-mass index (BMI), and chronic obstructive pulmonary disease (COPD) were identified to be the strongest risk factors for pneumonia [15]. Our study has also shown that COPD, ASA classification, number of comorbidities, cognitive impairment and preoperative dependent functional status were identified to be the independent risk factors for the development of POP. We considered that the smaller sample and ethnological difference might play a role in the discrepancy of the results. The impacts of pneumonia on clinical outcomes and rehabilitations in elderly patients with a hip fracture has been demonstrated, and it has been suggested that prevention of pneumonia is necessary to improve the outcome of hip fracture [15]. In a recent research,a nonrandomized, Quasi-experimental study was designed to evaluate an inpatient pulmonary rehabilitation program by comparing 2 sequential time periods in a same center [24], and a pulmonary rehabilitation program was demonstrated to be effective Page 7/25 to reduce the incidence of pneumonia. For elderly patients, prevention for complications is more important than that for cure. Thus, an effective prediction strategy is urgently needed clinically. However, previous researches have many limitations regarding the prediction strategies for POP, and prospective studies were rarely found in current literature. The main advantages of this study include: first, a nomogram was built for the prediction for the development of POP, with good accuracy according to the statistical verifications. As a graphical presentation of predictive model, nomogram has been widely used in the area of oncology [25–28], with great advantages such as easy operation, convenience and effectiveness. Previously, we have reported the predictive nomogram for the development of postoperative delirium in elderly patients with a hip fracture [29]. The nomogram created in this study is supposed to be used clinically as a prediction method in the preoperative assessment, thus further studies with prospective designs can be conducted based on the existing preliminary results. Second, we evaluated the clinical applicability of the nomogram through the method of decision curve analysis combining with the questionnaire of clinical practitioners. The decision curve showed that the nomogram was applicable when thresholds were in the range of 0.05 to 0.65 due to the net benefit compared with the null model for its added value, while the results of questionnaire showed 91.5% of the clinicians had a threshold of intervention when predictive risk of POP ranged from 5% to 50%, which slickly conformed to the net-benefit threshold interval produced by the decision curve. It gives the presentation of the clinical utility of a predictive model more intuitively, making up for the deficiency of the traditional receiver operator characteristic curve. The main limitations of this study include the following: firstly, it was a retrospective study, so the level of evidence was limited; secondly, it was programmed in a single institution, thus the sample size was small when compared to multi-center studies, and the selection of medical institution by patients might cause potential bias. Then, as the definition of the primary end point of this study—POP, the observation period of this study was limited to the duration of hospitalization, so the follow-up time was short. Conclusion This study showed that COPD, number of comorbidities, ASA classification > 2, preoperative dependent functional status and cognitive impairment were independent risk factors of POP. The predictive nomogram established in this study showed a good accuracy in predicting the risk of POP and was applicable for clinical practice. Abbreviations POP:postoperative pneumonia, BMI:body mass index, ASA:American Association of Anesthesiologists, COPD:chronic obstructive pulmonary diseases. Declarations Page 8/25 Authors’ contributions Dr. Da-ke Tong and Dr. Fang Ji conceived and designed this research. Data collection and extraction was performed by Dr. Xin Zhang and Dr. Xu-zhou Duan.Data was verifed by Dr.Kai-hang Xu and Dr.Rong-zhi He. Statistical analysis was performed by Dr. Xin Zhang and Dr. Xu-zhou Duan participated in drafting article. All authors gave fnal approval to the version submitted. All authors approved the fnal manuscript. Acknowledgements The authors of this research wish to thank the departments of Orthopedics and Record Room, Changhai Hospital affiliated to Second Military Medical University. Funding No Funding. Availability of data and materials Data associated with this study are retained at a central repository at the Orthopaedic Department, Changhai Hospital affiliated to Second Military Medical University. If there are any questions, please contact the corresponding author. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References 1. zMarks R. Hip fracture epidemiological trends, outcomes, and risk factors, 1970–2009. Int J Gen Med.2010, (3):1-17 2. Cummings SR, Rubin SM, Black D. The future of hip fractures in the United States: numbers, costs, and potential effects of postmenopausal estrogen. Clin Orthop Relat Res 1990; 252: 163-6 3. Swart E; Vasudeva E; Makhni EC; Macaulay W; Bozic KJ. Dedicated Perioperative Hip Fracture Comanagement Programs are Cost-effective in High-volume Centers: An Economic Analysis. 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Myocardial infarction after hip fracture repair: A population-based study. J Am Geriatr Soc 2012; 60: 2020-2026. 18. Carnevale Vincenzo, Fontana Andrea, Scillitani Alfredo, Sinisi Roberto, Romagnoli Elisabetta, Copetti Massimiliano. Incidence and all-cause mortality for hip fracture in comparison to stroke, and Page 10/25 myocardial infarction, a fifteen years population-based longitudinal study. Endocrine 2017, 58(2): 320-331 19. Durand, WM; Goodman, AD; Johnson, JP; Daniels, AH. Assessment of 30-day mortality and complication rates associated with extended deep vein thrombosis prophylaxis following hip fracture surgery. Injury 2018, 49(6): 1141-1148 20. Zhang Bin-Fei, Wei Xing, Huang Hai, Wang Peng-Fei, Liu Ping, Qu Shuang-Wei, Li Jia-Hao, Wang Hu, Cong Yu-Xuan, Zhuang Yan, Zhang Kun. Deep vein thrombosis in bilateral lower extremities after hip fracture, a retrospective study of 463 patients. Clin Interv Aging, 2018. 21. Lv H, Yin P, Long A, Gao Y, Zhao Z, Li J, Zhang L, Zhang L, Tang P. Clinical characteristics and risk factors of postoperative pneumonia after hip fracture surgery, a prospective cohort study. Osteoporos Int.2016, 27(10):3001-9 22. Patterson Joseph T, Bohl Daniel D, Basques Bryce A, Arzeno Alexander H, Grauer Jonathan N. Does Preoperative Pneumonia Affect Complications of Geriatric Hip Fracture Surgery? Am J Orthop (Belle Mead NJ).2017, 46(3): E177-E185 23. Kalil AC, Metersky ML, Klompas M, Muscedere J, Sweeney DA, Palmer LB, Napolitano LM, O'Grady NP, Bartlett JG, Carratalà J, El Solh AA, Ewig S, Fey PD, File TM, Restrepo MI, Roberts JA, Waterer GW, Cruse P, Knight SL, Brozek JL. Management of Adults With Hospital-acquired and Ventilatorassociated Pneumonia: 2016 Clinical Practice Guidelines by the Infectious Diseases Society of America and the American Thoracic Society. Clin Infect Dis. 2016, 63(5): e61-111 24. Chang Shih-Chieh, Lai Jiun-I, Lu Mei-Chin, Lin Kuan-Hung, Wang Wei-Shu, Lo Su-Shun, Lai Yi-Chun. Reduction in the incidence of pneumonia in elderly patients after hip fracture surgery, an inpatient pulmonary rehabilitation program. Medicine 2018, 97(33): e11845 25. Graesslin O, Abdulkarim BS, Coutant C, Huguet F, Gabos Z, Hsu L, Marpeau O, Uzan S, Pusztai L, Strom EA, Hortobagyi GN, Rouzier R, Ibrahim NK. Nomogram to predict subsequent brain metastasis in patients with metastatic breast cancer. : 2032-7 26. Tsoutsou Pelagia G, Jeanneret Sozzi Wendy, Matzinger Oscar, Ozsahin Mahmut. Nomograms predicting locoregional recurrence in the subtype era of breast cancer. J Clin Oncol. 2013, 31(5): 647648 27. Li J, Liu Y, Yan Z, Wan, X, Xia Y, Wang K, Liu J, Lau W Y, Wu M, Shen, F. A nomogram predicting pulmonary metastasis of hepatocellular carcinoma following partial hepatectomy. Br J Cancer. 2014, 110(5): 1110-1117 28. Semenkovich TR, Yan Y, Subramanian M, Meyers BF, Kozower BD, Nava R, Patterson GA, Kreisel D, Puri V. A clinical nomogram for predicting node-positive disease in esophageal cancer. Ann Surg, 2019(3): doi 10.1097/SLA.0000000000003450 29. Zhang Xin, Tong Da-Ke, Ji Fang, Duan Xu-Zhou, Liu Pei-Zhao, Qin Sheng, Xu Kai-Hang, Di-Li Xia-Ti. Predictive nomogram for postoperative delirium in elderly patients with a hip fracture. Injury, 2019, 50(2): 392-397 Page 11/25 Tables Table 1. Baseline Characteristics of patients Page 12/25 ALL No N=1285 N=1215 Yes N=70 Gender: 0.026 female 896 (69.7%) 856 (70.5%) 40 (57.1%) male 389 (30.3%) 359 (29.5%) 30 (42.9%) Age, years 80.0 (75.0-84.0) 79.0 (75.0-84.0) 82.0 (79.0-86.8) Age, years: 0.001 0.014 65-69 130 (10.1%) 127 (10.5%) 3 (4.29%) 70-74 176 (13.7%) 170 (14.0%) 6 (8.57%) 75-79 329 (25.6%) 317 (26.1%) 12 (17.1%) 80-84 330 (25.7%) 308 (25.3%) 22 (31.4%) ≥85 320 (24.9%) 293 (24.1%) 27 (38.6%) BMI，Kg/m2 p 23.4 (21.1-25.6) 23.4 (21.1-25.6) 23.4 (21.5-26.0) 0.326 0.021 BMI，Kg/m2: BMI ≤ 18.5 53 (4.12%) 48 (3.95%) 5 (7.14%) 18.5 < BMI ≤ 20.0 147 (11.4%) 143 (11.8%) 4 (5.71%) 20.0 < BMI ≤ 25.0 703 (54.7%) 664 (54.7%) 39 (55.7%) 25.0 < BMI ≤ 30.0 335 (26.1%) 320 (26.3%) 15 (21.4%) BMI > 30.0 47 (3.66%) 40 (3.29%) 7 (10.0%) Type of fracture: 0.136 femoral neck fracture 706 (54.9%) 661 (54.4%) 45 (64.3%) Intertrochanteric fracture 579 (45.1%) 554 (45.6%) 25 (35.7%) Cognitive impairment: <0.001 no 1239 (96.4%) 1180 (97.1%) 59 (84.3%) yes 46 (3.58%) 35 (2.88%) 11 (15.7%) Smoking: 0.582 never 984 (76.6%) 928 (76.4%) 56 (80.0%) current/former 301 (23.4%) 287 (23.6%) 14 (20.0%) Comorbidities: <0.001 Page 13/25 no 515 (40.1%) 508 (41.8%) 7 (10.0%) yes 770 (59.9%) 707 (58.2%) 63 (90.0%) Comorbidities: <0.001 <2 951 (74.0%) 932 (76.7%) 19 (27.1%) ≥2 334 (26.0%) 283 (23.3%) 51 (72.9%) Hypertension: 0.99 no 726 (56.5%) 687 (56.5%) 39 (55.7%) yes 559 (43.5%) 528 (43.5%) 31 (44.3%) Diabetes mellitus: 0.008 no 1074 (83.6%) 1024 (84.3%) 50 (71.4%) yes 211 (16.4%) 191 (15.7%) 20 (28.6%) COPD: <0.001 no 1236 (96.2%) 1186 (97.6%) 50 (71.4%) yes 49 (3.81%) 29 (2.39%) 20 (28.6%) Congestive heart failure: <0.001 no 1249 (97.2%) 1200 (98.8%) 49 (70.0%) yes 36 (2.80%) 15 (1.23%) 21 (30.0%) History of depression: 1 no 1279 (99.5%) 1209 (99.5%) 70 (100%) yes 6 (0.47%) 6 (0.49%) 0 (0.00%) hypotensive drugs: 0.99 no 726 (56.5%) 687 (56.5%) 39 (55.7%) yes 559 (43.5%) 528 (43.5%) 31 (44.3%) Hypoglycemic drugs: 0.171 no 1184 (92.1%) 1123 (92.4%) 61 (87.1%) yes 101 (7.86%) 92 (7.57%) 9 (12.9%) Cardiovascular drugs: 0.001 no 1189 (92.5%) 1132 (93.2%) 57 (81.4%) yes 96 (7.47%) 83 (6.83%) 13 (18.6%) Page 14/25 Respiratory drugs: <0.001 no 1267 (98.6%) 1204 (99.1%) 63 (90.0%) yes 18 (1.40%) 11 (0.91%) 7 (10.0%) Other drugs: 0.025 no 1218 (94.8%) 1156 (95.1%) 62 (88.6%) yes 67 (5.21%) 59 (4.86%) 8 (11.4%) Traction: 0.648 no 1184 (92.1%) 1121 (92.3%) 63 (90.0%) yes 101 (7.86%) 94 (7.74%) 7 (10.0%) ASA classification 2.00 (2.00-3.00) 2.00 (2.00-3.00) 3.00 (2.00-3.00) ASA(2): <0.001 <0.001 1-2 915 (71.2%) 888 (73.1%) 27 (38.6%) 3-4 370 (28.8%) 327 (26.9%) 43 (61.4%) Pre-operative RBC 3.75 (3.33-4.18) 3.76 (3.33-4.17) 3.66 (3.23-4.20) 0.284 Pre-operative WBC 8.20 (6.56-9.60) 8.20 (6.56-9.60) 8.21 (6.49-9.95) 0.955 Pre-operative ALB 36.0 (33.0-38.0) 36.0 (33.0-38.0) 34.0 (30.0-37.0) 0.002 Pre-operative ALB: 0.002 ≥35 807 (62.8%) 776 (63.9%) 31 (44.3%) <35 478 (37.2%) 439 (36.1%) 39 (55.7%) Pre-operative Cr 67.0 (56.0-82.0) 67.0 (56.0-81.5) 70.0 (61.0-90.5) 0.085 Pre-operative HGB 114 (102-125) 115 (102-125) 112 (98.0-124) 0.291 Pre-operative PLT 173 (140-214) 172 (140-214) 174 (142-214) 0.615 Time to surgery 4.00 (3.00-5.00) 4.00 (3.00-5.00) 4.50 (3.00-6.00) 0.181 Anaesthesia type: 0.582 Intra-spinal/nerve block 1068 (83.1%) 1012 (83.3%) 56 (80.0%) general 217 (16.9%) 203 (16.7%) 14 (20.0%) 80.0 (60.0-105) 80.0 (60.0-105) 87.5 (70.0-105) Operation time Type of operation: hemiarthroplasty 0.087 0.591 347 (27.0%) 325 (26.7%) Page 15/25 22 (31.4%) total hip arthroplasty 298 (23.2%) 280 (23.0%) 18 (25.7%) intramedullary nail 456 (35.5%) 437 (36.0%) 19 (27.1%) plate/Screw 153 (11.9%) 144 (11.9%) 9 (12.9%) multiple screw 31 (2.41%) 29 (2.39%) 2 (2.86%) 200 (100-350) 200 (100-350) Blood loss 200 (100-300) Preoperative anemia: 0.407 no 695 (54.1%) 661 (54.4%) 34 (48.6%) yes 590 (45.9%) 554 (45.6%) 36 (51.4%) Functional status: <0.001 independent 993 (77.3%) 956 (78.7%) 37 (52.9%) dependent 292 (22.7%) 259 (21.3%) 33 (47.1%) Transfusion > 2iu RBC: 0.983 no 690 (53.7%) 653 (53.7%) 37 (52.9%) yes 595 (46.3%) 562 (46.3%) 33 (47.1%) Postoperative ICU: 0.005 no 1066 (83.0%) 1017 (83.7%) 49 (70.0%) yes 219 (17.0%) 198 (16.3%) 21 (30.0%) Length of stay 0.264 10.0 (8.00-12.0) 10.0 (8.00-12.0) Table 2. Univariable logistic regression Page 16/25 11.5 (9.00-13.0) 0.006 Risk factors OR p. ratio female Ref. Ref. male 1.79 [1.09;2.92] 0.022 Age, years 1.06 [1.02;1.09] 0.002 65~69 Ref. Ref. 70~74 1.46 [0.36;7.43] 0.604 75~79 1.54 [0.47;7.18] 0.498 80~84 2.89 [0.97;12.87] 0.057 ≥85 3.72 [1.28;16.42] 0.013 1.05 [0.98;1.12] 0.196 BMI ≤ 18.5 Ref. Ref. 18.5 < BMI ≤ 20.0 0.27 [0.06;1.11] 0.069 20.0 < BMI ≤ 25.0 0.55 [0.22;1.68] 0.267 25.0 < BMI ≤ 30.0 0.44 [0.16;1.44] 0.163 BMI > 30.0 1.66 [0.48;6.17] 0.423 intertrochanteric fracture Ref. Ref. femoral neck fracture 1.50 [0.92;2.52] 0.107 no Ref. Ref. yes 6.32 [2.92;12.77] <0.001 never Ref. Ref. current/former 0.82 [0.43;1.45] 0.499 Ref. Ref. Gender: Age, years: BMI，Kg/m2 BMI，Kg/m2: Type of fracture: Cognitive impairment: Smoking: Comorbidities: no Page 17/25 yes 6.33 [3.07;15.42] <0.001 <2 Ref. Ref. ≥2 8.78 [5.18;15.51] <0.001 no Ref. Ref. yes 1.04 [0.63;1.68] 0.889 no Ref. Ref. yes 2.15 [1.22;3.65] 0.009 no Ref. Ref. yes 16.28 [8.51;30.77] <0.001 no Ref. Ref. yes 33.85 [16.50;71.32] <0.001 no Ref. Ref. yes 1.04 [0.63;1.68] 0.889 no Ref. Ref. yes 1.83 [0.82;3.63] 0.133 no Ref. Ref. yes 3.13 [1.58;5.80] 0.002 no Ref. Ref. yes 12.19 [4.28;32.43] <0.001 Comorbidities: Hypertension: Diabetes mellitus: COPD: Congestive heart failure: Hypotensive drugs: Hypoglycemic drugs: Cardiovascular drugs: Respiratory drugs: Other drugs: Page 18/25 no Ref. Ref. yes 2.56 [1.08;5.34] 0.034 no Ref. Ref. yes 1.35 [0.55;2.85] 0.486 2.83 [1.93;4.15] <0.001 1-2 Ref. Ref. 3-4 4.31 [2.63;7.18] <0.001 Pre-operative RBC 0.79 [0.55;1.13] 0.198 Pre-operative WBC 0.99 [0.89;1.09] 0.827 Pre-operative ALB 0.91 [0.87;0.95] <0.001 ≥35 Ref. Ref. <35 2.22 [1.37;3.64] 0.001 Pre-operative Cr 1.00 [1.00;1.01] 0.922 Pre-operative HGB 0.99 [0.98;1.01] 0.318 Pre-operative PLT 1.00 [1.00;1.00] 0.645 Time to surgery 1.08 [0.97;1.19] 0.165 intraspinal/nerve block Ref. Ref. general 1.26 [0.66;2.24] 0.471 Operation time 1.00 [1.00;1.01] 0.23 hemiarthroplasty Ref. Ref. multiple screw 1.08 [0.15;3.98] 0.918 plate/screw 0.93 [0.40;2.03] 0.865 total hip arthroplasty 0.95 [0.49;1.81] 0.88 intramedullary nail 0.64 [0.34;1.21] 0.172 Traction: ASA classification ASA(2): Pre-operative ALB: Anaesthesia type: Type of operation: Page 19/25 Blood loss 1.00 [1.00;1.00] 0.553 no Ref. Ref. yes 1.26 [0.78;2.05] 0.345 independent Ref. Ref. dependent 3.29 [2.01;5.37] <0.001 no Ref. Ref. yes 1.04 [0.64;1.68] 0.884 no Ref. Ref. yes 2.21 [1.27;3.72] 0.006 1.01 [1.00;1.03] 0.118 Preoperative anemia: Functional status: Transfusion: Postoperative ICU: Length of stay Table 3. Multiple Logistic Regression for risk factors of POP Page 20/25 Risk factors OR 95%CI COPD P value <0.001 No Ref. Yes 10.38 4.90-22.00 Number of comorbidities <0.001 <2 Ref. ≥2 5.50 3.07-9.84 ASA classification 0.001 1-2 Ref. 3-4 2.92 1.68-5.09 Preoperative functional status <0.001 independent Ref. dependent 3.33 1.90-5.84 Cognitive impairment 0.002 no Ref. yes 4.26 C-index: 0.8396355 (0.7838565-0.8954145) HL test: p = 0.8839044 Figures Page 21/25 1.69-10.70 Figure 1 Predictive nomogram Page 22/25 Figure 2 Calibration plot Page 23/25 Figure 3 Decision analysis curve Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Supplementaryinformation.docx Page 24/25 Appendix1.OnLineQuestionnair.docx Page 25/25 

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CONHECIMENTO CIENTÍFICO E AS FAKE NEWS: um obstáculo epistemológico no ensino de ciências e biologia? SCIENTIFIC KNOWLEDGE AND FAKE NEWS: are they an epistemological obstacle in science and biology teaching? CONOCIMIENTO CIENTÍFICO Y LAS FAKE NEWS: ¿una objeción epistemológica en la enseñanza de ciencias y lo a biología? Resumo: Este artigo apresenta uma análise teórica, de natureza qualitativa, por meio de pesquisa bibliográfica para tentar esclarecer se as fakes news podem ser caracterizadas como obstáculo epistemológico na aprendizagem do conhecimento científico, nas disciplinas de Ciências e Biologia, no contexto da pandemia da covid-19 e da disseminação de notícias falsas ou fake news. Para desenvolver essa proposta pretende-se identificar na obra de Gaston Bachelard a noção de obstáculo epistemológico ao reconstruir, de modo breve, a narrativa do autor, na tentativa de indicar caminhos para o conhecimento científico e as implicações das fake news nesse processo. Sabe-se que o excesso de informações, a infodemia, apresenta-se como barreira em função da dificuldade de critérios na seleção de informações relevantes e seguras como aliadas no processo educacional. Percebese um intenso movimento anticientífico, que não é novo, mas é possível observar seu crescimento mundial nos últimos anos. Será importante que o educador assuma uma postura investigativa e reflexiva para que o sentimento de fracasso não passe despercebido quando o comportamento instintivo não se apresente em sua prática pedagógica, por ser o detentor do conhecimento porque dessa forma todo conselho referente aos erros pedagógicos que cometem será absolutamente inútil. Sendo assim, as fake News, configuraram-se em um fenômeno do pensamento fraco ou em obstáculo epistemológico da experiência primeira, que significa deixar-se levar pelo impulso, pelo arrebatamento natural, causado pela informação, sem considerá-la criticamente, porque é na criticidade que o espírito científico se forma e se reforma, retificando saberes, quando não oferecemos resistência ao novo conhecimento. Palavras-chave: Conhecimento científico. Fake News. Obstáculo Epistemológico. Recebido em: 11/03/2023 Aceito em: 26/04/2023 Publicação em: 13/09/2023 Revista Espaço do Currículo ISSN 1983-1579 Doi: 10.15687/rec.v16i3.66091 http://periodicos.ufpb.br/ojs2/index.php Edilce Maria Balbinot Borba Mestra em Ensino de Ciências Professora da Universidade Tecnológica Federal do Paraná, Brasil. E-mail: edilcemb@gmail.com Orcid: https://orcid.org/0000-0001-61671375 Marcos Antonio Florczak Doutor em Astronomia Professor da Universidade Tecnológica Federal do Paraná, Brasil. E-mail: marcosflorczak@gmail.com Orcid: https://orcid.org/0000-0002-38741926 Como citar este artigo BORBA, E. M. B.; FLORCZAK, M. A. CONHECIMENTO CIENTÍFICO E AS FAKE NEWS: um obstáculo epistemológico no ensino de ciências e biologia? Revista Espaço do Currículo, v. 16, n. 3, p. 1-7, Ano. 2023. ISSN 1983-1579. DOI https://doi.org/10.15687/rec.v16i3.66091. REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V. 16, N. 3, P. 1-7, SET/DEZ. 2023. 1 BORBA, E. M. B.; FLORCZAK, M. A Abstract: This study presents a qualitative and theoretical analysis, based on bibliographical research to enlighten whether fake news can be characterized as an epistemological obstacle to the learning process of scientific knowledge, regarding Science and Biology subjects, during Covid-19 pandemic context and fake news dissemination. Thus, to develop this proposal, it aims at identifying the notion of epistemological obstacle in Gaston Bachelard's work by briefly reconstructing the author's narrative, in an attempt to indicate possibilities to scientific knowledge and implications of fake news along this process. It is known that an overflow of information, the infodemic, has been a barrier due to the difficulty of criteria to select relevant and safe information as allies in the educational process. There is an intense anti-scientific movement, which is not new, but it is possible to observe its worldwide increase in recent years. It will be important for educators to assume an investigative and reflective attitude so that the feeling of failure does not go unnoticed when instinctive behavior does not appear in their pedagogical practice, as they hold knowledge. Thus, any advice regarding pedagogical mistakes will be absolutely useless. At last, fake news was configured as a phenomenon of weak thought or an epistemological obstacle to the first experience, which means let you be carried away by impulse, by the natural rapture caused by information, without critically consider it, since criticalness helps on scientific background, reforms and rectifies knowledge, when we do not offer resistance to new knowledge. Keywords: Scientific Knowledge. Fake News. Epistemological Obstacle. Resumen: Este trabajo presenta un análisis teórico, de naturaleza cualitativa, por intermedio de pesquisa bibliográfica para intentar aclarar si las fake news pueden ser caracterizadas como objeción epistemológica en el aprendizaje del conocimiento científico, en las asignaturas de Ciencias y Biología, en el contexto de la pandemia de la covid-19 y de la diseminación de noticias falsas o fake news. Para desarrollar esa propuesta se pretende identificar en la obra de Gaston Bachelard la noción de objeción epistemológica al reconstruir, de pronto modo, la narrativa del autor, en la tentativa de indicar caminos para el conocimiento científico y las implicaciones de las fake news en ese proceso. Es percibido un intenso movimiento anticientífico, que no es nuevo, pero es posible observar su crecimiento mundial en los últimos años. Será importante que el educador haga cargo de una postura investigativa y reflexiva para que el sentimiento de fracaso no pase desapercibido cuando el comportamiento instintivo no se presente en su práctica pedagógica, por ser el detentor del conocimiento porque de esa forma todo consejo referente a los errores pedagógicos que perpetran será absolutamente inútil. Entonces, las fake News, se configuran en un fenómeno del pensamiento débil o en objeción epistemológica de la experiencia primera, que significa dejárselo llevar por el impulso, por el arrebatamiento natural, causado por la información, sin considerarla críticamente, porque es en la criticidad que el espíritu científico se forma y se reforma, rectificando saberes, cuando no ofrecemos resistencia al nuevo conocimiento. Palabras clave: Conocimiento científico. Fake News. Objeción Epistemológica. 1 INTRODUÇÃO A velocidade das informações veiculadas pelas mídias digitais, pelas redes sociais com circulação em volume global em tempo real é discrepante da velocidade com a qual a ciência produz afirmações confiáveis. Desta forma, a disseminação de fake news, notícias falsas, que implicam em desserviço à sociedade, gera confusão, desinformação e preocupações desmedidas em diversos temas, em especial os temas científicos. E o excesso de informações propagadas por essas redes pode gerar dificuldades no discernimento sobre o que é verdadeiro e falso (ABIB, 2020). Neste artigo, será utilizado o termo fake news, quando a referência for notícias falsas. Segundo Nunes 2020, o movimento anticientífico não é novo, mas é possível observar seu crescimento mundial nos últimos anos. Na corrida contra o tempo para conter a disseminação da doença, as fake news constantemente distorcem informações fundamentadas pela ciência e uma das formas para enfrentar a desinformação é o investimento em educação. Nessa perspectiva, pretende-se apresentar uma análise teórica, com base na pesquisa bibliográfica e na obra de Gaston Bachelard, para reconstruir, de modo breve, a proposta epistemológica do autor, no sentido de compreender o termo obstáculos epistemológicos na tentativa de indicar caminhos para superar tais obstáculos. Além de buscar a construção do conhecimento REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V. 16, N. 3, P. 1-7, SET/DEZ. 2023. 2 CONHECIMENTO CIENTÍFICO E AS FAKE NEWS: um obstáculo epistemológico no ensino de ciências e biologia? científico e as implicações das fake news nesse processo, em época da pandemia da covid-19, na educação. Dessa forma, a questão que norteia este texto é: as fake news podem ser caracterizadas como obstáculos na aprendizagem do conhecimento científico, nas disciplinas de Ciências e Biologia? Para desenvolver essa proposta, pretende-se, mais especificamente, alcançar o objetivo de relacionar os obstáculos epistemológicos de Bachelard que em tese foram constituídos pela disseminação de notícias falsas, as fake news, na construção do conhecimento científico. A pesquisa se caracteriza como sendo qualitativa, com revisão bibliográfica, segundo Flick (2009). 2 O QUE SÃO E COMO SE CARACTERIZAM AS FAKE NEWS A desinformação deliberada e a necessidade de inventar e divulgar fatos estão em pleno andamento, as mentiras e notícias falsas permeiam o debate público, controlando a discussão nos meios sociais, acadêmicos e políticos, servindo a causa populista. Neste sentido o autor Rolf Arnold em seu livro: Fake News in Science and Education, 2019, chama esse fenômeno emergente, (ARNOLD, 2019, tradução nossa). Segundo Arnold (2019), os cientistas tendem a ser muito mais céticos sobre as "verdades". No processo de formulação do raciocínio baseado em evidências, fatos incontestáveis não são deliberadamente ocultados, mas apresentados para debate aberto. A livre expressão de opinião, a imprensa livre e a liberdade de pesquisa e ensino garantem a discussão e o debate necessários para argumentar as evidências dos fatos em uma democracia. Uma característica importante da educação em nosso mundo moderno é orientar os estudantes para lidar com essa mudança, uma espécie de preparação para a insegurança de não saber o que está por vir e uma abertura que ameaça o conhecimento validado e comprovado, (ARNOLD, 2019, tradução nossa). Essa plausibilidade interna nos permite ver o que vemos: os “fatos” revelados em nossa pesquisa estão longe de ser objetivos, mas sim o resultado de nossa visão metódica, disciplinada, mas individual do que está acontecendo. A maneira como lidamos com os fatos geralmente mostra que gostamos ou não – mais sobre nós mesmos e nossos pontos de vista do que sobre o objeto de nosso interesse. A busca de fatos requer um olhar ampliado por trás dos fatos – uma reflexão metafactual, (ARNOLD, 2019, tradução nossa). Para Arnold (2019), esse tipo de reflexão nos ajuda a expandir a observação científica das condições para uma observação de segunda ordem, ou seja, questionar como observamos e reconhecemos nossa tendência a aceitar achados que não são necessariamente fatos baseados em evidências apenas porque os geramos e queremos que sejam "certezas". Se esta reflexão metafactual for omitida ao lidar com fatos, há mais em jogo do que apenas aceitar como fato o que parece ser o caso. Corre-se o risco de uma reprodução contínua da visão anterior das coisas, porque o “ponto cego” em sua percepção (e talvez em outras) não é reconhecido, e você considera sua maneira de lidar com as circunstâncias factualmente indiscutível, (ARNOLD, 2019, tradução nossa). Nesse sentido o autor também contribui, John Maynard Keynes disse uma vez: "Quando os fatos mudam, eu mudo de ideia... e o que você faz?" (citado em Chamberland, 2016, p. 191). Esta é uma boa pergunta que deve nos confrontar em nosso pensamento e interpretação. Somos realmente capazes de abandonar nossas opiniões duramente conquistadas quando a análise racional revela algo melhor? Que emoções nos perseguem quando temos que perceber que nos enganamos? Corrigimo-nos ou insistimos, tentando manter uma parte das nossas convicções anteriores? Ou argumentamos ignorando os fatos - inventando provas que não existem apenas para ganhar? (ARNOLD, 2019, p.xiii, tradução nossa) Para muitas pessoas, nem isso basta: não se limitam a inventar fatos, mas também sentem a necessidade de divulgá-los. Ao fazê-lo, essas proposições ganham significado social, não por sua veracidade, mas por sua repetição. No ciberespaço, não há mais nenhum controle de qualidade para garantir que haja evidências reais do que é divulgado. Os temas diante do público não são mais REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V. 16, N. 3, P. 1-7, SET/DEZ. 2023. 3 BORBA, E. M. B.; FLORCZAK, M. A determinados por fatos comprovados, mas por novidades generalizadas. A desinformação deliberada tornou-se um meio de controle da política e da opinião pública com consequências devastadoras para o clima e a cultura do diálogo em nossa sociedade (ARNOLD, 2019, tradução nossa). O termo fake News tornou-se nos últimos anos uma, uma expressão usada de forma exagerada, muitas vezes como uma explicação rápida e fácil para os problemas da sociedade atual. Fake news foi eleita a palavra do ano de 2017 pela editora inglesa Collins (BBC, 2017). O termo tornou-se extremamente corriqueiro e popular, empregado de forma generalizada e imprecisa, em geral pensada em associação com a ruptura progressiva das democracias liberais nesse início do século XXI. (MENESES, 2018; ALVES, MACIEL, 2020). Uma das característica das fake news é a fonte, sendo na internet que se criam: pela facilidade de publicação (qualquer pessoa, em qualquer parte do mundo, pode publicar), pela desregulação, não existindo controle e pela abundância de informações, são milhões de informações disponíveis, de todo o tipo, pela facilidade de confundir verdadeiro e falso, manipular uma foto ou um vídeo, e pela facilidade de obter retorno financeiro com publicidade. Certamente, a Internet e o crescimento das mídias sociais não inventaram o fenômeno da desinformação, mas criaram um ambiente propício para que houvesse uma difusão em massa de notícias falsas, em velocidade nunca vista na história da humanidade (MENESES, 2018; ALVES; MACIEL, 2020). Em suma, o fenômeno contemporâneo das fake news envolve algo mais do que a mera falsidade da notícia, presente também em formas simples e ingênuas de erros factuais ou equívocos involuntários. Nesse ponto, contudo, é que o fenômeno contemporâneo das fake news é mais amplo e, mais do que algo que envolve ações necessariamente insinceras e manipuladoras, pode ser compreendido de maneira mais adequada como algo que envolve desinformações produzidas em contextos de embate e disputa ideológica. Via de regra, as fakes news encontram seu motor não no desejo de negar a verdade, mas sim na vontade de vencer a disputa a qualquer preço, mesmo que para isso seja preciso falsear a realidade. As pessoas deixam de se perguntar se a notícia é verdadeira ou falsa.(ALVES; MACIEL, 2020). 3 OBSTÁCULOS EPISTEMOLÓGICOS A noção de obstáculo epistemológico pode ser estudada no desenvolvimento histórico do pensamento científico e na prática da educação. Em ambos os casos, esse estudo não é fácil. A história, por princípio, é hostil a todo juízo normativo. Pois, um obstáculo epistemológico se adere ao conhecimento não questionado. Hábitos intelectuais que foram úteis e sadios podem, com o tempo, obstruir a pesquisa (BACHELARD, 1996, p.19). Na obra ‘A Formação do Espírito Científico: contribuições para uma psicanálise do conhecimento’, Bachelard busca pelo entendimento de como ocorre o processo formativo do conhecimento e quais são as principais barreiras ou “obstáculos epistemológicos” que, para serem compreendidos, necessitam de uma psicanálise do conhecimento (BACHELARD, 1996; FÁVERO; TONIETO, 2017). É importante compreender tais aspectos para quem ensina e para quem aprende, pois implica olhar o seu processo formativo para compreender como o outro aprende e como é possível intervir pedagogicamente. Assim, é importante compreender a proposta de Bachelard sobre desenvolvimento do espírito científico sistematizada na “lei dos três estados para o espírito científico”, que está entrelaçada à “lei dos três estados da alma” e ao conceito de “paciência científica”, a partir dos quais o autor construirá o conceito de “obstáculo epistemológico” e apontará suas implicações para a epistemologia e para a educação (BACHELARD, 1996; FÁVERO; TONIETO, 2017). Na visão de Bachelard (1996), é preciso considerar que o dever de todo educador, em qualquer estágio de formação, deve ser o de criar e manter o interesse pela pesquisa. No entanto, o interesse pela pesquisa tem uma história e sua força se mostra no decorrer da construção da “paciência científica”, que não é a mesma paciência da erudição, já que esta trabalha com a acumulação e a repetição, enquanto aquela trabalha com a retificação. Segundo Bachelard (1996), “a paciência seria sofrimento, porém, com esse interesse, a paciência é vida espiritual”, isto é, o interesse pela pesquisa científica desinteressada torna comportamentos REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V. 16, N. 3, P. 1-7, SET/DEZ. 2023. 4 CONHECIMENTO CIENTÍFICO E AS FAKE NEWS: um obstáculo epistemológico no ensino de ciências e biologia? como calma, tranquilidade, serenidade, persistência, equilíbrio e constância sinônimos de paciência, aspectos vitais do espírito científico, já que, do contrário, tornaram-se comportamentos perturbadores e indesejados, por isso, eliminados pela adesão fácil às verdades primeiras. Nesse sentido, estabelecer a psicologia da “paciência científica” é, para Bachelard (1996), adicionar às leis do desenvolvimento do espírito científico a “[...] lei dos três estados da alma [já que] as forças psíquicas que atuam no conhecimento científico são mais confusas, mais exauridas, mais hesitantes do que se imagina quando consideradas de fora”, pois “[...] mesmo na mente lúcida, há zonas obscuras, cavernas onde ainda vivem sombras. Mesmo no novo homem, permanecem vestígios do homem velho” (BACHELARD, 1996; FÁVERO; TONIETO, 2017). Desse modo, reconhece que a dúvida é um movimento arriscado, já que trabalha com o desconhecido, porém não é ingênua, pois sabe que o dado não existe em si mesmo, mas é construído. É um projeto forjado pela “paciência científica”, já que todo saber científico deve ser constantemente reconstruído. Aqui a “paciência científica” ganha sua expressão máxima, por reconhecer que tudo o que é fácil de ensinar é inexato, e como tal, desafia quem ensina e quem aprende, já que inverte a “dialética prematura”, ou seja, deixa de operar sobre as coisas para operar a partir de axiomas, em que a experiência deixa de ser a legitimadora de toda e qualquer teoria (BACHELARD, 1979, p. 13-15). Dessa forma buscar as condições psicológicas do progresso científico é, na visão bachelardiana (1996, p. 17), reconhecer que “[...] é em termos de obstáculos que o problema do conhecimento científico deve ser colocado”. E tais obstáculos não são meramente objetivos (complexidade e fugacidade) ou subjetivos (fragilidade dos sentidos e do espírito humano), mas são intrínsecos ao ato de conhecer e se mostram como uma “espécie de imperativo funcional, lentidões e conflitos”, isto é, fazem parte do ato de conhecer como uma condição obrigatória prática, vagarosa e conturbada, na qual residem as causas da imobilidade e até do retrocesso, as quais são denominadas de “obstáculos epistemológicos” (BACHELARD, 1996; FÁVERO; TONIETO, 2017). O primeiro obstáculo a ser superado, em direção à cultura científica, é a opinião, pois ela “[...] pensa mal; não pensa: traduz a necessidade em conhecimentos. Ao designar os objetos pela utilidade, ela se impede de conhecê-los”, por isso, o primeiro passo é destruí-la. Não basta remodelá-la, pois compreendê-la cientificamente significa abster-se de opinar a respeito de questões que não conseguimos formular claramente (BACHELARD, 1996; FÁVERO; TONIETO, 2017). A partir deste posicionamento, é possível supor que é preciso saber formular problemas, o que não acontece de forma espontânea e, por isso, precisa de aprendizagem. Na visão de Bachelard (1996, p. 18), “[...] para o espírito científico, todo conhecimento é resposta a uma pergunta. Se não há pergunta, não pode haver conhecimento científico. Nada é evidente. Nada é gratuito. Tudo é construído.” Assim, a primeira atitude a ser aprendida é formular perguntas científicas. Esta é a primeira ação do espírito que lhe permite a superação da “alma pueril e mundana” e sair do “estado concreto”, superar o realismo ingênuo, reino das opiniões. Contudo, não é qualquer pergunta que provoca tal movimento, pois a “[...] pergunta abstrata e fraca se desgasta: a resposta concreta fica” (BACHELARD, 1996, p. 18), e causa a inversão da atividade espiritual e a instalação de um obstáculo epistemológico no conhecimento não questionado. Diante do exposto, considera-se que o primeiro obstáculo a ser superado nas aulas de Ciências e Biologia é o da experiência primeira, o aprendiz fica mais apegado à beleza do experimento do que à sua explicação científica. Nesse obstáculo, dá-se preferência às imagens e não às ideias. A ausência da explicação, no obstáculo citado anteriormente, faz com que haja uma generalização. Essa ocorre quando uma lei fica tão clara, completa e fechada, que dificulta o interesse pelo seu estudo mais aprofundado e pelo seu questionamento. 3 CONSIDERAÇÕES FINAIS: AS FAKE NEWS REPENSADAS NA EPISTEMOLOGIA DE BACHELARD Na educação, a noção de obstáculo epistemológico deveria ser mais discutida na formação dos professores, uma vez que esses conceitos ajudariam na elucidação das dificuldades na construção do conhecimento científico. Neste sentido, as fake news podem agir como instrumentos, pontes para esse conhecimento. REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V. 16, N. 3, P. 1-7, SET/DEZ. 2023. 5 BORBA, E. M. B.; FLORCZAK, M. A Devemos, segundo Bachelard (1996), levar sempre em consideração que os alunos chegam às aulas de ciências com conhecimentos pré-estabelecidos em suas estruturas de conhecimento, os quais são referentes aos seus conhecimentos empíricos. Esses podem, em algumas situações, ser obstáculos para o novo conhecimento que será ensinado pelo professor. Nesse sentido, para ensinar o aprendiz a “inventar, é bom mostrar-lhe que ele pode descobrir, e que descobrindo, ele pode perceber que errar não é um mal, mas uma oportunidade de aprendizagem”. O erro é positivo, normal e útil quando consequência de um interesse, de uma possibilidade de racionalização, já que a evolução do espírito é a retificação de seus erros pessoais e “[...] não há verdade sem erro retificado” (BACHELARD, 1996, p. 293). Para que seja possível construir uma nova compreensão, primeiro é necessário criticar e desfazer a teia de intuições primeiras, de conhecimentos empíricos, que pela sua segurança e simplicidade, impede a compreensão racional das leis científicas. Eis o primeiro desafio para a docência universitária, que busca formar melhor os futuros professores de Ciência e Biologia, ou seja, derrubar os obstáculos epistemológicos adquiridos e cultivados desde a mais tenra idade. Tal decisão implica levar os sujeitos da aprendizagem a se atentarem de quão limitadas e ingênuas são suas compreensões de mundo, e que, se não reconstruídas, serão obstáculos para a compreensão científica (BACHELARD, 1996; FÁVERO; TONIETO, 2017). Na educação científica, os obstáculos epistemológicos, as noções de obstáculos se constituem em obstáculos pedagógicos. Dessa forma, a cultura científica deve começar por uma catarse intelectual e afetiva. Resta, então, a tarefa mais difícil: colocar a cultura científica em estado de mobilização permanente, substituir o saber fechado e estático por um conhecimento aberto e dinâmico, dialetizar todas as variáveis experimentais, oferecer enfim à razão razões para evoluir. Tais observações podem, aliás, ser generalizadas: são mais visíveis no ensino de Ciências, mas aplicam-se a qualquer esforço educativo (BACHELARD, 1996; FÁVERO; TONIETO, 2017). O educador não tem o senso do fracasso justamente porque se acha um mestre. Quem ensina manda. Daí, a torrente de instintos. Há indivíduos para quem todo conselho referente aos erros pedagógicos que cometem é absolutamente inútil, porque os ditos erros são a mera expressão de um comportamento instintivo (BACHELARD, 1996; FÁVERO; TONIETO, 2017). Dessa forma as fake News, se configuram em obstáculo epistemológico da experiência primeira, que significa deixar-se levar pelo impulso, pelo arrebatamento natural, causado pela informação, sem considerá-la criticamente, porque é na criticidade que o espírito científico se forma e se reforma, retificando saberes, quando não oferecemos resistência ao novo conhecimento. (BACHELARD, 1996). Segundo Arnold (2019), algumas habilidades podem colaborar no pensamento crítico e ampliar os pontos de vista: a) refletir sobre suas próprias “construções emocionais da realidade”, b) oferecer linguagem fundamentada, c) expressar-se efetivamente, d) tolerar complexidade e confusão, e) aceitar argumentação factual e f) permanecer persuasível. Estas habilidades, requerem uma educação que o ensine a estar aberto aos outros. E aqueles presos em seus próprios medos estão fechados a pontos de vista, decisões e posições singulares que lhes proporcionam calorosas garantias de certeza, vagando em uma realidade particular, buscando impiedosamente realizar apenas sua própria certeza em cada ambiente social. (ARNOLD, 2019, tradução nossa). Nesse sentido, para Arnold (2019, tradução nossa) a eficácia da educação é uma questão importante a ser considerada. " Iluminismo " não designa apenas uma época da história intelectual (aproximadamente de 1650 a 1800), na qual os esforços para encontrar soluções razoáveis começaram a substituir a superstição, mas sim o início do exame racional pelo qual a ciência e a tecnologia, bem como a filosofia, e democracia, puderam assumir a forma que conhecemos hoje. A maneira tradicional de defender a validade de insights, avaliações ou conclusões foi substituída pela exigência de “buscar seu próprio entendimento”. Quer dizer com isso, é claro, que não se deve acreditar em tudo o que se ouve, "mas sim tomar uma postura de ceticismo e pedir evidências, contradições e provas. No entanto, essa é a única coisa que precisamos para desenvolver nossas visões de mundo e ações? Nossas respostas também não são criadas a partir de tentativas muito individuais de alcançar o reconhecimento e a necessidade de ser levadas a sério - mesmo nos casos em que não temos REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V. 16, N. 3, P. 1-7, SET/DEZ. 2023. 6 CONHECIMENTO CIENTÍFICO E AS FAKE NEWS: um obstáculo epistemológico no ensino de ciências e biologia? a expertise? séculos XX e XXI? (ARNOLD, 2019, tradução nossa). Nesse sentido, a superação de tal obstáculo não acontecerá sem a análise das fragilidades das fake news a luz do conhecimento científico, levando em conta o contexto histórico, político e social no qual estão inseridas. REFERÊNCIAS ABIB, R. Reciis aborda o discurso das fake news e o saber científico. Revista Eletrônica de Comunicação, Informação e Inovação em Saúde (Reciis), Rio de Janeiro, 01 abril 2020. Disponível em: https://portal.fiocruz.br/noticia/reciis-aborda-o-discurso-das-fake-news-e-o-saber-cientifico. Acesso em: 05 de maio de 2022. ALVES, M. A.S; MACIEL, E. R. H. Fenômeno das fake news: definição, combate e contexto. Internet&sociedade, jan. 2020. Disponível em: https://revista.internetlab.org.br/o-fenomeno-das-fakenews-definicao-combate-e-contexto/. Acesso em: 05 de maio de 2022. ARNOLD, R. Fake News in Science and Education, Leaving Weak Thinking Behind. Rowman & Littlefield Publishers, Incorporated, London, 2019. BACHELARD, G. A filosofia do não: filosofia do novo espírito científico. São Paulo: Abril Cultural, 1978. (Os pensadores). BACHELARD, G. A formação do espírito científico: contribuições para uma psicanálise do conhecimento. Rio de Janeiro: Contraponto, 1996. BACHELARD, G. Epistemologia. Barcelona: Anagrama, 1989. FÁVERO, A. A.; TONIETO, C. Docência Universitária e Formação do Espírito Científico: uma abordagem a partir da epistemologia de Gaston. REVISTA ROTEIRO, Joaçaba, jan./abr. 2017. Disponível em: https://www.ea2.unicamp.br/mdocs-posts/docencia-universitaria-e-formacao-do-espirito-cientifico-umaabordagem-a-partir-da-epistemologia-de-gaston-bachelard/. Acesso em: 05 de maio de 2022. FLICK, U. Introdução à pesquisa qualitativa. 3ª ed. Porto Alegre: Artmed, 2009. GOMES, S. F. Fake News Científicas: percepção, persuasão e letramento. Revista Ciência & Educação. Bauru, 29 Fevereiro, 2020. Disponível: https://www.scielo.br/scielo.php?script=sci_arttext&pid=S151673132020000100215&tlng=pt>. Acesso em: 05 de maio de 2022. MENESES, J.P. Sobre a necessidade de conceptualizar o fenómeno das fake News, 2018. Observatório (OBS*). Lisboa. Disponível em: https://www.researchgate.net/publication/328587253_Sobre_a_necessidade_de_conceptualizar_o_fen omeno_das_fake_news. Acesso em: 05 de maio de 2022. NUNES, F. Ciência contra desinformação: pesquisadora da UFF explica a importância do combate à anticiência em tempos de coronavírus. Rio de Janeiro, 09 abril 2020. Disponível: http://www.uff.br/?q=noticias/09-04-2020/ciencia-contra-desinformacao-pesquisadora-da-uff-explicaimportancia-do-combate. Acesso em: 05 de maio de 2022. Este obra está licenciado com uma Licença Creative Commons Atribuição-NãoComercial 4.0 Internacional. REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V. 16, N. 3, P. 1-7, SET/DEZ. 2023. 7 

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TYPE Original Research 15 August 2023 10.3389/fsufs.2023.1222455 PUBLISHED DOI OPEN ACCESS EDITED BY Subhasis Mandal, National Dairy Research Institute (ICAR), India REVIEWED BY László Vasa, Széchenyi István University, Hungary Raquel Ajates, Universidad Nacional de Educación a Distancia (UNED), Spain *CORRESPONDENCE Mingwei Yang 2020651906@email.ctbu.edu.cn RECEIVED 14 May 2023 August 2023 PUBLISHED 15 August 2023 ACCEPTED 03 CITATION Liu J, Yang M and Zhang Z (2023) Can rural cooperatives reduce poverty vulnerability of smallholder households? Evidence from rural Western China. Front. Sustain. Food Syst. 7:1222455. doi: 10.3389/fsufs.2023.1222455 COPYRIGHT © 2023 Liu, Yang and Zhang. This is an openaccess 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. Can rural cooperatives reduce poverty vulnerability of smallholder households? Evidence from rural Western China Jianzhong Liu 1, Mingwei Yang 2* and Zhiyong Zhang 2 School of Marxism Studies, Hunan International Economics University, Changsha, China, 2 Research Center for Economy of Upper Reaches of the Yangtse River, Chongqing Technology and Business University, Chongqing, China, 3 Qinghai University Library, Qinghai University, Xining, Qinghai, China 1 Introduction: Poverty eradication is one of the global challenges, and rural cooperatives provide an effective path to address smallholder households‘ poverty. However, the effect of poverty reduction can show heterogeneity depending on the economic capital, human capital, and social capital of households. Methods: Based on comprehensive research data on the poverty status of 1,622 smallholder households in four provinces in the less developed regions of western China, using OLS and PSM models, this paper empirically analyzes the impact and heterogeneous characteristics of rural cooperatives on the poverty vulnerability of smallholder households. Results/Discussion: The results show that rural cooperatives have a significant dampening effect on the poverty vulnerability of smallholder farmers, and the findings hold true after robustness tests using multiple methods. The impact of rural cooperatives on the poverty vulnerability of farming households differed significantly across smallholder households with different characteristics. Specifically, participation in cooperatives had a more pronounced effect on reducing poverty vulnerability among non-poor, higher human capital and higher income farm households compared to poor, lower human capital and lower income farm households. The results of the study can provide a useful reference for policy-making on rural mutual assistance and poverty reduction among farmers. KEYWORDS rural cooperatives, poverty vulnerability, smallholder households, rural mutual assistance, poverty alleviation 1. Introduction The alleviation and elimination of poverty is a common goal of human development and a worldwide challenge (Wang et al., 2022). In 2020, the Chinese government announced that it had achieved the goal of eradicating absolute poverty, which is an important milestone in the history of the fight against poverty in humanity. However, smallholder households in rural China, especially in the western region, are still at serious risk of poverty. On the one hand, China’s poverty standard is only roughly equivalent to the World Bank’s extreme Frontiers in Sustainable Food Systems 01 frontiersin.org Liu et al. 10.3389/fsufs.2023.1222455 poverty line,1 if the low-middle poverty line and the high-middle poverty line are applied, China still has a large number of low-income people; On the other hand, many families are at risk of returning to poverty. Nearly 2 million people who have escaped poverty are at risk of returning to poverty, and nearly 3 million of the marginal population are at risk of becoming poor (Xu and Li, 2023; Zhang et al., 2023). Reducing the risk of poverty among low-income groups is therefore the central task in China’s rural revitalization strategy. Between 1979 and 1984, China’s land system underwent a dramatic shift from collective farming based on production teams (equivalent to villages) to a family-based system of responsibility under the household joint production contract. The central feature of this system is the decentralization of the management of arable land from collective to family management. Under China’s family contract responsibility policy, farmers’ motivation to produce gets a boost. However, it also brings with it the difficulty of agricultural decentralization. Most farming households have a small landholding, with an average household size of less than 0.67 hectares (Yang et al., 2023). The ‘small scale’ nature of agricultural production activities makes it necessary for smallholder farmers to allocate their resources between rural and urban areas in order to obtain sufficient income to meet household consumption expenditure. This leads mainly to three distinct vulnerabilities of smallholder households (Zhang et al., 2016). How effective is education in fighting poverty? Researchers are still divided on this question. An analysis of Pakistani households found that educational attainment was negatively associated with the incidence of poverty among farming households, and that access to higher levels of education reduced the likelihood of farming households falling into poverty (Jia and Xu, 2021). However, some studies have found that some of the educational reform in Uganda designed for low income groups did not achieve poverty level reductions (Saz-Gil et al., 2021). Finally, social networks are an important part of social capital, enhances action by playing a role in instrumental and expressive action, with resources embedded in social networks. By embedding social capital in external social networks, cooperatives build close and strong relationships with other network actors and gain greater access to knowledge and information exchange to improve the efficiency of resource acquisition. Social capital plays a role in signaling, monitoring, steering, reducing inter-organizational transaction costs, ‘collateral’ substitution and risk reduction, and by reducing the level of mistrust between individuals, it improves collective cohesion and promotes cooperation (Person et al., 2017; Ajates, 2021). Small farmers, mainly left-behind farmers, have been integrated into the traditional social life of the countryside, where social relations are more closed (Liang et al., 2015; Ma and Abdulai, 2017). For most smallholder farmers, it is often difficult to effectively enhance their capacity for autonomous development simply by relying on their own efforts. Farmers’ cooperatives (hereafter referred to as cooperatives), as ‘self-organizations’ of farmers, have attracted the attention of many scholars in terms of increasing farmers’ incomes and reducing poverty (Deng et al., 2021; Cheng et al., 2022). Some scholars argue that cooperatives should be regarded as an efficient organizational innovation in rural poverty governance because of their ability to fundamentally improve the efficiency of the use of poverty reduction funds and to improve the income, capacity and rights poverty of farm households (Ma and Abdulai, 2017). Cooperatives convey agricultural knowledge while improving the market competitiveness and social adaptability of poor farmers by enhancing their individual capacities, and repairing the capacity deficits of farmers in the new economy (Bacon et al., 2014). The mechanisms inherent in the participation of poor farmers in cooperatives to reduce poverty and increase income are partly the result of the individual empowerment of members through their business, capital and management participation in the cooperative, and this capacity-enhancing effect is greater for middle- and highincome farmers (Bernard et al., 2008; Verhofstadt and Maertens, 2014). In general, most of the available studies confirm the positive role of cooperatives in reducing poverty and increasing income (Chagwiza et al., 2016; Cafer and Rikoon, 2018; Kumar et al., 2018). Cooperatives have an advantage over scattered smallholder farmers in terms of large-scale farming, use of advanced technologies, coping with market risks and access to policy subsidies, increasing the added value, profitability, labor productivity and employment of farmers engaged in agricultural production (Ito et al., 2012). Co-operatives not only help farmers reduce transaction costs in the procurement of agricultural materials and agricultural production services, but also improve their bargaining power in the sale of agricultural products; they also provide various types of training and activities to help farmers improve their ability to obtain information, express their needs and apply technology, thereby increasing their income (Kumar et al., 2018). However, some scholars have also found that farmer group differences have a key impact on the poverty-reducing effects of cooperatives. Some cooperatives have evolved into “self-run enterprises” that do not contribute to the development of their members or to the income of farmers as a whole (Bernard and Taffesse, 2012). The natural heterogeneity of smallholder farmers in terms of their initial resource endowments, such as production and management capacity, risk tolerance and household livelihood capital, may lead to “elite capture,” resulting in the diversion of poverty alleviation resources and misalignment of project implementation, creating new income inequalities (Beuchelt and Zeller, 2013). Some scholars also argue that small and medium-sized members of cooperatives are prone to “free-riding” behavior, unwilling to pay for the cooperative’s public services and enjoy the benefits without contributing much, affecting the efficiency of the organization’s operations and distributional equity (Tadesse et al., 2019; Ishak et al., 2020). The above-mentioned studies provide an important theoretical basis for this paper, but there are still shortcomings: firstly, although scholars have focused on the impact of cooperatives on poverty reduction among farmers, they have not yet reached a unanimous conclusion, and most of them are based on theoretical discussions, lacking qualitative and quantitative studies on the impact of cooperatives on farmers’ ability to reduce poverty. Secondly, few scholars have studied the heterogeneous effects of cooperatives on the future poverty reduction capacity of smallholder farmers from the perspective of farmer differentiation, especially the lack of discussion of groups of farmers with different poverty attributes and different human capital endowments. 1 In 2018, the World Bank used less than US$1.90, US$3.20 and US$5.50 per person per day as the extreme, low and medium poverty lines and the upper secondary poverty line. Frontiers in Sustainable Food Systems 02 frontiersin.org Liu et al. 10.3389/fsufs.2023.1222455 The main contribution of this paper is that it uses the cooperative empowerment dimension as an entry point to quantify the reduction effect of cooperatives on farmers’ poverty vulnerability and the differences in their effects on heterogeneous groups between groups, enriching the research framework on the “multidimensional pro-poorness” of cooperatives by using the Predominant Score Matching (PSM) method. The remainder of the paper is organized as follows: Section 2 proposes a theoretical analysis and four research hypotheses. Section 3 introduces the identification strategy, variables, and data for this study. Section 4 tests four hypotheses and presents the regression results and covers the heterogeneity analysis and robustness testing. Section 5 provides the discussions, conclusions and related policy implications. and the cost of control or supervision in management, ensures the smooth implementation of collective decisions, and minimizes opportunistic behavior on the part of cooperative managers (Bender, 1999; Wollni and Zeller, 2007; Ma and Abdulai, 2016). In general, cooperatives are effective in reducing the poverty vulnerability of smallholder farmers through market access, accelerated human (social) capital formation and empowerment of management. On this basis, we propose hypothesis 1. H1: Cooperatives can reduce the poverty vulnerability of smallholder households. From the beginning of their development, cooperatives have been characterized by an external market environment embedded in the vertical integration of agriculture and supply chain management (Verhofstadt and Maertens, 2015). Smallholder farmers are at a distinct disadvantage in terms of enjoying the benefits of global value chains due to their low sales volume and limited bargaining power, as well as the fact that smallholder farmers are often severely limited in their participation in markets by human capital and credit. Especially with the increasing trend toward globalization of agricultural markets and the need for higher management skills and logistics techniques to market agricultural products and meet higher standards of food safety certification, the problem of smallholder participation in integrating into global value chains has become more pronounced, and they even face the risk of being marginalized (An et al., 2015; Fan and Garcia, 2018; Ajates, 2020). As far as the internal environment is concerned, with socio-economic development and the expansion of cooperatives, the structure of group membership has stratified and the heterogeneity of members has increased significantly (Mojo et al., 2017). Therefore, when studying the impact of cooperatives on the poverty vulnerability of smallholder farmers, different group characteristic factors should be included in the examination. In terms of poverty attributes, on the one hand, poor farmers tend to have a strong will to escape poverty, but show vulnerability characteristics such as poor labor skills, sick and disabled members of the family, heavy child-rearing burden and few risk-averse means (Deng et al., 2021). The greater the vulnerability to poverty, the more risk-sensitive and risk-averse they are. They are reluctant to join a cooperative or even if they do join, they are reluctant to invest in shares, thus becoming passive or dormant members. The stratification between poor and non-poor farmers, and the resulting unequal power patterns, may constrain the accumulation of a virtuous cycle of poverty vulnerability reduction among poor farmers (Ma and Abdulai, 2016). On the other hand, from the perspective of cooperatives, although cooperatives are an effective way to reduce poverty through the organic combination of market and government mechanisms, and objectively have a poverty-reducing effect, some cooperatives do not have an obvious motivation to reduce poverty subjectively, let alone a mature concept of poverty alleviation (Grashuis and Su, 2019). Most of the leaders of co-operatives have a philosophy of serving their own economic performance, and their “deliberate care” for the poor tends to be weakened. Based on economic rationality, co-operatives are exclusive toward poor farmers who lack resources and have low development capacity, and tend to favor non-poor groups who are well endowed with large scale operations and dedicated investments in poor areas (Deng et al., 2010). Even when cooperatives open their membership to poor farmers in general, taking care of poor farmers 2. Theoretical analysis and research hypothesis The main reason for attracting farmers to join a cooperative is the economic return it can bring to them. Attached to the economic function, cooperatives also generate positive externalities by helping farmers to overcome barriers to market access, improve scientific and cultural literacy, increase social capital stock and empower management (Ito et al., 2012; Kumar et al., 2018). They also have positive externalities in terms of helping farmers overcome barriers to market access, improving scientific and cultural literacy, increasing social capital stock and empowering management, which in turn reduce the poverty vulnerability of smallholder farmers (Chagwiza et al., 2016; Zhang et al., 2023). With the development of the market economy, agricultural markets are becoming more and more mature. For decentralized smallholders, due to their weakness, asymmetric information, high transaction costs and low standardization of production, they face high barriers to market entry and lack sufficient competitiveness and voice in large markets, and are unable to connect effectively with markets (mainly high value-added markets) on their own (Loconto and Simbua, 2012; Richards and Mendez, 2014). Collective action, i.e., the formation of cooperatives, is an effective mechanism to help resolve the conflict between smallholders and large markets and to increase farmers’ participation in the market (Beuchelt and Zeller, 2013). Collective action provides relevant information and services to smallholders, including technical information and services (agricultural extension and research and development), educational services (production skills training, business skills training and general education), etc. It improves the efficiency and management of farmers’ access to agricultural technology and also promotes the sustainable and healthy development of the cooperative (Mavimbela et al., 2010; Meador et al., 2016; Tray et al., 2021). The human capital of the farmers is accumulated and the endogenous motivation for development is further stimulated. In terms of management participation, democratic management and control is the foundation and core principle of the cooperative system. The participation of members in general meetings, councils and supervisory boards, and the full expression of their views and demands, not only helps to safeguard their own property rights and interests and to obtain more residual claims, but also increases trust within the membership, helps to reduce conflicts in decision-making Frontiers in Sustainable Food Systems 03 frontiersin.org Liu et al. 10.3389/fsufs.2023.1222455 in a unified operation would mean lower returns for non-poor members (Wollni and Zeller, 2007). The more poor farmers a co-operative takes on, the greater the risk it may face of a decline in overall benefits. On this basis, we propose hypothesis 2. their resource endowments and effectively spill over, thus becoming the biggest beneficiaries of the development of the co-operative (Shi et al., 2019; Li et al., 2021). On this basis, we propose hypothesis 4. H4: Cooperatives are more effective in reducing the poverty vulnerability of middle- and high-income households than of low-income households. H2: The effect of poverty vulnerability reduction due to cooperative membership is higher on non-poor farming households than in poor farming households. Based on the above analysis, the theoretical analysis framework of this paper is shown in Figure 1. Poverty is a vague concept, but it has some basic characteristics, namely that it is mainly marked by “lack,” which appears as “low income” and “lack of material and services,” but in essence is a lack of “means,” “capabilities,” “rights” and “opportunities.” Education levels are closely linked to the ability of smallholder households to escape poverty (Yang et al., 2023). Better-educated farmers tend to have a greater ability to accept new knowledge and new things and to understand and learn, and their rich knowledge base makes them more likely to accept the organizational system, business philosophy and production techniques of the cooperative, which makes it easier for them to join the cooperative (Ito et al., 2012). Moreover, the stronger the ability to accept new knowledge and technology, the clearer the perception of the cooperative’s ability to enhance its own development. On this basis, we propose hypothesis 3. 3. Materials and methods 3.1. Data The data in this article comes from a comprehensive survey on the status of rural poverty in Southwest China, July–September 2021. The region covers four provinces, Yunnan, Guizhou, Sichuan and Chongqing. Including eight state-defined poor counties (cities) in Fengjie, Wanzhou, Yunyang, Xishui, Puding, Guang’an, Xuyong, and Dongchuan, 136 villages with 12 farmers per village, a total of 1,632 households in the sample. The research sample was selected based on a three-stage sampling: (1) Cluster analysis sampling. The original 592 state-defined poor counties were divided into three categories of overall poverty status, with experts empirically assessing the worst category and selecting sample provinces and counties in the worst category. (2) Probability Proportional Scale Sampling. Sample villages were selected in proportion to the size of the poor population. (3) Random sampling. A sample of 12 farmers was randomly selected in each village to answer the questionnaire. This sample data represents to a large extent the group of farming households that need the most attention in the less developed counties of China, and is representative and typical. Since the focus of this paper is on smallholder households, farmers with average arable land above 0.67 ha are excluded. After data cleaning and elimination of the 10 questionnaires that did not meet the requirements, the actual research population of this paper is 1,622 households. H3: Cooperatives are more effective in reducing the vulnerability to poverty of households with high human capital endowments than those with low human capital endowments. Institutional norms of rural cooperatives mainly include formal institutional arrangements and informal institutional norms. We have found that rural cooperatives in China tend to be member-based and rely closely on related enterprises. In terms of management, the cooperative has adopted the practice of “two brands and one set of staff ” with the enterprise. The day-to-day management, sales and technical guidance of the cooperative are all dependent on the relevant enterprise, with the core members responsible for the management of the enterprise and the ordinary members only involved in the business work. The heterogeneity of the membership structure of cooperatives is shaped by the differentiation of farming households (van Rijsbergen et al., 2016). This heterogeneity is reflected in the distinction between core members and general members of the cooperative. These two types of members have different levels of income and different levels of participation in the cooperative, resulting in different roles and division of labor, which leads to differences in their ability to improve their skills, showing typical asymmetrical characteristics (Valkila and Nygren, 2010). Compared to core members, general members are usually low-income, low-capital participation groups, and such groups often lack the interest and ability to participate in the public affairs of the cooperative, or even the opportunity to do so (Jitmun et al., 2020). They rarely participate in the day-to-day management and supervision of cooperatives, and are mostly limited to basic aspects such as participation in the purchase of agricultural inputs and materials, the sale of agricultural products, and access to specialized technical services and policy concessions. For core members, their material resource endowments are at an advantage, and they hold the majority of shares in the co-operative, control most of the residual control and residual claims, have more say in the daily production and management activities of the co-operative, and can make full use of Frontiers in Sustainable Food Systems 3.2. Method 3.2.1. Poverty vulnerability measurement Poverty vulnerability, which connects risk shocks to the degree of household welfare, is often seen as unobservable, dynamic, and forward-looking, with a focus on poverty generation expectations (Wang et al., 2022). Poverty vulnerability is the probability that a household or individual will fall into poverty or fail to escape from poverty as a result of exposure to uncertainty risk shocks. Poverty vulnerability is calculated as follows. (  ln z − X i βˆFGLS Φ Vˆi = Prob ( ln ci < ln z X i ) =  X i θˆFGLS  04 )    (1) frontiersin.org Liu et al. 10.3389/fsufs.2023.1222455 FIGURE 1 A theoretical model of the impact of cooperatives on poverty vulnerability of smallholder households. controls similar to the treatment group to compare their effects, thus effectively solving the endogeneity problem arising from sample selection bias (Yang et al., 2023). Since differences in farmers’ initial endowments can directly cause a “selective bias” in their willingness or behavior to join a cooperative, and whether or not to join a cooperative often reflects the ideological tendency of rational farmers to pursue optimal utility, a simple OLS regression of Equation (2), which estimates the capacity-enhancing effect of farmers ignores their own subjective initiative, yields only the conditional expectation effect of the explanatory variables on the explanatory variables, and the results obtained may be biased. The PSM propensity value matching method can effectively solve these problems by finding a control group (uninvolved farmers) with similar characteristics that can simulate the counterfactual state of the treatment group (involved farmers), thus maximizing the elimination of endogeneity problems due to self-selection bias. The specific steps are as follows. Step 1: we used a logistic model to calculate the conditional probability of a household Participating in rural cooperatives, i.e., the propensity score. Step 2: based on the propensity scores obtained through three methods: nearest neighbor matching, radius matching and kernel matching, we found a sample of farmers in the control group with propensity scores as similar as possible to those in the treatment group, in order to control and eliminate selectivity errors. Step 3: PSM model requires that the variables used for matching meet the common support domain assumption and the balance test, and after the sample has been matched and the matching effect has been achieved, we calculate the average treatment effect (ATT). The ATT is calculated as shown below. Where Vˆi is an estimate of the probability of future poverty for farmer i, ci is the value of per capita household consumption, z is the delineated poverty line, Φ is the cumulative distribution function of the normal distribution, βˆFGLS and θˆFGLS denote the expected value and variance of future household consumption estimated by the FLGS method, respectively. Xi is an observable variable, referring to Wang et al. in their examination of poverty vulnerability by introducing household characteristics variables (including household income, household size, land assets, liabilities, agricultural machinery, etc.) and household head characteristics variables (including age, gender, education, etc.). 3.2.2. Econometric model We constructed an OLS model to examine the impact of cooperatives on poverty vulnerability of smallholder households. The OLS model is set up as in Equation (2): yi = α + β Cooperativesi + θ Xi + ε i  (2) Among them, y i is the poverty vulnerability of smallholder households. Cooperatives i represents the participation in a rural cooperative, and X i indicates a series of control variables, mainly including family characteristics, village characteristics and head of household Characteristics. 3.2.3. Propensity score matching (PSM) model The propensity score matching method is a counterfactual inference method, the basic idea of which is to find a sample of Frontiers in Sustainable Food Systems 05 frontiersin.org Liu et al. 10.3389/fsufs.2023.1222455 PSi = Pr Di = 1|X i  = E Di = 0|X i  4. Empirical results (3)  4.1. Benchmark regression results ATT = 1 Nt ∑i∈I ∩S {Yi − ∑ j∈I ∩SWijY j } t c  (4) Table 2 reports the results of the benchmark regression of the impact of rural cooperatives on the poverty vulnerability of smallholder households. In Model 1, we only control for the characteristic variables of the household head. In Model 2, we further controlled for household characteristics variables of smallholder farmers. In Model 3, we included household head characteristics, household characteristics and village residence characteristics as control variables. The results show that, controlling for a range of variables, participation in cooperatives can significantly reduce the poverty vulnerability of smallholder farmers. The impact factor is −0.0162, and is significant at the 5% level, which basically supports hypothesis 1 that cooperatives can reduce the poverty vulnerability of smallholder farmers. The coefficients and signs of the control variables remain consistent with existing studies. The level of education of the household head, the net household income, social capital and the distance from the village to the county have a significant positive impact on the reduction in poverty vulnerability of smallholder farmers. Age of head of household and level of household poverty have a significant negative effect on reduction in poverty vulnerability of smallholder farmers. In addition, the number of laborers and migrant workers also show a negative impact on the reduction of poverty vulnerability, which may be closely related to the demographic disadvantage of smallholder households. Among them, N t is the number of samples, I t is the sample set of the disposal group (Participating in rural cooperatives), I c is the sample set of the control group (Not Participating in rural cooperatives), Yi is the observed value of the sample of the disposal group, and Y j is the sample of the control group. The observations of j, S is the common support domain set, Wij is the matching weight, and ATT is the average disposition effect. 3.3. Variables 3.3.1. Dependent variables To forecast household poverty vulnerability, this article uses household per capita consumption. One reason for using consumption to define poverty is that income is easily underestimated in micro-surveys, whereas consumption can better reflect the level of family welfare, and the other is that using income as an explanatory variable can easily lead to strong endogenous problems in the measurement model. Regarding the choice of the poverty line, there are primarily two standards of per capita daily consumption of US$1.9 and US$3.1 proposed by the World Bank in 2015, which we convert into ¥2,800 and ¥4,570 per capita annual consumption based on China’s average purchasing power and CPI index (Wang et al., 2022). In the subsequent analysis, we focus on ¥4,570 as the poverty standard line. 4.2. Robustness tests of the PSM model The benchmark regression results show that joining a rural cooperative can significantly reduce the poverty vulnerability of smallholder farmers. However, there is also a potential problem that OLS regression results are susceptible to sample selection bias, and those factors that are not observed may affect the precision of the estimates. In order to ensure the credibility and robustness of the regression results, we further used the come PSM model to verify the poverty vulnerability reduction effect of cooperatives on farm households. We have selected control variables that were significant in the baseline regression model for the propensity score matching, in order to eliminate the variability of the characteristic variables between the two sample groups. After propensity score matching, the question of conditional independence between the two sample groups needs to be checked, i.e., there are no significant differences in the characteristics variables between the matched sample groups, except for differences in the poverty vulnerability of the farmers. Table 3 reports the results of the conditional independence hypothesis tests for the explanatory variables before and after PSM matching. After PSM matching, the pseudo R2 decreases from 0.013 before matching to 0.001–0.003 after matching. LR chi2, B-values and mean bias-value have all fallen substantially. All p values are greater than 10%. Thus, after matching by the PSM model, we significantly eliminate systematic differences in the distribution of explanatory variables between the treatment and control groups, minimize sample selection bias, and propensity score estimation and sample 3.3.2. Independent variables The core independent variable is whether or not one participates in a cooperative. The ability of cooperatives to bring significant capacity enhancement effects to farmers at different stages of agricultural production depends on whether or not farmers participate in cooperatives. The core explanatory variable is “whether or not the farmer participates in a cooperative,” which describes the impact of cooperatives on the poverty vulnerability of smallholder households. 3.3.3. Control variables With reference to existing studies, this paper introduces three types of control variables, namely, variables on individual household head characteristics, variables on household characteristics and variables on village characteristics (Scripcariu et al., 2020; Yin et al., 2020; Ma and Jin, 2022). Household head characteristics include gender, age, and education level; Household characteristics include the number of household labors, net household income per capita, level of household poverty, whether there are family members working in the city, total productive assets and annual gift expenses; village characteristics include village transportation conditions and economic status. The descriptive statistical characteristics of the specific variables are shown in Table 1. Frontiers in Sustainable Food Systems 06 frontiersin.org Liu et al. 10.3389/fsufs.2023.1222455 matching are more successful, significantly weakening estimation bias due to self-selection. In Table 4, we used five PSM methods to estimate ATT, ATU and ATE for the impact of cooperatives on the poverty vulnerability of smallholder farmers. Among them, ATT represents the average treatment effect of the treatment group; ATU represents the average treatment effect of the control group; ATE is the average treatment effect for the overall sample. The results show that the five matching methods ATT, ATU and ATE all passed the test at the 1% significance level, which indicates that the results of matching between samples are relatively robust. The mean value of ATT is −0.0252, which suggests that cooperatives have a significant dampening effect on the poverty vulnerability of farm households. In other words, the poverty vulnerability of farmers who joined cooperatives was reduced by 0.0252 compared to those who did not join cooperatives. 4.3. Results of the heterogeneity analysis 4.3.1. Heterogeneity analysis based on educational level of household heads The level of education of the household head is largely representative of the overall human capital endowment of the smallholder household. Therefore, we examine the heterogeneity of the effect of cooperatives on reducing the vulnerability of farm households to poverty in terms of the educational attainment of the TABLE 1 Definition and descriptive statistics of the variables involved. Variables Definition Mean SD 0.1414 0.2089 0.2429 0.4289 48.8199 11.3194 0.6374 0.4808 5.6535 4.3273 8.6364 1.2595 3.3083 1.6904 0.5720 0.3775 0.5758 0.4943 Dependent variables Vulnerability Poverty vulnerability of smallholder households Independent variables Cooperative Whether or not to participate in a cooperative Control variables Age Gender Education Income Labor Poverty Age of the head of household (Years) Gender of head of household. Female = 0; male = 1 Years of education of the head of household Logarithm of net household income per capita Number of members in household aged 15–64 Whether the household is households registered as living under the poverty line. Yes = 1; No = 0 Migrant Whether there are family members working in the city. Yes = 1; No = 0 Assets Logarithm of total productive assets 11.9661 1.1318 S-capital Logarithm of total gift expenses 7.3053 2.0172 51.9343 40.824 4.1003 1.0108 0.3218 0.1509 0.1849 0.2514 0.3817 0.3499 0.1116 0.1724 Distance Economic SiChuang YuNan ChongQing GuiZhou Observation Frontiers in Sustainable Food Systems Distance between the settlement and the county (km) Settled village economic status assessment Whether the province is SiChuang. Yes = 1; No = 0 Whether the province is Yunnan. Yes = 1; No = 0 Whether the province is ChongQing. Yes = 1; No = 0 Whether the province is Guizhou. Yes = 1; No = 0 1,622 07 frontiersin.org Liu et al. 10.3389/fsufs.2023.1222455 TABLE 2 Baseline regression results of the impact of rural cooperatives on the poverty vulnerability of smallholder households. Variables (1) (2) Cooperatives (3) −0.0443 *** (0.0112) −0.0182 ** (0.0072) −0.0162** (0.0076) Age 0.0032*** (0.0004) 0.0036 *** (0.0002) 0. 0036*** (0.0002) Gender 0.0424*** (0.0103) 0.0282*** (0.0067) 0.0246*** (0.0060) −0.0135 *** (0. 0011) −0.0050 *** (0.0007) −0.0039*** (0.0006) Education −0.0897 *** (0. 0046) −0.0763*** (0.0041) Labor 0.0580*** (0.0018) 0.0595 *** (0.0164) Poverty 0.1332*** (0.0112) 0.0452*** (0.0163) S-capital −0.0039*** (0.0007) −0.0035 *** (0.006) Migrant 0.0198*** (0.0066) 0.0175*** (0.0059) Income Economic −0.0072 (0.0199) Distance −0.0294*** (0.0014) Province-FE Yes Yes Yes N 1,622 1,622 1,622 R2 0.1464 0.6433 0.7165 The standard error is shown in parentheses. ** and *** are statistically significant at 5%,1% levels, respectively. Province-FE represents provincial fixed effects. TABLE 3 Results of conditional independence hypothesis tests for explanatory variables before and after matching. Matching method Ps R2 LR chi2 MeanBias B-value Value of p 0.013 24.27 7.8 37.9 0.002 0.001 1.01 2.1 7.2 0.998 Radius matching 0.000 0.54 1.4 5.2 0.997 Kernel matching 0.000 0.51 1.4 5.1 0.999 Mahalas matching 0.001 1.30 1.5 8.1 0.996 0.002 3.81 1.6 7.6 0.874 Unmatched Nearest neighbor matching (k = 4) Partial linear regression matching household head. China has a 9-year compulsory education system. Based on China’s school system, this paper classifies the years of education for heads of households into two categories, namely lower education group (0–9 years), and higher education group (more than 9 years). As shown in Table 5, the effect of cooperatives on reducing poverty vulnerability is 2.05 times greater in the high quality group (−0.0387) than in the low quality group (−0.0188) for the education level of the household heads, which suggests that farmers with high quality human capital endowments are more likely to improve their poverty status after joining a cooperative than those with low quality. Hypothesis 3 was tested. The reduction effect of cooperatives on poverty vulnerability of non-poor households (0.0570) is 4.37 times greater than that of poor households (0.0130). This shows that non-poor households are more likely to benefit from co-operative seeds than poor households. Hypothesis 2 is verified. 4.3.3. Heterogeneity analysis based on the household incomes This paper classifies farm households into lower-income and higher-income groups based on their median per capita income levels, and removes the variable of net household income from the regression. As shown in Table 7, the reduction effect of cooperatives on poverty vulnerability of higher income households is greater than that of lower income households. Overall, the cooperatives had a dampening effect on poverty vulnerability for both the lower and higher income groups of farmers, but there were differences in the magnitude of the effect, with a greater reduction effect for the higher income group than for the lower income group (−0.0406 > −0.0184), and hypothesis 4 is tested. 4.3.2. Heterogeneity analysis based on the level of household poverty We divided the sample into two groups according to whether the households were registered as living under the poverty line or not. As shown in Table 6, cooperatives have a negative impact on the poverty vulnerability of farmers across different poverty attributes, but there are differences in the magnitude of the effect. Frontiers in Sustainable Food Systems 08 frontiersin.org Liu et al. 10.3389/fsufs.2023.1222455 TABLE 4 Propensity score matching estimates of the impact of cooperatives on the poverty vulnerability of smallholder farmers. Matching method ATT ATU ATE Nearest neighbor matching (k = 4) −0.0239** −0.0299*** −0.0265*** Radius matching −0.0235*** −0.0284*** −0.0272*** Kernel matching −0.0222*** −0.0377*** −0.0340*** Mahalas matching −0.0238** −0.0335*** −0.0288*** Partial linear regression matching −0.0324*** −0.0229*** −0.0276*** −0.0252 −0.0305 −0.0288 Average value The standard error is shown in parentheses. ** and *** are statistically significant at 5 and 1% levels, respectively. Significance tests for ATT, ATU, and ATE values were obtained using the bootstrap method of repeated sampling 500 times. TABLE 5 Results of heterogeneity analysis on the relationship between the cooperatives and educational level of household heads. Variables ATT lower education (0–9 years) higher education (more than 9 years) Nearest neighbor matching (k = 4) −0.0191* −0.0463*** Radius matching −0.0179* −0.0337** Kernel matching −0.0180* −0.0440*** Mahalas matching −0.0121 −0.0326*** −0.0271** −0.0369*** −0.0188 −0.0387 Partial linear regression matching Average value The standard error is shown in parentheses. *, ** and *** are statistically significant at 10, 5, and 1% levels, respectively. Significance tests for ATT, ATU, and ATE values were obtained using the bootstrap method of repeated sampling 500 times. 5. Discussion and conclusions have an advantage over scattered smallholder farmers in terms of large-scale operation of farmland, use of advanced technology, coping with market risks and access to policy subsidies, increasing the added value, profitability, labor productivity and employment rate of farmers engaged in agricultural production (Sun et al., 2009; Babiarz et al., 2010). Co-operatives not only help farmers reduce transaction costs in the procurement of agricultural materials and agricultural production services, and increase their bargaining power in the marketing of agricultural products; they also provide a variety of training and activities to help farmers improve their viable capacity to access information, express their needs and apply technology, which helps reduce the poverty vulnerability of smallholder households (Dao et al., 2023). In practice, some researchers have focused on the alienation of cooperatives caused by differences in groups of farmers (Dai et al., 2023; Zeren et al., 2023). Some co-operatives have evolved into ‘selfrun enterprises’ that do not contribute to the development of their members, nor do they contribute to the incomes of farming households as a whole (Wilmsen et al., 2022; Dong et al., 2023). The natural heterogeneity of smallholder farmers in terms of their initial resource endowments, such as production and management capacity, risk tolerance and household livelihood capital, may lead to “elite capture,” resulting in deviated resources for poverty alleviation and misplaced project implementation, creating new income inequalities (Gilcrease et al., 2022). Some scholars also argue that small and medium-sized members of cooperatives are prone to “free-riding” behavior, unwilling to pay for the cooperative’s public services and enjoy the benefits without contributing much, affecting the efficiency of the organization’s operations and distributional equity (Ito et al., 2012). 5.1. Discussions Our explorations of the heterogeneous characteristics of smallholder farmers leads to a topic worth exploring, namely whether cooperatives in reality can meet the development aspirations of a wide range of disadvantaged groups and whether they really have the desired organizational effectiveness in driving smallholder farmers. According to classical co-operative theory, alleviating the inherent tension between smallholders and the larger market is the purpose of forming a cooperative for ‘weak’ smallholders (Lennard-Jones and Devonshire, 1939; Bleaney, 1963; Elliott et al., 1971). However, an important prerequisite for the effective operation of cooperatives is a high degree of homogeneity in membership. In reality, farmers are highly heterogeneous, and this is difficult to eliminate in the short term (Cai, 2002). As a result, the organizational objectives of cooperatives deviate from the assumptions of classical cooperative theory, and the organizational performance is biased toward members with superior resource endowments (Cai, 2002; Wagstaff et al., 2009). Thus, the organizational objectives of cooperatives deviated from the assumptions of classical cooperative theory, and organizational performance was biased in favor of members with superior resource endowments, so that the development and profitability of small farmers was reduced. Cooperatives are an important vehicle for industrial poverty alleviation (Bernard and Spielman, 2009). The vast majority of studies have affirmed the positive role of cooperatives in reducing poverty and increasing income (Sun et al., 2009; Mojo et al., 2017). Cooperatives Frontiers in Sustainable Food Systems 09 frontiersin.org Liu et al. 10.3389/fsufs.2023.1222455 TABLE 6 Results of heterogeneity analysis on the relationship between cooperatives and the poverty levels of farmers. Variables ATT Under the poverty line Above the poverty line Nearest neighbor matching (k = 4) −0.0133** −0.0407* Radius matching −0.0128** −0.0548** Kernel matching −0.0068*** −0.0623*** Mahalas matching −0.0132*** −0.0655** Partial linear regression matching −0.0191** −0.0617** −0.0130 −0.0570 Average value The standard error is shown in parentheses. *, ** and *** are statistically significant at 10, 5 and 1% levels, respectively. Significance tests for ATT, ATU, and ATE values were obtained using the bootstrap method of repeated sampling 500 times. TABLE 7 Results of heterogeneity analysis on the relationship between cooperatives and the household incomes. Variables ATT Lower income families Higher income families Nearest neighbor matching (k = 4) −0.0173*** −0.0458 *** radius matching −0.0181** −0.0384** kernel matching −0.0168*** −0.0423*** Mahalas matching −0.0202** −0.0355** partial linear regression matching −0.0195** −0.0413** −0.0184 −0.0406 Average value The standard error is shown in parentheses. ** and *** are statistically significant at 5 and 1% levels, respectively. Significance tests for ATT, ATU, and ATE values were obtained using the bootstrap method of repeated sampling 500 times. Heterogeneous characteristics make a difference in both the motivation of farmers to join and their factor inputs, resulting in differences in the impact of cooperatives on farmers’ incomes (Gorczyca et al., 2022). It has been generally agreed that farmers with better resource endowments and more factor inputs are more likely to seek more control over their surplus. To accurately detect the impact of co-operatives on the poverty vulnerability of farm households, it is necessary to distinguish between the heterogeneity of farm households and focus on which groups co-operatives work more significantly for. The findings of this paper also confirm this phenomenon (Fernandes and Silva, 2022). Farmers whose heads have higher levels of education, non-poor families and higher household incomes have gained a more pronounced reduction in their poverty vulnerability after joining the cooperative. In research in less developed areas, poverty alleviation work generally suffers from strong policy input but weak endogenous motivation enhancement, etc. Most cooperatives only objectively absorb poor farmers into their societies as a matter of policy, but subjectively they do not pursue the effectiveness of bringing poverty, and are not willing to absorb poor farmers. In addition, the risk-averse nature of poor farmers with inherent lack of production endowments and social network resources often makes them reluctant to join the society, or even if they do, the shares they put in are low due to financial constraints, which inevitably leads to the problem that the cooperatives are “pro” the capable rural people and “anti” the Frontiers in Sustainable Food Systems disadvantaged (Bernard and Spielman, 2009; Chagwiza et al., 2016). The problem is that cooperatives are inevitably “pro” rural people and “pro” disadvantaged groups. However, it is also important to note that the value of cooperatives in benefiting the poor should not be dismissed because of their “affinity” to the rural and disadvantaged groups (Xu and Li, 2023). The empirical results also show that even if poor farmers find it difficult to participate in co-operatives because they are ‘excluded’ or less willing to join them, they can still benefit indirectly through the spillover effects of co-operatives. Additionally, Farmers in different income brackets and education levels have different levels of involvement in the operations and management of the cooperative, leading to differences in their poverty vulnerability reduction. Farmers with less physical capital and less learning capacity have less control and say in the day-to-day operations of the cooperative, and tend to be in a lower position in the cooperative than those with more material resources and higher levels of education (Bouichou et al., 2021). Accordingly, cooperatives are also less effective in reducing their poverty vulnerability than members with superior physical capital and high levels of education. Conversely, farmers with strong economic resource endowments usually have sufficient accumulation of their own resource factors and can make full use of and effectively spill over their economic resource endowments (Alam et al., 2021). As a result, the decision to join the society is more effective and more rewarding for these farmers. 10 frontiersin.org Liu et al. 10.3389/fsufs.2023.1222455 5.2. Conclusion the cooperatives themselves. The variables in this paper are selected from the perspective of farmers only, and are based on a single dimensional characteristic of farmers, without detailed descriptions and statistics of cooperatives. Second, the paper does not consider the willingness of cooperatives to take on board. Whether farmers can become members of cooperatives is not only based on whether they have a demand for membership, but also on whether cooperatives have the willingness to open up membership to the public, which is the result of a combination of demand and supply factors. However, on the supply side, the willingness of cooperatives to take up membership varies depending on the organizational model, governance and other characteristic factors of cooperatives in less developed western regions. Thirdly, social capital and the governance model of cooperatives play an important role in the poverty reduction effect of rural cooperatives. However, we did not conduct an in-depth analysis of these two areas due to the availability of data. Finally, the paper does not include the factors of policy intervention in cooperatives in its examination. If the policy is to support excellence and strength, cooperatives will choose to exclude the rural disadvantaged because they want to improve their competitiveness; in contrast, if the policy is to regulate the development of cooperatives and advocate their pro-poor attributes, then the relevant policy interventions will affect the exclusion decision of cooperatives as well as the demand of farmers to join the society. Policy interventions affect both the willingness of co-operatives to take in and the demand of farmers to join. The construction of an analytical framework that incorporates policy interventions, cooperative and farmer characteristics is an important direction for future research. Based on micro-survey data from smallholder farmers in eight counties in four provinces in the underdeveloped regions of western China, this paper analyses the impact of farmers’ membership in cooperatives on their poverty vulnerability and further explores the differences in the poverty reduction effects of cooperatives on groups with different poverty attributes, different human capital endowments (education level of the household head), and different income class heterogeneity. The main conclusions are as follows. (1) Cooperatives have a significant dampening effect on the poverty vulnerability of smallholder farmers. Cooperatives have a positive external impact in terms of helping farmers to overcome barriers to market access, accelerating the formation of human (social) capital and empowering management, which in turn has a combined effect on improving the ability of farmers to develop themselves, which has a combined effect on the improvement and enhancement of farmers’ capacity for autonomous development. (2) After overcoming the sample selection bias using the PSM model, the results show that participation in cooperatives still reduces the poverty vulnerability of smallholder farmers by an average of −0.0252, and the result remains robust to multiple tests of the methodology. (3) The impact of cooperatives on the poverty vulnerability of smallholder farmers is significantly heterogeneous across groups. Specifically, participation in cooperatives has a more pronounced effect on poverty reduction among non-poor, higher human capital endowment and higher income bracket households than among poor, lower human capital endowment and lower income bracket households. Data availability statement The validated conclusions outlined above can contribute and assist in emerging policy enlightenment. Firstly, enhancing the linkages between the interests of rural ‘elite’ figures and ‘weak’ small farmers. Policy makers should guide and encourage farmers to join or start cooperatives and support the development of cooperatives as an effective initiative to reduce poverty among smallholder farmers, but they should also see the limitations of the effectiveness of policy implementation. A top-down push for cooperative development and the pursuit of incremental growth in order to achieve an increase in the ability of farmers to reduce poverty will likely lead to a further widening of the gap in the ability of farmers to escape poverty in the future. The government should, on the basis of cultivating the stock of cooperatives, keenly identify the fit between “elite” figures and “weak” small farmers in terms of business areas and cooperative relationships, and strengthen the linkage between the interests of cooperatives and small farmers in order to minimize the negative effect of “elite capture.” Secondly, cooperatives are an effective way for smallholder farmers in less developed areas to escape poverty. Even if poor farmers find it difficult to participate in cooperatives because they are ‘excluded’ or have a low willingness to join, they can still benefit indirectly through the spillover effects of cooperatives. There are still many limitations in this paper, which can be seen in the following aspects: Firstly, we have focused more on the heterogeneity of farmers and neglected the heterogeneity of Frontiers in Sustainable Food Systems The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. Ethics statement Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the participants was not required to participate in this study in accordance with the national legislation and the institutional requirements. Author contributions MY: conceptualization, validation, data curation, and supervision. ZZ: methodology and resources. MY and JL: software, writing—original draft preparation, writing—review and editing, and visualization. JL, MY, and JL: formal analysis. JL: investigation. All authors have read and agreed to the published version of the manuscript. 11 frontiersin.org Liu et al. 10.3389/fsufs.2023.1222455 Funding Conflict of interest This research was financially supported by the National Social Science Fund of China (Grant/Award Number: 17XTQ011); the MOE Project of Key Research Institute of Humanities and Social Sciences in Universities of China (Grant/Award Number: 16JJD790063); the National Social Science Foundation Youth Project of China (Grant/ Award Number: 16JJD79006321CJY044). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Journal of Management Sciences Vol. 4(1): 27-53, 2017 DOI: 10.20547/jms.2014.1704102 GSP Plus Status and Income Distribution: A CGE Model for Pakistan Muhammad Shahzad Iqbal ∗ Sofia Anwar † Muhammad Aamir Khan Kakali Mukhopadhay ‡ § Abstract: The purpose of the study is to investigate the impact of the Generalised System of Preferences (GSP) plus on a household income of Pakistan. The European Union, the largest trading partner of Pakistan, granted the status of GSP plus to Pakistan in December 2013. The study has used MyGTAP, developed by Minor and Walmsley (2013) to calculate the impact at household level. This MyGTAP model uses the data of the latest available Social Accounting Matrix (SAM) to make changes in the standard GTAP by including multiple types of household and labor. The main findings of the simulations, run under MyGTAP model show a positive change in real GDP, merchandise imports and terms of trade while the first simulation shows a negative change in merchandise exports. Similarly, EBA status of Pakistan in the EU28 shows an increase in the household income with maximum gain by the household of rural Sindh with no agricultural land and a positive change in real wages of most of the factors. However, the large and medium agricultural household types show a negative change in household income in case of first simulation. Comparatively low improvement over urban and non-farm household of rural areas. Keywords: Economic growth, trade, GSP Plus, European Union, CGE model, real GDP, terms of trade, real investment, household income. Introduction For many years EU is the largest importer of Pakistani products. Total exports from Pakistan to the EU during the year 2014 were US$ 8.13 billion which accounted 29 percent of the total exports. It was 21.5 percent in 2012 and 24 percent in 2011. Although the EU is considered to be the dominant importer in Pakistan, but the country always showed a sluggish export growth, especially in terms of commodity diversification. On the other hand, penetration into the EU market remained overdue comparing to other competitors. Pakistan is a member of the preferential trading system of the EU, ever since its evolution. The examination evident that external trade relations of the EU with the developing countries has been conducted with a number of different channels, principally with the African, Caribbean and Pacific (ACP) states through the Lome Convention, Mediter∗ PhD Scholar, Department of Economics, GC University Faisalabad. E-mail:shahzad.iqbaal@gmail.com † Chairman, Department of Economics, GC University Faisalabad. ‡ Senior Researcher, Department of Natural Resource Sciences, McGill University. § Assistant Professor, COMSATS Institute of Information Technology Islamabad. 27 Journal of Management Sciences ranean countries through the Global Mediterranean Policy (GMP) and with the rest of Latin America and Asian developing countries including Pakistan through the GSP scheme. This is evident from the EU’s complex network of discriminatory tariff through generalized and country-specific or region-specific trade preferences. While, trade relations with most industrialized countries have been based on most-favoured-nation treatment (Rashid, 2006). The common commercial policy uses a spread of instruments to regulate trade among the EU and its trade partners. It covers not only tariff, but other trade instruments as well. A complicated system of trade advantages, differentiated according to specific groups of countries, has drawn up to a hierarchy of trade preferences called as ‘pyramid’ of trade preferences. The examination of EU’s trade regime practice worked out over the years points out that it uses fairly complicated procedures and a very elaborate panoply of instruments. Although the system has some of the economic effects hoped for and has been established for political reasons; it seems advisable to simplify considerably in order to expand its benefits largely according to ‘trade not aid’ principle (Persson & Wilhelmsson, 2016). On December 2013, European Union granted GSP plus status and since January 2014, Pakistan has been enjoying this status. It is expected from the very beginning that the exports from Pakistan are expected to increase in the EU market under GSP plus status. This status will substantially increase Pakistan’s exports to the EU-28 especially in textile, wearing apparel and leather sectors. Everything But Arms (EBA) is a status that allows almost everything to be exported to the EU by the beneficiary country. It is considered as a most generous scheme for developing countries like Pakistan, but Pakistan has not been granted this status yet. It is expected that the EBA status may bring more benefits for Pakistan than any other status. The European Union is the largest export destination for Pakistan. Recently, the EU granted GSP plus status to Pakistan. Our main research problem is to investigate the likely impact of GSP Plus on Pakistan’s exports to the European Union and hence its resulting positive effects on economic development. The impact can be underestimated or overestimated if one tries to capture the impact of the FTA like GSP plus by applying partial equilibrium analysis. As all the sectors of the economy are interlinked, so any shock in one sector leads to changes in other sectors. So, the main objective of the study is to find out the impact of GSP plus status of Pakistan in the EU - on Economic growth of the country. To capture the impact of these measures, Computable General Equilibrium models are an ideal tool. The main objective of the study is to calculate the potential impact of GSP plus on the household income and real wages by applying MyGTAP. The study also aimed to calculate the potential economic gains from GSP plus and EBA in terms of GDP and merchandise exports. The study wants to investigate the possible outcomes from a series of different policy experiments and suggest some policy recommendations. 28 Journal of Management Sciences Is GSP Plus Different from Normal GSP? The basic and foremost objective of the preferential system known as the Generalized System of Preferences (GSP) is to help the economies to reduce the poverty, promote good governance and sustainable economic growth. These preferences enable the economies to increase their role in the international trade and especially the exports to the EU that ultimately help them to reduce the poverty and maintain a sustainable development. The GSP of EU covers the following regimes (European, 2013). • The standard/normal GSP that covers more than 6300 tariff lines, transports preferences in 90 developing countries that have been reduced from 177 in 2013. • GSP Plus that brings special arrangements to promote good governance and sustained development in addition to offering duty free access to more goods from the vulnerable economies including Pakistan. The list includes 25 countries, adding 9 more to the previous 16. The beneficiary economies have to implement and ratify certain international conventions. • The most attractive arrangements for the 50 Least Developed Economies (LDCs) called Everything But Arms (EBA), provides duty free and quota free access to nearly all commodities. In addition to the preferential agreements, EU has established trade relations on the basis of Most Favored Nation (MFN) treatment, allowing all industrialized countries outside the EU to trade with. Identification of ‘high potential’ products of Pakistan in EU after the GSP plus status is first step that should be followed by the identification of potential competitors with same status or even better. The EU has a range of agreements with different countries, including GSP, GSP plus, Every Thing, but Arms (EBA), Overseas Countries and Territories (OCT), Economic Partnerships Agreement (EPA) and some more. While considering the competition among developing economies, EBA is considered to be more attractive than the GSP plus status (Carbone & Orbie, 2016). Although, achieving the status of zero tariff on export of all products is a huge opportunity, but the exports from Pakistan may not observe abrupt jump. It is because Pakistan will continue to face a tough competition from countries enjoying the same or better treatment in the EU market. The countries with GSP plus status will face an annual capping mechanism while others with EBA status like Bangladesh will enjoy the tariff free access throughout the year. In addition to such status in the EU market, the commodity price, production capabilities and demand for the products will also play key role in such a competitive environment. Table 1 below summarizes the position of Pakistan and its competitors with similar status in the EU market. Literature Review Many authors have observed the literature on CGE models applied to less developed and developing countries. 29 Journal of Management Sciences Naqvi (1998) developed a CGE model and used the SAM 1983-84 aiming to analyse the economy wide impact of energy policy. The results found by the simulation show that a change in energy tax has varied influences on different commodities, i.e. If distortions are removed from taxes on petroleum products, it fulfills the objective of social equity while Table 1 Comparison of Imports by the EU (28) with GSP Plus Beneficiaries (US $ Million) S.No 1 2 3 4 5 6 7 8 9 10 Source: Exporters Peru Pakistan Paraguay Costa Rica Ecuador Georgia Armenia Bolivia Mongolia Cabo Verde International 2008 2009 2010 5757.8 4606.1 6964.3 6068.4 5295.4 5918.3 732.3 509.9 1313.1 5446.4 4516.8 5310.1 3569.8 3078.3 3047.4 908 604.6 886.9 478.1 241.4 323.2 474.4 420.6 569.5 95.4 75.7 135.9 40.4 39.1 50.6 Trade Centre (ICT) 2011 9044.2 7485.8 1657.4 6077.3 3712.9 923.8 443.1 592.2 114.9 63.9 2012 8245.5 6093.2 1225.1 6790.6 3601 762.9 330.8 603.7 93.3 68.5 2013 7319.7 6868.2 1574.6 6394.6 3897.1 916.3 329.4 761.4 105.6 64.4 2014 6829.2 7220.7 1527.6 6119.1 4003.4 907.5 335.9 879.9 115.8 78.5 removal of distortion in taxes on electricity has no impact on the consumption of rural household while it showed a negative impact on urban household. On the other hand, applying a tax on natural gas brought a negative impact on the real consumption of the household. It was further discovered that removing distortions not only increase the real GDP, but also bring a positive change in the trade balance. The model was simply a static model aiming comparative analysis and had nothing to do with the forecasting. Ghosh (2004) developed a CGE model for Pakistan by using GTAP 4 to investigate the impact of roads and transport infrastructure on the economy. The study proposed a multiregional CGE model in order to check the impact of a new road network between Karachi and Peshawar. The simulation results discovered that new road will bring positive change in the industrial sector of Punjab and NWFP (North West Frontier Province, Now KPK (Khyber Pakhtunkhwa) provinces of Pakistan. The road network will increase the real income of households that ultimately will increase the utility level in both provinces. The study further concluded that this network will bring positive change of 16% to the GDP of the economy. Butt (2006) utilized the CGE model for Pakistan to calculate the impact of tariff cuts on the regional disparities, output, employment and exports by keeping in view different regions of the country. The study developed a PAKREG database by utilizing the I-O table of 1990-91 developed by the Federal Bureau of Statistics (FBS 2001). The study in this way helped the GTAP to recognize Pakistan as a separate country. The results of the study revealed a positive impact of trade liberalization on all regions of Pakistan in terms of improvement in output, exports and employment. The results further discovered a positive relationship between trade liberalization and regional disparities during the military regimes and opposite in the case of democratic governments. The cross border tariff cuts seemed to help the increment in real GDP slightly in the short run but significant in the long run. Ahmed and O’Donoghue (2008) described the welfare effect of the external balances on Pakistan economy. The study used a CGE model to capture economy wide impact of policy 30 Journal of Management Sciences simulation. Social accounting was used as a database and GAMS software was used to run the model. The study encompassed12 agriculture sector 16 industrial sector and 6 services sector. Households have been distributed in rural and urban. The rural households have been further distributed into 17 categories. The experiment was performed through trade liberalization simulations. The simulations were concerned with a 50 percent increase in foreign savings, a 10 percent increase in overall import prices and 10 percent increase in the import prices of petroleum etc. The results of the study suggested that the external oil prices possessed the high potential to affect Pakistan socio economic conditions. Increase in foreign saving decrease poverty in the country. The analysis suggested that poverty is increasing with the increase in import prices. Shaikh and Rahpoto (2009) has studied the SAFTA implication on Pakistan economy using the GTAP model. This study used 10 regions and 10 commodities. The experiments were based on the unilateral trade liberalization (uniform tariff rate 15 percent), regional trade liberalization, and unilateral trade liberalization (15 percent) for the rest of the world. The study used GTAP model to investigate the benefits and costs of granting MFN (Most Favored Nation) status to India and SAFTA. The results highlighted the potential industries which need to be expanded or contracted. Pakistan gained highest welfare in case of SAFTA with 15percent uniform external tariff. There is high demand in the international trade for Pakistani dates, leather and garments etc. The study identified a variety of industries in which a high potential existed. The SAFTA role is important by giving opportunity for member countries to achieve economies of scale, diversify their exports net and improve competitiveness. The study further explored that if SAFTA is fully integrated and Pakistan gets a tariff cut of 15%, it would bring highest welfare gain for the people. Hussain (2010) applied CGE model using SAM 2002 to investigate the fiscal strictness and the trade liberalization impact on household welfare and inequality. The study explored that there are two principal effects of export taxes and tariffs. Firstly, they reduce the trade volumes on both the import and export sides. Secondly, they impose economic costs by inducing resource mis-allocation. Therefore, if trade related taxes are eliminated, an economy can avoid production and consumption distortions. It is an established fact that free trade leads to enhanced efficiency The case of efficiency for free trade is the converse process to the tariff’s cost benefit analysis. The study further discovered that for a small country like Pakistan, imposing a tariff does not allow it to influence world prices. However, prices for domestic consumers and domestic producers do arise as a result. Consequently, imports and consumption are reduced and the production of import substitute increased. Dorosh and Rashid (2013) utilized CGE models to measure the latest terms of trade for the agriculture sector, comparing it to the industrial sector in Pakistan during the years of 2000-2010 and to study the impact of agriculture income tax on Pakistan economy by using a social accounting matrix (SAM 2002). The result of the experiment showed a 5% and 10% increase in the government revenue through the imposition of agriculture tax. The study further elaborated that manufacturing and imports flourished while construction and exports faced decline. The labor demand in the non - agriculture sector raised, whereas the demand for labor in agriculture sector reduced due to increase in the agriculture income tax. 31 Journal of Management Sciences Siddig, Aguiar, Grethe, Minor, and Walmsley (2014) studied the Nigerian socioeconomic policy impacts of reducing fuel subsidies while employing countrywide framework using the newly developed MyGTAP model. This model incorporated country-specificinformation covering 12 groups of households to examine domestic policies. The study encompassed 13 regions and 21 commodities. My GTAP incorporated government budget in two folds; income and expenditure. It is considered that lowering the fuel prices accompanied by a low level of living cost can be the outcome of subsidies incorporated in the imported goods of the petroleum sector. The study supported a reduction in fuel subsidy policy because it has non-decreasing effects on the household income-especially the poor one-hence leading to alleviate poverty in Nigeria. Kuiper and Shutes (2014) studied the impact of food and nutrition security on multiple households in Ghana by employing MyGTAP database of Minor and Walmsley (2013). The study involved a multiple household to study the effects of food policy on the most vulnerable sector of the society, helping the government to design intervention in order to provide relief to the poor segment of the society. The study embedded 19 commodities and 9 households. In the study, the following three approaches were used to incorporate multiple household data in GTAP data base. Firstly, user weights has been assigned to household and incorporated in GTAP. Secondly, the study included household data through national SAM. Thirdly, they directly place household survey in GTAP analysis. The result suggested that the removal of export subsidy is useful to the poor people of Ghana. Khan, Saboor, Mohsin, et al. (2015) developed a CGE model to investigate the impact of agriculture trade liberalization (the elimination of import tariff and the removal of export subsidies) on income inequality of Pakistan. The study adopted the newly developed MyGTAP model developed by Minor and Walmsley (2013). The model used a two kind of data base i.e. GTAP and SAM (2007-08). This study deeply analyzed the impact of agriculture, trade liberalization on multiple households. The study encompassed 18 households, 12 regions and 37 sectors. The results of agricultural trade liberalization suggested that income inequality in Pakistan is increased by 0.49% from the baseline. Medium and large household types are aided, and there is a nominal increase in the real wages of medium and large agricultural labors. The labor intensive crops are replaced by capital intensive and cheap imported products that ultimately helped to enhance the income inequality in Pakistan. Rationale for a New CGE Study on Pakistan The majority of the CGE studies conducted in Pakistan includes the following issues, changes in tax, food policies, government expenditures and investments, policies related energy crisis, performance of public sector organizations, trade liberalization, Dutch disease effect, unequal distribution of wealth, and poverty conditions in Pakistan. All of the previous studies conducted in Pakistan using CGE have some common characteristics that include some behavior related phenomenon like producer behavior, consumer behavior and international trade are some common features used by many researchers. Secondly, most of these researchers used Neo classical comparative static CGE model in their studies. These are single country models except (Butt, 2006) who developed PAKREG a 32 Journal of Management Sciences regional CGE model in the study to discuss the tariff cuts, exports and regional disparities, which aimed to help Global Trade Analysis Project (GTAP) for the trade issues relating to Pakistan. Methodology MyGTAP Model The CGE model in its global version is supported by the Global Trade Analysis Project (GTAP) model as it provides the modeling framework as well as the database to the CGE model. That is; the main source of data for the global CGE model is the GTAP database. The model of GTAP is the most commonly used and known software for the multi country trade analysis. It is a multi-region, multi-country and multi- sector CGE model which assumes perfectly competitive markets and return to scale (Burfisher, 2011). GTAP 09 with reference years 2004, 2007 and 2011 to 140 regions, 57 sectors and 244 countries has been used to link the Pakistan economy with the rest of the world in general and the European Union (EU28) in particular. The study has linked the latest available comprehensive Social Accounting Matrix (SAM) 2007-08 developed by International Food Policy Research Institute (IFPRI) to a latest extension of the standard GTAP to make it MyGTAP. The MyGTAP developed by Minor and Walmsley (2013) is briefly explained here. MyGTAP is developed on the basis of the standard GTAP model which is commonly used for trade analysis focusing the tariffs (Hertel & Hertel, 1997). A regional household is represented in the standard GTAP which combines the income coming from factors of production (land, labor and capital) and taxes and then distributes it among the private household, savings and government expenditures on the basis of their shares. The standard GTAP provides the details of the structure of the expenditures in the economy. It combines the income from all sources and then determine the expenditures, but it does not require the information that which agent receives the income, taxes or transfers. The study under consideration not only requires the income to be split into private household and government, but also split the household into further categories (poor and non-poor household). The newly developed MyGTAP explains that government collects revenue from taxes and foreign aid to spend on government expenditures, funding foreign governments, providing subsidies and transferring to households. Budget deficit or surplus is decided on the basis of difference between government spending and income. Income sources for private households on the other hand, are a function of returns to factor endowments (land, labor and capital), net rent on foreign capital and foreign remittances, transfer payments made by the government and other households. The household net income is either spent or save. The impact of gain in trade i.e GSP plus status of Pakistan in the EU28 will be noted upon the change in income of different households, but not on the basis of change in consumption of the household. 33 Figure 1 Flows of Income and Expenditures in MyGTAP Model Journal of Management Sciences 34 Journal of Management Sciences Relationships in MyGTAP Model The study has already explained the source of data base in MyGTAP. As discussed earlier, the standard GTAP is modified to make it MyGTAP where the single regional household is replaced with the multiple private households along with a separate government sector for one region (Walmsley, Minor, et al., 2013). Just like in case of standard GTAP, the private households receive income from factors, but in MyGTAP, it also incorporates foreign remittances and capital, which is further used for consumption and saving purpose. The new model assumes that the government sector gains income from taxes and foreign aid in. Government income is consumed on the transfers to the private household and foreign aid out. Moreover, government entertains the receiving of foreign aid instead of the direct acceptance by the private households (Minor & Walmsley, 2013). MyGTAP Model Closure Model closures are the starting point of this model that assumes perfect competition in all sectors of the economy. Capital and labor as factors of production are considered to be fully mobile among different sectors of the economy and land along with natural resources is immobile. The economy of Pakistan faces the problem of high unemployment rate, so we assumed that unskilled labor (LASKU) are unemployed. Similarly, it is assumed that the factors prices influence the foreign income flows in the respective country. Trade balance is endogenous and expected rate of return determines the investment as in case of standard GTAP model and total domestic savings by the government budget deficit and sum of the private household savings. Any country that gets GSP Plus status in the EU face the annual growth capping mechanism for products with higher growth rates. The study incorporated the capping mechanism with quota restrictions. Data Base and Analysis Social Accounting Matrix (SAM) for MyGTAP Pakistan Institute of Development Economics (PIDE) developed the first Social Accounting Matrix (SAM) for Pakistan in 1985 with base year 1979. Federal bureau of Statistics (FBS) under Project Improvement of National Accounting System (INAS) with collaboration with the Netherland government developed the second SAM that was limited to only single household. Siddiqui and Iqbal (1999) constructed a new SAM for Pakistan with base year 1989-90 and aggregated the industrial classification of Input-Output (IO) table into SAM with five production accounts. Dorosh, Niazi, and Nazli (2004) built a broad SAM with 34 production accounts and 19 household groups with base year 2001-02. These household groups were disaggregated across provincial basis, hence most suited for policy analysis that target particular households. Khan et al. (2015) produced a financial SAM for the year 1999-2000. They disaggregated the workings of the loanable funds market into disaggregated payments related to physical and financial flows among institutions. 35 Journal of Management Sciences The latest available SAM that depends heavily on concomitant National Accounts and house-hold data was developed by Debowicz, Dorosh, Haider, and Robinson (2007) with base year 2007-08, under the Pakistan Strategy Support Program (PSSP) funded by USAID aiming to support the Government of Pakistan with evidence-based policy reform for pro-poor economic growth and enhanced food security. To implement a CGE model with an income distribution component, a consistent data base is required. MyGTAP in the study pursues the SAM (2007-08) desegregation of activities, commodities, factors and institutions. The model follows the framework developed by Lofgren et al. (2001). This model is a standard static model rather than dynamic CGE model. Therefore the second period effects of changes in investment expenditures are not taken into account. Moreover, the model neither specific about the time horizon of the adjustment nor how the adjustment is sequenced. Otherwise stated, the model cannot resolve whether adjustment from the base to a new equilibrium takes place over any particular length of time, or whether a large part of the adjustment occurs in a particular year. SAM provides actual values for the coefficients in these equations through the calibration process. The model will be solved primarily for equilibrium to make sure that the base year dataset is reproduced. Afterward, it would be possible to shock the model with a change in the value of one of the exogenous variables. The model will be resolved for equilibrium and the changes in the values of the endogenous variables. Moreover these values will be compared to those of the base-year equilibrium to establish the impact of the exogenous shock. To simplify the things, the study aggregated the regions and sectors. The details are presented in appendix. Simulations The study used MyGTAP in order to calculate the impact of two simulations on the real wage rate and household primarily. The base year used in MyGTAP is 2007, as the latest available SAM for Pakistan is of year 2007-08. In addition to calculating the impact of simulations on household income and real wage rate, the study have also discussed some other areas of the economy, taking into account the overall and regional characteristics of the production sectors and factors allocation. Although the evidence is available that trade liberalization is good for the growth, but how it impact on household income and real wage rate is less clear. Pakistan is a developing country and is more concerned about the effects of EU’s GSP plus and EBA status on household income in Pakistan. There are many studies that identified the positive and negative impact of trade liberalization (Hertel & Hertel, 1997; Winters, McCulloch, & McKay, 2004; Vanzetti, Huong, et al., 2006). Calculating the impact of GSP plus and EBA status on poor household and different types of labor is important to formulate the policy and planning for sustainable growth. The study have only used the two following simulations for this purpose. Simulation I: EU-28 GSP Plus status with quota restriction: What would happen if quota restriction is applied on Pakistan to incorporate the capping mechanism of the EU-28? 36 Journal of Management Sciences Simulation II: Potential EU28-EBA with Competitors: What would happen if Pakistan gets the Everything But Arms (EBA) status in the EU-28 with no Capping mechanism/Quota restriction, with main competitor Bangladesh that already enjoying the EBA status? The results of these simulations are presented below. Results of the Simulations GDP and Production The results of the both simulations are presented in Table 2 which show positive change in real GDP of Pakistan. In case of first simulation, the GDP of Pakistan increases by US$ 21.594 million which is 0.015 percent positive change from the base line value. The results of second simulation also very encouraging with positive change of US$ 884.047 million in GDP. Table 2 GDP Quantity Index EU, Constant 2007 Prices (Percent and Millions US$) Simulations GSP Plus status with Quota Restrictions EBA Status Base Value (Millions US$) Post Shock Effects Change in GDP Percentage Change 143169.594 143169.594 143191.188 144053.641 21.594 884.047 0.015 0.617 Similarly, Table 3 presents the changes in real output of different sectors of Pakistan after both simulations. The results of both simulations reveal mixed effects on the real output of commodities. The results of the first simulation show that there are 13 sectors out of 38 where output level is increased with maximum increase in services sector (US$ 0.383million). The other major winning sectors are construction US$ 0.356 million, sugar US$ 0.199 million, vegetable, fruit and nuts US$ 0.196 million, livestock and meat products US$ 0.125 million. While there is decrease in real output in rest of the sectors. The prominent sectors with decrease in output include machinery and equipment US$ -2.117 million, metals and products US$ -1.875 million, leather products US$ -1.777 million and oil seeds with US$ -1.722 million. The results of the second simulation show increase in real output in 25 sectors of Pakistan economy. The results reveal that paddy rice show maximum gain with US$ 2.080 million followed by construction sector (US$ 1.14 million), services sector (US$ 1.26 million), mineral products nec (US$ 0.96 million) and processed food (US$ 0.93 million). While there is deterioration in the 11 sectors. The prominent sectors with decrease in output include leather products with US$ -1.99 million, plant based fibers with US$ -1.60 million and textiles with US$ -1.99 million. The results of second simulation are more encouraging than Simulation 1 which means that if Pakistan is allowed to export in EU28 without any restriction, the real output level will increase in most of the sectors of the economy. 37 Journal of Management Sciences Exports and Imports The duty free and quota free entry of Pakistan into the EU28 is expected to bring positive effects on the exports of Pakistan. Similarly, the flow of imports will also increase due to increased demand of foreign inputs and resultant higher prices of many goods. Figure 2 explains the results of the simulations. The results of both simulations show an increase in imports and reduction in exports resulting disturbance in the trade balance. The exports of Pakistan to EU28 reduced by -1.79 percent in case of first simulation while in case of second simulation, the reduction is -1.282 percent. This reduction is export is due to Table 3 GDP Quantity Index EU, Constant 2007 Prices (Percent and Millions US$) Commodity Base Value (Millions US$) pdr wht gro vf osd Sugar pfb ctl Animalprod frs fsh minerals oil Meatfood vol mil sgr bt tex wap lea Wood pc crp nmm Metals Autoparts ele ome omf utilities cns trd Transport cmn All services 1489.76 2616.37 181.39 7201.54 365.22 5895.7 2953.45 4271.85 20862.25 291.25 879.47 630.01 1468 5029.13 3506.79 4373.73 5333.85 3618.37 23984.3 4404.37 1203 2418.66 8120.48 4289.23 5498.41 2107.05 2733.24 2672.3 420.68 1111.31 22303.91 16914.6 21665.48 18768.37 3009.44 55321.8 GSP Plus with EU Capping (Quota) Changes in Change in Value Percent -0.205 -0.01 -0.102 0 -0.02 -0.01 0.196 0 -1.722 -0.47 0.087 0 -0.796 -0.03 0.016 0 0.125 0 0.027 0.01 0.076 0.01 -0.396 -0.06 -0.318 -0.02 -0.357 -0.01 -0.528 -0.02 0.073 0 0.199 0 0.117 0 -1.208 -0.01 -1.296 -0.03 -1.777 -0.15 -0.628 -0.03 -0.24 0 -1.07 -0.02 0.119 0 -1.875 -0.09 -0.569 -0.02 -0.795 -0.03 -2.117 -0.5 -1.151 -0.1 -0.216 0 0.356 0 -0.133 0 -0.036 0 0.058 0 0.383 0 Potential EBA Status Changes in Change in Value Percent 2.08 0.14 -0.12 0 0.46 0.26 0.17 0 -0.76 -0.21 -0.11 0 -1.6 -0.05 0.23 0.01 0.25 0 -0.07 -0.02 0.27 0.03 0.2 0.03 -0.23 -0.02 0.93 0.02 -0.21 -0.01 0.62 0.01 0.65 0.01 0.67 0.02 -1.11 0 -0.91 -0.02 -1.99 -0.17 0.28 0.01 0.53 0.01 0.22 0.01 0.96 0.02 0.19 0.01 0.55 0.02 0.58 0.02 -0.94 -0.22 0.28 0.03 0.66 0 1.14 0.01 0.39 0 0.64 0 0.76 0.03 1.26 0 production capacity of Pakistan in 2007 which was adversely affected by load shedding. The adverse effects of energy crises increased the production cost in Pakistan resulting into decline in exports. Due to this reason, the results of the simulations produced negative impacts. The results of both simulations show a positive increase in the imports of Pakistan. The increase is 0.558 percent in case of first simulation, while in case of second simulation, the increase is 1.153 percent. This increase in imports is also a result of increased cost of production in Pakistan. Similarly, in order to increase the production, Pakistan would also 38 Journal of Management Sciences require more inputs to import. Figure 2 shows that the trade balance is highly deteriorated in case of first simulation while in case of second simulation, increase in imports is slightly less than the decrease in exports. This is because the quota restriction will restrict exports to EU28, leaving more products available for the domestic consumer to consume. Hence imports increased at lower rate. While in case of EBA status, there is no restriction to export resulting into reduced availability of domestic products. The reduction in availability and increased demand of imported inputs, increase the overall imports of the country. Figure 2 Merchandise Exports and Imports of Pakistan (Percent) Impact on Pakistan’s Terms of Trade Terms of trade is define as the ratio of prices that a country receives and pays in exchange of its exports and imports. It is considered important to understand the impact of change in price on the welfare of public generally. Current study investigated the impact of two different simulations on the change in price of imports and exports. Pakistan has already achieved the status of GSP plus in EU28, the restriction free exports from Pakistan in case of GSP plus and EBA, may increase the export price of Pakistani products. Similarly, applying quota restriction may increase the price level at lower rate. Figure 2 explains the effects of different simulations performed on the Pakistan’s termsof-trade. The results of both simulations are positive but very different. In case of first simulation the export prices that Pakistan receives from EU28 are 0.019 percent higher than the import prices that Pakistan pays to the EU28. Highest gain is seen in the results of simulation 2, assuming that if Pakistan gets the status of EBA in EU28 just like Bangladesh. Due to this status, the exports from Pakistan may increase rapidly resulting an increase in export prices. Hence, the results of this simulation show that Pakistan is receiving 1.834 percent higher export price than it is paying for its imports from EU28. 39 Journal of Management Sciences Changes in Household Income The study has discussed the impact of two simulations by using MyGTAP focusing on the issues of trade, GDP, output and prices. A unique feature of the MyGTAP model used in this study is the capability to disaggregate the regional household into both private and government entities (Minor & Walmsley, 2013). The study disaggregated the regional household of standard GTAP model into 18 types to conduct a detailed analysis. The simulations used in the study will calculate the effects on household income distribution and expenditures. The data and weights required were obtained from the latest comprehensive Pakistani Social Accounting Matrix (SAM) 2007-08. Figure 3 Changes in Term of Trade (TOT) of Pakistan, Constant 2007 Prices, (Percent) Changes in Household Income The study has discussed the impact of two simulations by using MyGTAP focusing on the issues of trade, GDP, output and prices. A unique feature of the MyGTAP model used in this study is the capability to disaggregate the regional household into both private and government entities (Minor & Walmsley, 2013). The study disaggregated the regional household of standard GTAP model into 18 types to conduct a detailed analysis. The simulations used in the study will calculate the effects on household income distribution and expenditures. The data and weights required were obtained from the latest comprehensive Pakistani Social Accounting Matrix (SAM) 2007-08. While conducting the welfare analysis, the studies that employ CGE models, normally show all household are equally affected due to any change in the trade policy. In case of MyGTAP, the households are distributed into categories aiming to calculate the impact on the marginalized population before designing a trade policy. Any change in wage rate 40 Journal of Management Sciences is considered a change in the household income. The household income consist of income coming from different factors, so any change in income of factors means a change in income of household. The results of both simulations are summarized in Figure 4 which show a positive change in overall household income. There is a change of 0.74 percent in case of first simulation but in case of second the change is 2.17 percent which means that if Pakistan is allowed to export duty free and quota free into the EU28, the household income in Pakistan will rise. Figure 4 Changes in Households Income in Pakistan, Constant 2007 Prices (Percent) The results of both simulations are summarized in Figure 4 which show a positive change in overall household income. There is a change of 0.74 percent in case of first simulation but in case of second the change is 2.17 percent which means that if Pakistan is allowed to export duty free and quota free into the EU28, the household income in Pakistan will rise. The results below show change in all 18 categories of households. The regional household is divided into three categories, household in Punjab, household in Sindh and household in rest of the Pakistan. The results further reveals that that every household is not equally affected. There are some household better off and vice versa. Household Income of Large and Medium Farm Table 4 represents the results of both simulations in order to check their impact on the income of large and medium household of Pakistan. The results reveal that in case of first simulation, the household other than Sindh and Punjab have positive change of 0.029 percent in its income while the income of household in Sindh is reduced by -0.478 percent and in Punjab by -0.239 percent. On the other hand, the income of all household increases with maximum 2.347 percent increase in household of rest of the Pakistan (excluding Sindh and Punjab provinces). 41 Journal of Management Sciences Table 4 Changes in Household Income of Large and Medium Farm, Constant 2007 Prices (Percent) Household Types Large and medium farm Sindh Large and medium farm Punjab Large and medium farm other HH Code Population (millions) Income shares (percent) GSP Plus with Quota Potential EBA Status H-MF1 H-MF2 H-MF3 0.8 2.4 0.6 1.5 6.1 0.8 -0.478 -0.239 0.029 1.831 1.613 2.347 Income of Small Farm Household The results of the both simulations are presented in Table 5 which show the impact on the small household living in Pakistan. The results reveal that small farmer living anywhere in Pakistan is befitted in both cases. The results further reveal that the farmer living in parts other than Sindh and Punjab is benefitting maximum in case of both simulations while the small farm household in Sindh is getting minimum benefits in both cases. The maximum benefit that small farm household of other Pakistan is getting is 2.253 percent which is in case of Simulation 2 (if Pakistan gets the EBA status in EU28) and small farm household of Sindh is getting minimum benefit of 0.331 percent in case of first simulation (applying quota restrictions to justify the capping mechanism of EU28 after GSP plus status of Pakistan). Table 5 Changes in Household Income of Small Farmers, Constant 2007 Prices (Percent) Household Types HH Code Population (millions) Income shares (percent) GSP Plus with Quota Potential EBA Status H-SF1 H-SF2 H-SF3 3.1 16 5.6 1.8 11.5 3.3 0.331 0.487 0.885 2.146 2.179 2.253 Small farm Sindh Small farm Punjab Small farm other Pakistan Income of Landless Farmer Household In this section, we will discuss the rural household that is farmer but do not own a piece of agriculture land in any area of Pakistan. The results of both simulations are presented in Table 6 that show a positive change in income of all rural households that are landless but are farmers. In case of first simulation landless farmer of Sindh is gaining minimum (0.405 percent) while the landless farmer of rest of the Pakistan is gaining maximum (0.954 percent). The results of second simulation show that landless farmer of Punjab is gaining maximum (2.452 percent) while the landless farmer of Sindh is gaining minimum (2.08 percent). Table 6 Changes in Household Income of Landless Farmers, Constant 2007 Prices (Percent) Household Types Landless farmers Sindh Landless farmers Punjab Landless farmers other Pakistan HH Code Population (millions) Income shares (percent) GSP Plus with Quota Potential EBA Status H-OF1 H-OF2 H-OF3 2.5 3.6 1.7 1.4 1.8 0.7 0.405 0.68 0.954 2.08 2.452 2.323 42 Journal of Management Sciences Income of Landless Labor The household living in rural areas of Pakistan working in agriculture farms as a laborer and having no land is included in landless labor. The results showing the impact of both simulations on the household income of landless agriculture labor of Pakistan are presented in Table 7. The results show maximum gain for the landless labor of whole Pakistan in both simulations as compared to any other household. In case of first simulation maximum gain is shown in for the landless agriculture labor of Sindh (1.563 percent) while the landless agriculture labor of Punjab is getting minimum gain (1.432 percent). In case of second simulation, the results are quite similar with maximum gain again in case of landless agriculture labor of Sindh (4.136 percent) while the landless agriculture labor of Punjab is getting minimum gain (3.512 percent). Table 7 Changes in Household Income of Rural Agricultural Labor, Constant 2007 Prices (Percent) Household Types HH Code Population (millions) Income shares (percent) GSP Plus with Quota Potential EBA Status Landless agri. Lab Sindh Landless agri. Lab Punjab Landless agri. Lab other Pakistan H-AGW1 H-AGW2 H-AGW3 3 3.3 0.4 1.5 1.4 0.2 1.563 1.432 1.498 4.136 3.512 3.944 Income of Rural Non-farm Household In rural areas of Pakistan, there are households that have no direct connection with agriculture farming. The results shown in Table 8 reveal the impact of both simulations on the income of non-farm rural households. The rural non-farm household of Sindh gets minimum gain (0.994 percent) in case of first simulation while rest of the rural non-farm households in Pakistan gain (0.997 percent). Similarly, in case of second simulation, rural non-farm households of Sindh get maximum gain (1.569 percent) and minimum gain (1.356 percent) in case of rural non-farm households of rest of the Pakistan. Table 8 Changes in Household Income of Rural Non-farm Household, Constant 2007 Prices (Percent) Household Types HH Code Population (millions) Income shares (percent) GSP Plus with Quota Potential EBA Status Rural non-farm quintile 1 Rural non-form quintile 2 Rural non-farm quintile other H-NFQ1 H-NFQ2 H-NFOTH 8.2 8.9 27.7 2.8 3.3 17.3 0.994 0.997 0.997 1.569 1.539 1.356 Income of Urban Household Table 9 show the impact of both simulations on the urban household of Pakistan. The urban household of Sindh is showing maximum gain in both simulations (0.935 percent and 1.342 percent respectively) while minimum gain is seen in the income of urban household of rest of the Pakistan in case of both simulations (0.86 percent and 1.162 percent respectively). 43 Journal of Management Sciences Table 9 Changes in Household income of Urban Household, Constant 2007 Prices (Percent) Household Types Urban quintile 1 Urban quintile 2 Urban other HH Code Population (millions) Income shares (percent) GSP Plus with Quota Potential EBA Status H-UQ1 H-UQ2 H-UOTH 8.6 8.6 25.7 2.6 3.4 38.7 0.935 0.927 0.86 1.342 1.265 1.162 Overall, factor income remains positive for almost all households. If there is no tariff and quota restriction from EU28, the income of every household type will increase but that increase. Primarily, it is because, the increased exports of Pakistan will definitely increase the economic activities in the economy and the backward and forward linkages of the industry will bring positive change in the income of every household. Effects on Real Returns to Factors Increased trade and especially exports increase the rate of return to factors. The HeckscherOhlin model is although workable only in the economies where amount of goods and the number of production factors is equal. The products are assumed homogenous in the model, despite many markets in the world are represented in a better way with differentiated products (Suranovic, 2010). In case of Pakistan, the model suggests that there will be decreases in the return to capital as country is already capital deficient as compare to its competitors (Khan et al., 2015). Labor force of Pakistan is more than 65 million. Unemployment rate in Pakistan is about 6 percent (Government of Pakistan, 2015). Despite tremendous Government efforts for ensuring minimum wages in Pakistan like “Minimum Wages Ordinance 1961, the Punjab Minimum wages for unskilled Workers Ordinance 1969, Minimum Wages Board,” etc ensure that Government is dedicated to support low income groups. The model used for the calculation of effects on real returns to factors is the extension of standard GTAP model and uses the Armington assumption that categories the products on the basis of country of origin. Furthermore, assimilation could disturb the rate of return on capital by virtue of the prices of transitional and capital goods. The results of both simulations show the change in factor prices with respect to the price index for private consumption expenditure. However, it fails to consider the impact of changes in government’s revenue, and government’s capacity to redistribute tax income to individuals, whether it is through transfer payments or provision of public goods (Khan et al., 2015). Wages of Large Agriculture Land Owned Labor The results of both simulations are presented in the Table 10 showing changes in the wages of household that owns large agricultural land. The results reveal that in case of first simulation, the wage of labor decrease by -1.45 percent while in case of second simulation, it increased by 0.283 percent. 44 Journal of Management Sciences Table 10 Changes in Household income of Urban Household, Constant 2007 Prices (Percent) Factor RF Code Description Labor LA-AGL Labor - agric (own)-large GSP Plus with Quota Potential EBA Status -1.45 0.283 Wages of Medium Agriculture Land Owned Labor The results in Table 11 show the changes in the wages of labor that own medium size piece of agriculture land, after performing both simulations. The results show that in case of first simulation, the wage of labor with medium sized agriculture land decreased by -1.335 percent in the region of Punjab. The results of second simulation show that wage of all households with medium sized agriculture land increased with maximum increase in the case of labor living in parts of Pakistan other than Punjab and Sindh and that is 1.487 percent. Table 11 Change in Real Wages of Medium Agriculture Land Owned Labor (Percent) Factor RF Code Description Labor LA-MF1 LA-MF2 LA-MF3 Labor - agric (own)-med Sindh Labor - agric (own)-med Punjab Labor - agric (own)-med OPak GSP Plus with Quota Potential EBA Status -1.244 -1.335 -0.956 1.184 0.285 1.487 Wages of Small Agriculture Land Owned Labor In case of farmers having small area of agriculture land in all areas of Pakistan, the results seem quite similar to the case of farmers having medium sized agriculture land. The results of both simulations are shown in Table 12. According to the results, the wages of the labor with small size agriculture farm decreased everywhere in Pakistan when country faces quota restrictions in the EU while a positive change can be seen in case of Simulation 2 i.e. if Pakistan gets the status of EBA in the EU28. Table 12 Change in Real Wages of Small Agriculture Land Owned Labor (Percent) Factor RF Code Labor LA-SF1 LA-SF2 LA-SF3 Description GSP Plus with Quota Potential EBA Status -1.227 -1.017 -0.454 1.136 0.851 1.577 Labor - agric (own)-sm Sindh Labor - agric (own)-sm Punjab Labor - agric (own)-sm OPak Wages of Skilled and Unskilled Labor One additional simulation is added in the study by assuming unskilled labor is unemployed in the model and then performing both simulations to check what would be the impact on real wages of other types of labors. The impact of both simulations on the wage of skilled and unskilled labor is shown in the Table 13. The results show that in case of first simulation, the wage of agriculture labor in general is increased by 1.355 percent, in case of 45 Journal of Management Sciences unskilled non-agriculture labor, the wage increased by 0.286 percent and in case of skilled non-agriculture labor, it is increased by 0.304 percent. The results of the second simulation are quite different where the wage of non-agriculture unskilled labor is decreased by -1.851 percent while a maximum increase is seen in the agriculture labor (5.366 percent) Table 13 Change in Real Wages of Skilled and Unskilled Labor (Percent) Factor RF Code Description Labor LA-AGW LA-SKU LA-SK Labor - agric (wage) Labor - non-ag (unsk) Labor - non-ag (skilled) GSP Plus with Quota Potential EBA Status 1.355 0.286 0.304 5.366 -1.851 0.075 It is worthy to note that the supply of labor is fixed in the agriculture sector, so any decrease in demand may decrease the wage of the agriculture labor. The results of the simulations show that the increase in wage of skilled labor is greater than the increase in wage of unskilled labor. Similarly, the supply of production labor is also fixed which results into increase in wage rate as the production demand increases. The results of the simulations show that increased exports will increase the wage rate which opposes the theory that liberalized trade may reduce the wage rate. For further information, please see (Stiglitz, 1970; Davis, 1996; Feenstra & Hanson, 1997; Topalova, 2005; Harrison, 2006). Majority of the exports from Pakistan are textile and agricultural products so increased volume of exports due to GSP plus and EBA status in the EU28, might shift labor from the agriculture to industry. This is primarily due to the fact that land is sector specific but labor is inter-sectorally mobile. So this offset effect might lead industrialization in Pakistan. Real Return to Land of Large Agriculture Farms After labor, land is another factor of production, the rent paid to land is also affected by certain changes in the trade. The source land is fixed, so any change in demand for the production of goods requiring more land, may result into change in return to land. The results shown is Table 14 reveal the effects of both simulations on the large land farms. The results of first simulation show a negative change in the real return to land in case of large farms everywhere in Pakistan but in case of Simulation 2, the large land farm of Punjab is losing in return while a positive change is seen in the farms of Sindh (0.464 percent) and rest of the Pakistan (2.053 percent) which is maximum gain. Table 14 Change in Real Return to Land of Large Farms (Percent) Factor RF Code Description Land LN-LG1 LN-LG2 LN-LG3 Land - large- Sindh Land - large- Punjab Land - large - OthPak GSP Plus with Quota Potential EBA Status -1.252 -1.639 -1.085 0.464 -0.175 2.053 46 Journal of Management Sciences Real Return to Land of Medium Agriculture Farms The case of change in real return to the land of medium farms is a little bit different than the previous case. The results of both simulations are presented in Table 15 that show a reduction in return to the land of medium farms in case of first simulation. The maximum reduction in return is seen in the province of Punjab (-1.338 percent). While in case of second simulation, the results are quite encouraging. There is gain in return to the land of medium farms in all areas of Pakistan with maximum gain in return of 1.467 percent in the region other than Punjab and Sindh. Table 15 Change in Real Return to Land of Medium Farms (Percent) Factor RF Code Description Land LN-MD1 LN-MD2 LN-MD3 Land - irrigated - med Sindh Land - irrigated - med Punjab Land - irrigated - med OthPak GSP Plus with Quota Potential EBA Status -1.228 -1.338 -0.951 1 0.359 1.467 Real Return to the Land of Small Agriculture Farms The results of both simulations relating to change in return to the land of small farms are presented in Table 16 which are quite similar to the results of medium size farms. The gain is negative in case of first simulation for all regions of Pakistan with maximum loss in return in the province of Sindh (-1.215 percent). The results of the second simulation are opposite to the first simulation that show a gain in return to the land of small farms in all areas of Pakistan. The maximum gain is seen in the areas other than provinces of Punjab and Sindh that is 1.613 percent. Table 16 Change in Real Return to Land of Small Farms (Percent) Factor RF Code Description Land LN-SM1 LN-SM2 LN-SM3 Land - irrigated - sm Sindh Land - irrigated - sm Punjab Land - irrigated - sm OthPak GSP Plus with Quota Potential EBA Status -1.215 -1.022 -0.399 0.977 0.942 1.613 Real Return to the Land of Non-Irrigated Agriculture Farms There are certain areas in Pakistan where traditional canal system is not activated to irrigate the agriculture land. Those farms are called non-irrigated farms. The results of both simulations are presented in Table 17 to show the return to the land of non-irrigated farms. Interestingly the results are very similar to the results of previous two cases (land of medium and small farms). In case of first simulation, there is negative gain in all areas of Pakistan where non-irrigated farms exist with loss of -1.12 percent in the province of Sindh while in rest of the Pakistan the non-irrigated land suffered from loss of -1.337 percent in return. The case of second simulation shows gain in return of the non-irrigated land with maximum gain in the Sindh province (0.821 percent) whereas in rest of the Pakistan, the non-irrigated land gain 0.708 percent in return. 47 Journal of Management Sciences Table 17 Change in Real Return to Land of Non-Irrigated Farms (Percent) Factor RF Code Description Land LN-DR1 LN-DR2 LN-DR3 Land non-irrig - sm/m Sindh Land non-irrig - sm/m Punjab Land non-irrig - sm/m OthPak GSP Plus with Quota Potential EBA Status -1.12 -1.337 -1.337 0.821 0.708 0.708 It is interesting to note that the results of first simulation suggest a negative gain in return to land which also support the results of (Bradley & Norman, 1977) while working on US economy. While these are contradictory with the findings of Hong (1990) which suggested that protection increases the return as resources shift towards agricultural productivity. The gain is not possible without technonolgy whether economy is facing restrictions or not (Ghosh, 2004). The Stolper Samuelson Theorem that is based on the famous Heckscher-Ohlin model also supports the results that liberalized trade lead to increase in return to land (Leamer et al., 1995). Similarly, a large majority of researchers support the results that return on land increases if there is no restrictions on the exports of an economy, for example see (Runge & Halbach, 1990; Chang, 1979). Real Return to the Capital We have already discussed the impact of both simulations on the return to labor and land. In this section, we will focus on the factor of capital. The study has divided the capital into four categories and results along with categories are presented in Table 18. The results of both simulations show gain in return to capital in most of the types except capital other than agriculture (-1.106 percent in case of quota restriction on Pakistan) and capital formation (-0.054 percent in case if Pakistan gets EBA status in EU28). In case of both simulation, the maximum gain is seen in capital livestock, 1.818 percent when quota is applied on exports from Pakistan to justify the capping mechanism of EU28 and 6.24 percent gain in return if Pakistan gets the status of EBA in EU28. Table 17 Change in Real Return to Land of Non-Irrigated Farms (Percent) Factor Capital RF Code Description K-LVST K-AGR KFORM KINF Capital Capital Capital Capital GSP Plus with Quota Potential EBA Status 1.818 -1.106 0.105 0.28 6.24 0.798 0.105 -0.054 livestock other agriculture formal informal The results of both simulation suggest that there is gain in return to capital in most of the cases which support the work of researchers like (Chang, 1979; Hong, 1990; Thompson, 2016). Walmsley et al. (2013); Khan et al. (2015) also produced the same results while studying the Mozambique and Pakistan economy respectively. Although Ghosh (2004) also support this but adds that gain in return to the capital can be maximized with continuous improvement in the technology. 48 Journal of Management Sciences Limitations of the Study Parallel to many empirical studies, this study was constrained by a variety of factors which could be considered as limitations. The first and the most important limitation is the database, similar to most other studies which adopt CGE models. Current study used GTAP v 9.0 and Social Accounting Matrix with base year 2007-08. This extension of standard GTAP model is known as MyGTAP which provided the parameters related to trade elasticity but these parameters are not estimated econometrically. It is noted that household welfare results are sensitive to parameter values assumed in the model. Likewise, a superior understanding of implications at household level could have been achieved if we had been able to use more disaggregated data at household level. Despite the above mentioned limitations, the global CGE model (standard GTAP) generated reasonable good results at macro level for the country and the MyGTAP model with the most latest constructed SAM (2007-08) for Pakistan. The results could be more practical with current data. Conclusion and Policy Recommendations The study has attempted to calculate whether the GSP plus status of Pakistan in the EU28 produce positive change not only at aggregate level but also at household income and real wages. The study attempted to calculate the effects of different potential and current opportunities for Pakistan by using standard MyGTAP. The MyGTAP employed the latest available Social Accounting Matrix (SAM) with base year 2007-08. The descriptive analysis of the results of different simulations MyGTAP reveal that there is overall increase in the GDP of Pakistan. The incentive to export in the EU28 will increase the production level in the Pakistan. Similarly, the improvement in production also increase the real wages and household income. Similarly, EBA status of Pakistan in the EU28 show an increase in the household income with maximum gain by the household of rural Sindh with no agriculture land and a positive change in real wages of most of the factors. Comparatively low improvement over urban and non-farm household of rural areas. Therefore, empirical evidence in terms of the household welfare from this study supports the overall view that Pakistan can gradually gain from GSP plus status. The policy implications for the study are straightforward and related to trade policy which is also the main concern of the research. The competition after the GSP plus implementations have been improved, so the country needs to improve the competitiveness. For this purpose, it should be acknowledged that it is firm that can bring success for the country only if it works under right strategies and clear vision about how and why to produce and for which target market it is producing. Therefore, the firms wishing to enter into tough competition require very supportive role from the public authorities in the Pakistan. The public authorities should ensure the provision of safe and healthy working environment that may enable firms to compete at domestic and finally in the world market. The production activities of the country are concentrated towards the sectors of textiles, wearing apparel, beverages and tobacco and leather products that need to be diversified with cost effective methods. Moreover, Pakistan can focus on those markets where its 49 Journal of Management Sciences competitors have a small share. 50 Journal of Management Sciences References Ahmed, V., & O’Donoghue, C. (2008). Welfare impact of external balance in Pakistan: CGE-micro simulation analysis (Tech. Rep.). Marina Congress Centre in Helsinki, Finland. Bradley, I. G., & Norman, G. (1977). Welfare effects of trade restrictions. Elsevier, Netherlands. Burfisher, M. E. (2011). Introduction to computable general equilibrium models. Cambridge University Press, England. Butt, M. S. (2006). Impact of tariff cuts on Pakistan: a computable general equilibrium analysis with particular focus on main exports and regional disparities (Unpublished doctoral dissertation). Griffith University, Australia. Carbone, M., & Orbie, J. (2016). The trade-development nexus in the European Union: differentiation, coherence and norms. Routledge, United Kingdom. Chang, W. W. (1979). Some theorems of trade and general equilibrium with many goods and factors. Econometrica, 47 (3), 709. doi: 10.2307/1910416 Davis, D. (1996). Trade liberalization and income distribution (Tech. Rep.). Retrieved from http://dx.doi.org/10.3386/w5693 Debowicz, D., Dorosh, P., Haider, H. S., & Robinson, S. (2007). A 2007-08 social accounting matrix for Pakistan. SSRN Electronic Journal . Retrieved from http://dx.doi.org/ 10.2139/ssrn.2161402 Dorosh, Niazi, M., & Nazli, H. (2004). A social accounting matrix for Pakistan, 2001-02: Methodology and results. a background research paper for the Pakistan rural factor markets study, World Bank (Tech. Rep.). Dorosh, & Rashid, S. (2013). Domestic Terms of Trade for Agriculture in Pakistan and Agricultural Income Tax. IFPRI. (Tech. Rep.). Retrieved from http://pssp.ifpri.info/files/2013/05/Domestic-tems-of-trade -for-agriculture-in-Pakistan-and-agriculture-income-tax.pdf European, C. (2013). The EU’s new Generalised Scheme of Preferences (GSP) (Tech. Rep.). Retrieved from http://trade.ec.europa.eu/doclib/docs/2013/ february/tradoc 150582.pdf Feenstra, R. C., & Hanson, G. H. (1997). Foreign direct investment and relative wages: Evidence from Mexico’s maquiladoras. Journal of International Economics, 42 (3), 371–393. Ghosh, N. (2004). Impact of trade liberalization on returns from land: A regional study of Indian agriculture. In The WTO, developing countries and the Doha development agenda (pp. 92–129). Springer Nature. Retrieved from http://dx.doi.org/ 10.1057/9780230523265 5 doi: 10.1057/9780230523265 5 Harrison, A. (2006). Globalization and poverty (Tech. Rep.). National Bureau of Economic Research, University of Chicago. Hertel, T. W., & Hertel, T. W. (1997). Global trade analysis: modeling and applications. Cambridge University Press, England. Hong, W. (1990). Export-oriented growth of Korea: a possible path to advanced economy. 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EPJ Web of Conferences 146, 02022 (2017) DOI: 10.1051/epjconf/201714602022 ND2016 Evaluation of excitation function for 186 W(d,2n)186 Re reaction Jimin Wanga , Xi Tao, Mengxiao Kang, and Xiaolong Huang, and Youxiang Zhuang China Nuclear Data Center, China Institute of Atomic Energy, PO Box 275(41), 102413 Beijing, China Abstract. The excitation function of the 186 W(d,2n)186 Re reaction was evaluated and the thick target yield (TTY) of the therapeutic radioisotope 186 Re was calculated. The available experimental data from the EXFOR library and literature were analyzed and corrected with the g-ray branching ratio, radioactive decay constant and standard cross sections. The excitation function of the 186 W(d,2n)186 Re reaction was recommended below 50 MeV on basis of the least-squares fit with polynomial and the theoretical calculation with TALYS code. The TTY was calculated using the recommended excitation function of the 186 W(d,2n)186 Re reaction. 1. Introduction The charged particle excitation functions are the important part of the nuclear data, applied to the activation analysis, nuclear medicine investigation, nuclear theory, calculation of integral thick target yield, and so on. Rhenium-186 is an important medically radionuclide, and regarded very suitable for radiotherapy and radioimmunotherapy due to its attractive properties which include emission of high-energy β-rays 1.07 MeV, low-abundance (9.42%) gamma emission at 137 keV, and 90.64 h half-life. The 186 Re has been produced using nuclear reactors through 185 Re(n,γ )186 Re reaction, but the specific activity is medium, not in no-carrier-added form. In order to radiolabel antibodies more efficiently, production of nocarrier-added 186 Re with high specific activity is required. There are two major routes for the production of nocarrier-added 186 Re by cyclotron, namely 186 W(p,n)186 Re and 186 W(d,2n)186 Re. In the present work, the excitation function of 186 W(d,2n)186 Re reaction was evaluated. The available experimental data include the latest reports [1, 2] that are not considered in previous works [3, 4] were analyzed and corrected. The nuclear model calculation was done using TALYS [5]. The TTY of 186 Re was calculated using the recommended excitation function of 186 W(d,2n)186 Re reaction, and compared with the TTY from 186 W(p,n)186 Re reaction [6]. 2. Evaluation of experimental data The activation method and stacked foil technique are often used in the charged particle excitation functions measurement. The formula [7] of charged particle excitation functions is as follows: eλtd M c 1 1.6 × 10−19 ti · · · · · 1024 (b) nεpa N0 t χ Q 1 − e−λti (1) Where, M is the target molecular weight, nis the target nucleus number in a molecule, ε denotes the detection σ = a e-mail: jmwang@ciae.ac.cn efficiency of detector at full energy peak, p is the g-ray branching ratio, a denotes the isotope abundance, N0 is the Avogadro constant, c/tis the accounting g-ray number at full energy peak in a unit time, χ denotes the target weight in a unit area, ti denotes the irradiation time, Qis the total integrated beam current (in Coulomb), td denotes the cooling time (start from stop irradiation) and λ is the radioactive decay constant. From the mentioned-above formula, the cross sections are proportional to the radioactive decay constant, and inversely proportional to the g-ray branching ratio. Moreover, if the experiment is relative measurement, the cross sections are proportional to the standard cross sections. According to these relations, σ can be corrected by the new p, λ and standard cross sections. Eight experiments [1, 2, 7–12] of the 186 W(d,2n)186 Re reaction were founded in the EXFOR library and literature, listed in Table 1 and shown in Fig. 1. The experimental data were normalized to 100% enrichment of 186 W, considering the given enrichment of the target in the literature and the isotopic composition of natural tungsten, i.e., 180 W (0.12%), 182 W (26.50%), 183 W (14.31%), 184 W (30.64%) and 186 W (28.43%). In the measurements of F. Tarkanyi et al. [11], K.Ochiai et al. [12], S. Manenti et al. [1] and C. Duchemin et al. [2], the samples were thick natural W-metal foils, so the normalization factor is 3.517. In the measurement of F.W. Pement et al. [8], the sample was WO3 powder, enriched to 97.2% in 186 W, so the normalization factor is 1.0288. In the measurement of Tao Zhenlan et al. [7], the g-ray branching ratio of 137.16 keV are 0.092, and the new value is 0.0942 [13]. The sample was WO3 powder, enriched to 99% in 186 W, so the corrected factor is 0.9813. The experimental data after normalization and correction are shown in Fig. 2. 3. Nuclear model calculations Several nuclear model code have been developed, in the evaluation of data are very helpful. In the process of evaluation, the parameters of the nuclear model c The Authors, published by EDP Sciences. This is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0
(http://creativecommons.org/licenses/by/4.0/). EPJ Web of Conferences 146, 02022 (2017) DOI: 10.1051/epjconf/201714602022 ND2016 Table 1. Measurements of 186 W(d,2n)186 Re reaction. Author F.W. Pement et al. [8] Sample WO3 enriched to 97.2% S.J. Nassiff et al. [9] — Tao Zhenlan et al. [7] WO3 enriched to 99% N.S. Ishioka et al. [10] natural F. Tarkanyi et al. [11] natural K. Ochiai et al. [12] natural S. Manenti et al. [1] natural C. Duchemin et al. [2] natural Method STTA STTA, ACTIV STTA, ACTIV STTA, ACTIV STTA, ACTIV STTA, ACTIV STTA, ACTIV STTA, BCINT Figure 3. The results of nuclear model calculation using TALYS. Figure 4. The results of fitting from threshold to 14 MeV. Figure 1. Original experimental data. rvsdjust was set to 1.2. The pairing energy of 186 Re was set to 0.0. The theoretical results are shown in Fig. 3. 4. Fitting of experimental data After normalization and correction, the experimental data are consistent besides the partial data of S.J. Nassiff et al. [9] and S. Manenti et al. [1]. S.J. Nassiff et al. reported six cross section values from 7.3 to 16.7 MeV, but the errors not given. The data are higher than the calculated values using TALYS and those reported by other authors between 10 to 20 MeV. The data of S. Manenti et al. are highter than the results of calculation and other measurements at 11.76, 12.38, 12.54 and 13.14 MeV. F.W. Pement et al. [8] reported nine data points but not given the errors also. So these data were neglected in fitting of experimental data. On basis of least-squares fit with polynomial, the evaluated experimental data were fitted from threshold to 14 MeV and from 14 to 50 MeV. The curves of fitting are shown in Fig. 4 and Fig. 5. The function of fitting from threshold to 14 MeV as follws: σd,2n = a − bE d + cE d2 − d E d3 (2) Figure 2. The experimental data after normalization and correction. code need to be adjusted to reproduce the experimental data. In this work, the cross sections were calculated using TALYS, a nuclear model code, developed by Koning et al. [5]. The nuclei were considered non-spherical in shape. The compound nucleus contribution was considered in the frame of Moldaner model. The contributions of direct reactions were taken into account by ECIS. The back-shifted Fermi gas model (BFM) was used for level densities (ldmodel 2). The OM parameters were adjusted, Where, a = 4.2176, b = 1.4378, c = 0.1555, d = 0.0052. The function of fitting from 14 to 50 MeV as follws: σd,2n = exp(a − bE d + cE d2 ) Where, a = 1.8248, b = 0.2544, c = 0.0031. 2 (3) EPJ Web of Conferences 146, 02022 (2017) DOI: 10.1051/epjconf/201714602022 ND2016 function of 186 W(d,2n)186 Re reaction are agree with the experimental data, the maximum value is slightly higher than the value of IAEA TRS 473, and little lower than the value of M. Hussain et al. The thick target yield (TTY) of 186 Re was calculated using the recommended excitation function of 186 W(d,2n)186 Re reaction, and compared with the TTY from 186 W(p,n)186 Re reaction [6], together with the measurements [1, 14, 15] are shown in Fig. 7. The TTY of 186 W(d,2n)186 Re reaction are much higher than the value of 186 W(p,n)186 Re reaction above 15 MeV. So the 186 W(d,2n)186 Re reaction may be more suitable for production of no-carrier-added 186 Re for medical application, if the deuteron beams are available by cyclotron and the energy is high enough. Figure 5. The results of fitting from 14 to 50 MeV. References [1] S. Manenti, E. Persico, K. Abbas, M.L. Bonardi, L. Gini, F. Groppi, U. Holzwarth, F. Simonelli, Radiochimica Acta 102, 669 (2014) [2] C. Duchemin, A. Guertin, F. Haddad, N. Michel, V. Metivier, Applied Radiation and Isotopes 97, 52 (2015) [3] M. Hussain, S. Sudar, M.N. Aslam, R. Ahmad, A.A. Malik, S.M. Qaim, Radiochimica Acta 98, 385 (2010) [4] S.M. Qaim, F. Tarkanyi, R. Capote, IAEA-TRS-473, 328 (2011) [5] A.J. Koning, S. Hilaire and M.C. Duijvestijn, EDP Sciences, 211 (2008) [6] Mengxiao Kang, Xiaolong Huang, Lile Liu, Atomic Energy Science and Technology, (in Chinese), (to be published) [7] Zhenlan Tao, Fuying Zhu, Huiyuan Qiu, Gongqing Wang, Chinese Journal of Nuclear Physics 3, 242 (1981) [8] F.W. Pement, R.L. Wolke, Nuclear Physics 86, 429 (1966) [9] S.J. Nassiff, H. Munzel, Radiochimica Acta 19, 97 (1973) [10] N.S. Ishioka, S. Watanabe, A. Osa, M. Koizumi, H. Matsuoka, T. Sekine, Journal of Nuclear Science and Technology, Supplement 2, 1334 (2002) [11] F. Tarkanyi, S. Takacs, F. Szelecsenyi, F. Ditroi, A. Hermanne, M. Sonck, Nuclear Instruments and Methods in Physics Research B 211, 319 (2003) [12] K. Ochiai, M. Nakao, N. Kubota, S. Sato, M. Yamauchi, N.H. Ishioka, T. Nishitani, C. Konno, EDP Sciences, 1011 (2008) [13] Xiaolong Huang, Zhigang Ge, Zhendong Wu, Guochang Chen, Jimin Wang, Xi Tao, Jianhui Li, V.P. Chechev, S.A. Badikov, T.V. Golashvilli, O.O. Patarakin, V.I. Rachkov, Nuclear Characteristics of Nuclides, (China Atomic Energy Press, 2013) [14] S. Mukhammedov, A. Vasidov, E. Pardaev, Soviet Atomic Energy 56, 56 (1984) [15] M.L. Bonardi, F. Groppi, S. Manenti, E. Persico, L. Gini, Applied Radiation and Isotopes 68, 1595 (2010) Figure 6. The recommended excitation function for 186 W(d,2n)186 Re reaction, compared with experimental data and results published earlier. Figure 7. The TTY of 186 W(d,2n)186 Re reaction, compared with the TTY of 186 W(p,n)186 Re reaction. 5. Results and discussion The excitation function for 186 W(d,2n)186 Re reaction was recommended from threshold energy to 50 MeV after comprehensive evaluation, take into account the results of calculation and fitting. The recommended curve is given in Fig. 6, together with the results of M. Hussain et al. [3] and IAEA TRS 473 [4]. The recommended excitation 3 

W2528671231.txt

2 - ORIGINAL ARTICLE WOUND HEALING Adipose tissue stromal vascular fraction in the treatment of full thickness burns in rats1 Alexandre Lamaro CardosoI, Maria Márcia. BachionII, Júlia de Miranda MoraisIII, Marcelo Silva FantinatiIV, Vera Lúcia Lima de AlmeidaV, Ruy Souza Lino JúniorVI DOI: http://dx.doi.org/10.1590/S0102-865020160090000002 Fellow Master degree, Faculty of Medicine, Universidade Federal de Goiás (UFG), Goiania-GO, Brazil. Intellectual and scientific content of the study, conception and design of the study, manuscript writing. II PhD, Full Professor, Faculty of Nursing UFG, Goiania-GO, Brazil. Acquisition, analysis and interpretation of data. III PhD, Associate Professor, Faculty of Medicine, UFG, Goiania-GO, Brazil. Acquisition, analysis and interpretation of data. IV PhD, Full Professor, ESEFFEGO, Universidade Estadual de Goiás (UEG), Goiania-GO, Brazil. Acquisition, analysis and interpretation of data. V Biomedic, Institute of Tropical Pathology and Public Health, Experimental Pathology Laboratory, UFG, Goiania-GO, Brazil. Acquisition of data. VI PhD, Associate Professor, Institute of Tropical Pathology and Public Health, Experimental Pathology Laboratory, UFG, Goiania-GO, Brazil. Scientific and intellectual content of the study, analysis and interpretation of data, statistical analysis, manuscript preparation. I ABSTRACT PURPOSE: To analyze the healing effects of stromal vascular fraction (SVF) application compared to wound dressing with 2% silver sulfadiazine in full thickness burn wounds in rats. METHODS: Animals were divided into two groups: 2% silver sulfadiazine group and SVF group. Both groups received occlusive bandages while the first one was treated with 2% silver sulfadiazine and the latter was treated with injections of SVF prepared from adipose tissue extracted from an animal donor. The animals were accompanied through 3, 7 and 30 days for evaluation of macroscopic, microscopic and morphometric aspects. RESULTS: On day three, a significant increase (p<0.05) of infiltration of polymorphonuclear, fibrin formation and fibroblasts migration in SVF group was observed. On the 7th day the mononuclear infiltrate, angiogenesis, collagen and fibroblasts were significantly increased in the SVF group (p<0.05). At 30 days significantly increased collagen deposition was observed in the SVF group (p<0.05). CONCLUSION: Adipose tissue derived stromal vascular fraction injections promotes better wound repair than 2% silver sulfadiazine in the treatment of full thickness burn in rats during the evaluated experimental period. Key words: Burns. Wound Healing. Rats. 578 - Acta Cirúrgica Brasileira - Vol. 31 (9) 2016 Adipose tissue stromal vascular fraction in the treatment of full thickness burns in rats Introduction cell cultivation techniques, injured tissue and patient’s immune response8. Of particular importance is the recognition of abundant Full thickness burns are characterized by destruction of sources of cells, with great therapeutic potential9. all skin layers. The destruction of the free nerve endings in the The separation of adipose tissue components by dermis makes these lesions painless, and grafting procedures enzymatic digestion and subsequent centrifugation of the material are often needed to optimize tissue repair that usually evolves raised new possibilities for using this tissue in regenerative with formation of hypertrophic scars, severe fibrosis and tissue therapy. While initially the focus was turned to the use of mature 1 adhesion . adipocytes, located in the upper region of tissue solution after The global incidence of burns requiring medical centrifugation, different studies have explored the therapeutic intervention in 2004 was about 11 million people. Although the majority of cases throughout the world are not fatal, fire related burns are among the leading causes of disability in low and middle income countries2. The Brazilian Burn Society estimates that millions of burn cases occur every year in Brazil. Approximately two hundred thousand are treated at emergency wards and forty thousand require hospitalization. Estimates presented in a study conducted in the Federal District of Brazil showed a mortality rate of 6.2% among hospitalized burns victims in emergency wards. Among violent causes of death in Brazil burn injuries represent the third most important cause after road accidents and homicides3. The techniques conventionally used in the treatment of burn wounds vary according to the extent of the lesion, affected body area, age and previous individual health status. Prevention of infections, removal of necrosis, and grafting procedures are routinely used to treat such wounds4. In full thickness burn injuries surgical debridement followed by occlusive dressing are generally used in most clinical centers dedicated to the treatment of these lesions. Moisturizing agents such as glycerin and microbicides as 2% silver sulfadiazine are incorporated into the occlusive dressing to promote the healing process and prevent infection . 5 applications of stromal vascular fraction (SVF)10. Endothelial cells, lymphocytes, myelocytes, pericytes, pre-adipocytes and adipose derived stem cells (ADSCs) are part of its composition and, depending on the stimuli in the tissue microenvironment, present considerable plasticity, and the capacity to proliferate and differentiate into distinct cell lines11. Studies suggest that therapeutic effects of SVF are primarily a result of the activity of ADSCs7,12 such as modulation of inflammatory response13, increased migration of fibroblasts14, collagen production15 an angiogenesis16-18. Considering the need to develop interventions which address the reduction of fibrosis, restrictions in tissue mobility, aesthetic deformities and functional limitations in full thickness burn injuries, this study aimed to compare the microscopic, macroscopic and morphometric effects between the treatments with SVF applied intradermally and occlusive bandages with 2% silver sulfadiazine in experimentally induced full thickness burn wounds in rats. Methods The study was approved by the Ethics Committee on the use of animals from the Universidade Federal de Goiás (protocol While the outcomes in burn wound injury treatments Number 035/13). increased significantly in the last decades, as a result of developments in surgical, nursing and rehabilitation procedures, Stromal vascular fraction preparation and injection severe functional and cosmetic limitations are still highly prevalent procedure in burn patient populations6. The development of interventions morphological One day after the burn procedure, euthanasia of the reconstruction and functional recovery are necessary in order to donor animal was performed (lethal injection of thiopental sodium achieve better results . 100mg/kg) followed by the removal of the visceral adipose tissue to reduce hypertrophic scarring, promote 7 Cell therapy is one of the most promising approaches to by surgical dissection. The material was conducted to laminar flux morphological reconstruction and functional restoration of skin in transport solution (Sterile Phosphate Buffer Solution at 2% burn damage. The effective application of cell populations that (PBS) and 1% antibiotics: 105 units/ml penicillin and 105ug/ml optimize the repair processes and tissue regeneration in different streptomycin) washed in PBS to remove debris and red blood cells clinical conditions depends on several factors such as type of cell, and minced with fine tissue scissors. The fragmented tissues were Acta Cirúrgica Brasileira - Vol. 31 (9) 2016 - 579 Cardoso AL et al. incubated with type I collagenase (1.5 mg/ml) and kept in water Animals and treatment bath at 37ºC for 50 minutes. After enzymatic digestion, the tissue was filtered using a 70µm filter and collagenase was removed by dilution with phosphate buffer solution (PBS). The cell suspension was centrifuged at 363g (1800 rpm)/7 minutes and the supernatant, composed primarily of mature adipocytes, was removed. The pellet was formed by cells which were re-suspended in PBS. The counting of viable cells was performed using the Tripan Blue stain exclusion method. Injections were prepared adding PBS and a SVF volume equivalent to 5 x105 cells/ml in 1 ml syringes. Four injections of 0.2 ml were made in each wound edge and dripping of 0.2 ml covering the wound bed. Full thickness burn procedure After local preparation of the dorsal skin, shaving and antisepsis, the animals were anesthetized with 0.01ml/g of Ketamine 10% and 0.03ml/g of Xilazine 2% via intraperitoneal. Afterwards, the full thickness thermal injury was induced by the immersion of this region in boiling water as described by Morais et al.19. Briefly, each animal was placed inside a PVC (polyvinyl chloride) plastic cylinder with a 2x2 cm opening and sealed extremities, Thirty male adult Wistar rats weighing between 300 and 350g were used in this study. Adipose tissue extraction was made once in each experimental series with durations of 3, 7 and 30 days, with a total of three animals used as donors. Each experimental group was composed by five animals. Each experimental group received treatment after the debridement procedure, 2 days after the injury induction. The 2% silver sulfadiazine group (CT) received occlusive bandages with sterile gauze impregnated with glycerine and 2% silver sulfadiazine. The stromal vascular fraction treated group (SVF) received occlusive bandages with sterile gauze impregnated with glycerine and injections of stromal vascular fraction at a concentration of 5 x105 cells/ml. The injection procedure was made only once after debridement in SVF group and bandages were change in each group daily during the experimental series. The animals euthanasia was performed at each experimental day with a lethal injection of thiopental sodium 100 mg/kg followed by collection of material for microscopic examination and photographic register of the injury for morphometric and macroscopic analysis. Macroscopic evaluation the uncovered area was immersed in water at 95 for 14 o seconds. The accomplishment of a full thickness burn by the cited method was early verified by histological analysis with hematoxylin-eosin preparation showing complete destruction of the epidermis, dermis and hypodermis confirming a full thickness burn wound with necrosis and disarrangement of collagen fibers. On the day that followed the burn procedure, animals received surgical debridement under general anesthesia (0.06 ml/Kg of 10% ketamine, 0.01 ml/Kg distilled water and 0.03 ml/Kg of 2% xylazine applied intraperitoneally) to remove necrosis. Debridement procedure initiated by dissection of skin with a Metzembaum scissor in panniculus carnosum, at the suprafascial level. The other edges were sectioned with a straight Mayo scissor always in the internal border of demarcated area. A full After euthanasia, wounds were photographed and the component phases of tissue repair such as inflammation, proliferation and maturation19 were macroscopically examined. Parameters of hyperemia, necrosis, hemorrhage, crust formation, tissue granulation and fibrosis were graded as absent (score 0), discrete (up to 25% of committed area, score 1), moderate (from 26 to 50% of committed area, score 2) and accentuated (more than 50% of committed area, score 3) in a semi-quantitative form. Examination was performed by the same researcher, blind to group allocation. The results were expressed in mean ± standard deviation. Morphometric evaluation pain management was performed by inclusion of 2 mg/ml Wound photographs were analyzed using the ImageJ software (National Institute of Health; version 1.3.1). Wound areas were circulated by the researcher without knowing the group allocation, and the contraction level was calculated using the following equation: CL= (area T0 – area Teuthanasia day)x 100/area T0,were paracetamol in the rats’ drinking water. CL=contraction level; T0= day of burn injury induction; Teuthanasia thickness skin (4 cm2) was excised from burn area by removing the epidermis and dermis layer with a surgical blade (0.1 mm) until the subcutaneous fat,avoiding the panniculus carnosum and the muscle layer. Early post operative and three days post burn 580 - Acta Cirúrgica Brasileira - Vol. 31 (9) 2016 Adipose tissue stromal vascular fraction in the treatment of full thickness burns in rats = days after injury induction. The morphometric evaluation day was performed at days 7 and 30 due to the absence of wound contraction at the early stage of the healing process (3 days). Microscopic evaluation After euthanasia, the wound tissue and adjacent skin were removed. This material was incubated with 10% formaldehyde, included in paraffin blocks, prepared with sections of 4 µm and stained with hematoxylin-eosin (H&E). Tissue slides were analyzed by optical microscopy of entire wound area at magnifications of x10 and x40 and parameters of fibrin, hemorrhage, polymorphonuclear infiltration, mononuclear infiltration, hyperemia, angiogenesis, necrosis, fibroblasts and collagen were graded as absent (score 0), discrete (up to 25% of committed area, score 1), moderate (from 26 to 50% of committed area, score 2) and accentuated (more than 50% of committed area, FIGURE 1 - Macroscopic evaluation of third degree burn wounds experimentally induced in rats treated with 2% silver sulfadiazine and adipose tissue stromal vascular fraction. (CT - control group) 2% silver sulfadiazine. (SVF) adipose tissue stromal vascular fraction treated group. (A) Arrow indicates discrete hyperemia. (B) Arrow indicates accentuated granulation tissue. (C) Arrow indicates discrete fibrosis. (D) Arrow indicates accentuated necrosis at wound edges. (E) Arrow indicates moderate necrosis. (F) Arrow indicates accentuated fibrosis. score 3) in a semi-quantitative form. Examination was performed by the same researcher, blind to group allocation. The results were expressed in mean ± standard deviation. Statistical analysis Statistical analysis was performed with the SigmaStat 2.3 software. Variables were tested for normal distribution and homogeneous variance (Kolmogorov-Smirnov test). The macroscopic and microscopic statistical analyses were performed through the Mann-Whitney test. The morphometric analysis was performed through the Student T test. Differences were considered significant when p<0.05. Results Macroscopic results General pathologic process associated with inflammation, proliferation and maturation phases were observed in days 3, 7 and 30 respectively (Figure 1). Statistically significant differences were found between the CT group and SVF group regarding necrosis formation, which was significantly increased in the CT group at day 3 (p<0.05). Other parameters such as crust, hemorrhage, hyperemia, granulation tissue and fibrosis did not present a macroscopic difference when comparing both treatments TABLE 1 - Macroscopic evaluation (mean ± standard deviation) of general pathological processes in third degree burn wound in rats treated with 2% silver sulfadiazine and adipose tissue stromal vascular fraction at days 3, 7 and 30. Pathological CT SVF DAI P Process (n = 5) (n = 5) 3 p=0.01* 1.0 ± 0.6 0.3 ± 0.8 Necrosis 7 0.3 ± 0.6 1.3 ± 0.4 p=0.183 30 0.0 ± 0.0 0.0 ± 0.0 p=1.00 3 0.3 ± 0.6 0.0 ± 0.0 p=0.183 Crust 7 0.3 ± 0.6 0.0 ± 0.0 p=0.183 30 0.0 ± 0.0 0.0 ± 0.0 p=1.00 3 0.3 ± 0.6 1.3 ± 0.4 p=0.42 Hemorrhage 7 0.3 ± 0.6 1.3 ± 0.4 p=0.183 30 0.0 ± 0.0 0.0 ± 0.0 p=1.00 Hyperemia Granulation tissue Fibrosis 3 0.3 ± 0.6 2.6 ± 0.4 p=0,.57 7 30 3 7 30 3 7 30 0.3 ± 0.6 0.0 ± 0.0 0.0 ± 0.0 0.3 ± 0.6 0.3 ± 0.6 0.0 ± 0.0 0.0 ± 0.0 3.0 ± 0.0 2.3 ± 0.4 0.0 ± 0.0 0.0 ± 0.0 2.6 ± 0.4 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 3.0 ± 0.0 p=0.42 p=1.00 p=1.00 p=0.057 p=1.00 p=1.00 p=1.00 p=1.00 SVF: stromal vascular fraction group. CT: 2% silver sulfadiazine group. n: number of animals in each group. DAI: days after injury induction. *Statistically significant (p<0.05 – Mann-Whitney). (Table 1). Acta Cirúrgica Brasileira - Vol. 31 (9) 2016 - 581 Cardoso AL et al. Microscopic results Fibrin formation, polymorphonuclear infiltration and fibroblast content were significantly increased in the SVF group compared to the CT group at day 3 (p<0.05) (Figure 2). The mononuclear infiltration, angiogenesis, fibroblast content and collagen deposition were significantly increased in the SVF group compared to the CT group at day 7 (p<0.05). 30 days after burn injury induction hyperemia and collagen deposition were significantly increased in the SVF group compared to the CT group (p<0.05) (Table 2). Collagen disruption and hemorrhage did not present significant differences between the treated groups during the experimental days. FIGURE 2 - Microscopic evaluation of third degree burn wounds experimentally induced in rats treated with 2% silver sulfadiazine and adipose tissue stromal vascular fraction. (CT - control group) 2% silver sulfadiazine. (SVF) adipose tissue stromal vascular fraction treated group. (A) Arrow indicates moderate fibroblastic proliferation. (B) Arrows indicate accentuated angiogenesis. (C) Arrows indicate accentuated collagen formation. (D) Arrow indicates discrete polymorphonuclear cells inflammatory infiltration. (E) Arrow indicates accentuated mononuclear cells inflammatory infiltration. (F) Arrow indicates moderate mononuclear cells inflammatory infiltration (x400 hematoxylin & eosin. Scale Bar indicates 20 µm). TABLE 2 - Microscopic evaluation (mean ± standard deviation) of general pathological process in third degree burn wound in rats treated with 2% silver sulfadiazine and adipose stromal vascular fraction at days 3, 7 and 30. Pathological DAI CT (n = 5) SVF (n =5) P Process 3 3.0 ± 0.0 3.0 ± 0.0 p=1.00 Collagen 7 1.0 ± 0.0 0.3 ± 0.6 p=0.183 disruption 30 0.6 ± 0.5 0.0 ± 0.0 p=0.422 3 0.0 ± 0.0 0.0 ± 0.0 p=1.00 Hemorrhage 7 1.8 ± 0.7 1.0 ± 0.0 p=0.183 30 0.2 ± 0.4 0.3 ± 0.4 p=1.00 3 1.6 ± 0.4 1.6 ± 0.9 p=0.741 Hyperemia 7 0.6 ± 0.8 2.0 ± 0.81 p=0.225 30 p=0.03* 0.2 ± 0.4 2.0 ± 0.0 3 p=0.03* 0.3 ± 0.6 3.0 ± 0.0 Fibrin 7 0.0 ± 0.0 0.0 ± 0.0 p=1.00 30 0.0 ± 0.0 0.0 ± 0.0 p=1.00 3 p=0.03* 0.0 ± 0.0 1.6 ± 0.4 PMN infiltra7 0.6 ± 1.2 1.0 ± 0.0 p=1.00 tion 30 0.0 ± 0.0 0.0 ± 0.0 p=1.00 3 0.6 ± 0.48 1.0± 0.0 p=0.183 MN infiltra7 p=0.01* 0.6 ± 0.4 3.0 ± 0.0 tion 30 0.8 ± 0.7 2.0 ± 0.0 p=0.183 3 0.0 ± 0.0 1.3 ± 0.4 p=0.057 Angiogenesis 7 p=0.01* 0.3 ± 0.6 3.0 ± 0.0 30 0.3 ± 0.6 2.0 ± 0.0 p=1.00 3 p=0.03* 0.3 ± 0.6 2.0 ± 0.0 Fibroblast 7 p=0.03* 0.3 ± 0.6 2.0 ± 0.0 30 0.3 ± 0.6 3.0 ± 0.0 p=0.42 3 0.0 ± 0.0 0.0 ± 0.0 p=1.00 Collagen 7 p=0.03* 0.3 ± 0.6 2.0 ± 0.0 30 p=0.03* 0.3 ± 0.6 3.0 ± 0.0 SVF: stromal vascular fraction group. CT: 2% silver sulfadiazine group. PMN:plymorphonuclear cells; MN: mononuclear cells. DAI: days after injury induction. *Statistically significant (p<0.05 – Mann-Whitney). Morphometric results No significant difference was observed between the SVF group and the CT group regarding the wound contraction levels at days 7 and 30 (Table 3). 582 - Acta Cirúrgica Brasileira - Vol. 31 (9) 2016 Adipose tissue stromal vascular fraction in the treatment of full thickness burns in rats TABLE 3 - Morphometric evaluation of wound contraction area in third degree burn wound in rats treated with 2% silver sulfadiazine and adipose tissue stromal vascular fraction at days 7 and 30. Results expressed in percentage ± standard deviation. Experimental group 7 DAI 30 DAI CT 14.476 ± 0.4 88.207 ± 2.6 SVF 15.023 ± 0.5 88.203 ±5.8 p p= 0.676 p= 0.149 CT: 2% silver sulfadiazine group. SVF: stromal vascular fraction group. DAI: days after injury induction. Statistical analysis: Student T test. Discussion This study demonstrated that the stromal vascular fraction removed from the adipose tissue improved the microscopic and macroscopic aspects of the healing process of full thickness burn wounds experimentally induced in rats. These improvements were observed in all phases of the healing process, inflammatory, proliferative and maturation. Stromal vascular fractions are composed of different cell populations such as mesenchymal stem cells, T regulatory cells, endothelial precursor cells, preadipocytes and macrophages. All of them are capable of influencing the wound microenvironment in many ways as they share similarities with bone marrow stromal cells, including the capacity of multilineage differentiation20. In this study, an amplification of the inflammatory response evidenced by increased migration of polymorphonuclear and fibrin deposition was observed at day 3. Studies applying SVF or ADSCs previously expanded in culture on wound models observed a reduction of the inflammatory response21. These different observations suggest that SVF could modulate inflammation in a bi-directional manner. Studies have shown that controlled amplification of initial inflammatory episodes has been associated with a shift in repair processes of different tissues towards more regeneration and less scar formation22-24. Trials designed to elucidate how different SVF preparations influence burn wound repair, regarding the type of adipose tissue utilized and dose-response curves for application of different SVF concentrations are needed to clarify the reasons for variable experimental findings. A significant increase of fibrin and fibroblasts in the SVF group compared to the CT group at day 3 was also observed. Evidence suggests that fibronectin binding to fibrin25 and ADSCs paracrine secretion of growth factors such as platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) could be possible associated mechanisms leading to the observed effect26. At day 7, angiogenesis (p=0.01), fibroblasts (p=0.03), mononuclear infiltrate (p=0.01) and collagen production (p=0.03) showed a marked increase in the SVF group when compared to the control group at the same experimental day. The secretome repertoire of ADSCs has been reported to be composed by many cytokines and growth factors which are able to influence these parameters12. Expression of potent angiogenic agents such as vascular endothelial growth factor (VEGF), fibroblast migration and activity stimulators like Transforming Growth Factor (TGF) and macrophages chemotaxis agents were expressed by ADSCs in studies performed in different models8,14. Collectively, this process could accelerate the proliferative phase and build better conditions for wound repair as observed in our results. Regarding mononuclear infiltration, classically activated macrophages have been known to express high levels of pro-inflammatory cytokines like IL-1, IL-6 and IL23 and under its influence, Th17 cells have released IL-17 which induces increased recruitment of polymorphonuclear cells towards the injury site24. It was possible to observe an increase in the mononuclear infiltration at day 7 with no increase in polymorphonuclear infiltration. This result suggested that the cytokines released by SVF cells could change the macrophage activation profile from classic to regulatory which improved the wound healing profile. Corroborating this hypothesis is the observation of marked collagen formation in the SVF group. Activated regulatory macrophages have been known to increase the production of extracellular matrix components and the release of anti-inflammatory cytokines such as IL-10, controlling the extension and intensity of inflammation, therefore promoting better conditions for the occurrence of the proliferative process24. Similarities between ADSCs in vivo morphology and fibroblasts could also contribute to the observation of increased fibroblast. Histological preparations with H&E did not allow the differentiation between the two cell types, nor the detection of fibroblasts that differentiate directly from ADSCs27. The initial stimulation of fibroblasts at 3 and 7 DAI in the SVF treated group resulted in increased collagen deposition at day 30. These results are in accordance to the Acta Cirúrgica Brasileira - Vol. 31 (9) 2016 - 583 Cardoso AL et al. description that ADSCs up-regulate type I and III collagen and fibronectin and down-regulate metalloproteinase 1(MMP-1), significantly accelerating healing from the wound edges and increasing the tensile strength of scar tissue28. Studies approaching scar tissue tensile strength in burn wounds treated with SVF and isolated ADSCs could better elucidate the specific contributions of these different cell populations regarding the functional outcomes in the burn wound healing process and guidance of the clinical decision-making process about which procedure would have a better cost-benefit relationship. SVF acquisition in humans using procedures such as lipoaspiration have been reported as a method that does not cause marked morbidity and have been easy to apply, particularly in conditions requiring immediate medical interventions such as in burn injuries18. Conclusions Intradermally applied SVF have 3. 4. 5. 6. 7. 8. 11, 12. 13. 14. 15. improved References 2. 10. 16. full thickness burn wound healing in rats when compared to occlusive bandages with 2% silver sulfadiazine through amplification of the initial inflammatory episode, have increased fibroblast migration and collagen production. The improved wound healing process observed in this study showed that therapeutic application of cells extracted from adipose tissue could have a significant role in burn wound care. 1. 9. Warner PM, Coffee TL, Yowler CJ. Outpatient burn management. Surg Clin North Am. 2014;94:879–92. doi: 10.1016/j. suc.2014.05.009. Peck MD. Epidemiology of burns throughout the world. Part I: Distribution and risk factors. Burns. 2011;37:1087–100. doi: 10.1016/j.burns.2011.06.005. Vale ECS. Inicial management of burns : approach by dermatologists. An Bras Dermatol. 2005;80:9–19. doi: 10.1590/ S0365-05962005000100003. Sheridan RL. Special problems in burns. Surg Clin North Am. 2014;94:781–91. doi: 10.1016/j.suc.2014.05.002. Wolf SE, Tompkins RG, Herndon DN. On the horizon: research priorities in burns for the next decade. Surg Clin North Am. 2014;94:917–30. doi: 10.1016/j.suc.2014.05.012. Tredget EE, Levi B, Donelan MB. Biology and principles of scar management and burn reconstruction. Surg Clin North Am. 2014;94:793–815. doi: 10.1016/j.suc.2014.05.005. You HJ, Han SK. Cell therapy for wound healing. J Korean Med Sci. 2014;29:311–9. doi: 10.3346/jkms.2014.29.3.311. Cherubino M, Rubin JP, Miljkovic N, Kelmendi-Doko A, Marra KG. Adipose-derived stem cells for wound healing applications. Ann Plast Surg. 2011;66:210–5. doi: 10.1097/SAP.0b013e3181e6d06c. 584 - Acta Cirúrgica Brasileira - Vol. 31 (9) 2016 17. 18. 19. 20. 21. 22. 23. 24. Gimble JM, Guilak F, Bunnell BA. Clinical and preclinical translation of cell-based therapies using adipose tissue-derived cells. Stem Cell Res Ther. 2010;1:19. doi: 10.1186/scrt19. Tabit CJ, Slack GC, Fan K, Wan DC, Bradley JP. Fat grafting versus adipose-derived stem cell therapy: distinguishing indications, techniques, and outcomes. Aesthetic Plast Surg. 2012;36:704–13. doi: 10.1007/s00266-011-9835-4. Gentile P, Orlandi A, Scioli MG, Di Pasquali C, Bocchini I, Cervelli V. Concise review: adipose-derived stromal vascular fraction cells and platelet-rich plasma: basic and clinical implications for tissue engineering therapies in regenerative surgery. Stem Cells Transl Med. 2012;1:230–6. doi: 10.5966/sctm.2011-0054. Kapur SK, Katz AJ. Review of the adipose derived stem cell secretome. Biochimie. 2013;95:2222–8. doi: 10.1016/j. biochi.2013.06.001. Lee SH, Lee JH, Cho KH. Effects of human adipose-derived stem cells on cutaneous wound healing in nude mice. Ann Dermatol. 2011;23:150–5. doi: 10.5021/ad.2011.23.2.150. Kokai LE, Marra K, Rubin JP. Adipose stem cells: biology and clinical applications for tissue repair and regeneration. Transl Res. 2014;163:399–408. doi: 10.1016/j.trsl.2013.11.009. Lim JS, Yoo G. Effects of adipose-derived stromal cells and of their extract on wound healing in a mouse model. J Korean Med Sci. 2010;25:746–51. doi: 10.3346/jkms.2010.25.5.746. Wang X, Liu C, Li S, Xu Y, Chen P, Liu Y, Ding Q, Wahafu W, Hong B, Yang M. Hypoxia precondition promotes adipose-derived mesenchymal stem cells based repair of diabetic erectile dysfunction via augmenting angiogenesis and neuroprotection. PLoS One. 2015;10:e0118951. doi: 10.1371/journal.pone.0118951. Uysal CA, Tobita M, Hyakusoku H, Mizuno H. The effect of bonemarrow-derived stem cells and adipose-derived stem cells on wound contraction and epithelization. Adv Wound Care. 2014;3:405–13. doi: 10.1089/wound.2014.0539. Atalay S, Coruh A, Deniz K. Stromal vascular fraction improves deep partial thickness burn wound healing. Burns. 2014;40:1375– 83. doi: 10.1016/j.burns.2014.01.023. Broughton G, Janis JE, Attinger CE. The basic science of wound healing. Plast Reconstr Surg. 2006;117:12S-34S. doi: 10.1097/01. prs.0000225430.42531.c2. Fraser JK, Hicok KC, Shanahan R, Zhu M, Miller S, Arm DM. The Celution® system: automated processing of adipose-derived regenerative cells in a functionally closed system. Adv Wound Care. 2014;3:38–45. doi: 10.1089/wound.2012.0408. Gentile P, De Angelis B, Pasin M, Cervelli G, Curcio CB, Floris M, Di Pasquali C, Bocchini I, Balzani A, Nicoli F, Insalaco C, Tati E, Luarini L, Palla L, Pascali M, De Logu P, Di Segni C, Bottini DJ, Cervelli V. Adipose-derived stromal vascular fraction cells and platelet-rich plasma: basic and clinical evaluation for cellbased therapies in patients with scars on the face. J Craniofac Surg. 2014;25:267–72. doi: 10.1097/01.scs.0000436746.21031.ba. Wada K, Arita M, Nakajima A, Katayama K, Kudo C, Kamisaki Y, Serhan CN. Leukotriene B4 and lipoxin A4 are regulatory signals for neural stem cell proliferation and differentiation. FASEB J. 2006;20:1785–92. doi:0.1096/fj.06-5809com. Kyritsis N, Kizil C, Zocher S, Kroehne V, Kaslin J, Freudenreich D, Iltzsche A, Brand M. Acute inflammation initiates the regenerative response in the adult zebrafish brain. Science. 2012;338:1353–6. doi: 10.1126/science.1228773. Gourevitch D, Kossenkov AV, Zhang Y, Clark L, Chang C, Showe LC,Heber-Katz E. Inflammation and its correlates in regenerative wound healing: an alternate perspective. Adv Wound Care. 2014;3:592–603. doi: 10.1089/wound.2014.0528. Adipose tissue stromal vascular fraction in the treatment of full thickness burns in rats 25. Bainbridge P. Wound healing and the role of fibroblasts. J Wound Care. 2013;22:407–8, 410–2. doi: 10.12968/jowc.2013.22.8.407. 26. Kim WS, Park BS, Sung JH. Protective role of adipose-derived stem cells and their soluble factors in photoaging. Arch Dermatol Res. 2009;301:329–36. doi: 10.1007/s00403-009-0951-9. 27. Harasymiak-Krzyżanowska I, Niedojadło A, Karwat J, Kotuła L, Gil-Kulik P, Sawiuk M,Kocki J. Adipose tissue-derived stem cells show considerable promise for regenerative medicine applications. Cell Mol Biol Lett. 2013;18:479–93. doi: 10.2478/s11658-0130101-4. 28. Kim WS, Park BS, Sung JH, Yang JM, Park SB, Kwak SJ,Park JS. Wound healing effect of adipose-derived stem cells: a critical role of secretory factors on human dermal fibroblasts. J Dermatol Sci. 2007;48:15–24. doi: 10.1016/j.jdermsci.2007.05.018. Acknowledgement To Vânia Beatriz Lopes for help in technical analysis. Correspondence: AlexandreLamaro Cardoso Faculdade de Medicina - UFG Rua 235, 1a s/n, Setor Universitário Goiânia-GO Brasil Tel/Fax: (55 62)3209-6165 alexandrelamaro@hotmail.com Received: May 21, 2016 Review: July 19, 2016 Accepted: Aug 18, 2016 Conflict of interest: none Financial source: FAPEG (grant number 2013/0267001091) Research performed at Laboratory of Experimental Pathology, Institute of Tropical Pathology and Public Health, Universidade Federal de Goiás (UFG), Goiania-GO, Brazil. 1 Acta Cirúrgica Brasileira - Vol. 31 (9) 2016 - 585 

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Bangladesh Journal of Medical Science Vol. 20 No. 02 April’21 Original article: Impact of Socio-demographic factors on Quality of life in Medical Students of Eastern Saudi Arabia Mohammad Ayoob1, Abdulrehman Alsultan2, Nurjahan Begam3, Saleh Al Sumaih4, Hamad Waleed Albuali5 Abstract Background: Quality of life of medical students may influenced by different socio-demographic components and disrupt their capability to study medicine. An in-depth exploration of these elements may encourage enhancement in learning and retention of medical students. Aim: This study was designed to identify the role of socio-demographic factors in quality of life in undergraduate medical students. Material and Method: A cross-sectional study was conducted among 223 undergraduate medical students (male = 125, females = 98) at King Faisal University, Saudi Arabia between February and September 2019. An Arabic version of WHOQOL-BREF was used to explore the QOL of medical students. Regression analysis were employed to evaluate association between QOL and socio-demographic factors including age, sex, academic year, family type, area of residence, monthly income, parental education and housing status. Result: The results showed a significant relationship between various socio-demographic variables and quality of life. The findings of the study revealed that gender, academic year, marital status, monthly income and family type were found significant predictors of quality of life among medical students. Conclusion: The present study has explored the impact of socio-demographic factors on the quality of life of medical students. Keywords: socio-demographic; quality of life; medical students; Saudi Arabia Bangladesh Journal of Medical Science Vol. 20 No. 02 April’21. Page : 250-257 DOI: https://doi.org/10.3329/bjms.v20i2.51531 Introduction The World Health Organization (WHO) defined Quality of Life (QoL) as “an individual’s perception of their position in life, in the context of the culture and value systems in which they live, and in relation to their goals, expectations, standards and concerns”1 . Quality of life focused all aspects of life, including physical health, family, security, status, education, beliefs, and the environment. Quality of life is influenced by many factors such as, biological, behavioral, psychological and demographical2. However, the link between these elements and QOL are varying among different communities and population3. Generally, medical students experience more stress than general population.4-6 The causes of their stress could be due to the academic burden, less leisure time, more material to be retained, high parental expectation, peer pressure, and repeated formative and summative examinations.7 Previous research reported that poor quality of life among medical students is linked with poor attention and concentration, increased incidence of errors, negligence, absenteeism, self-medication, and cheating during examinations.8 1. Mohammad Ayoob, Assistant Professor, Department of Clinical Neurosciences, College of Medicine, King Faisal University, Saudi Arabia, Email: ayooblone08@gmail.com; 2. Abdulrehman Alsultan, Assistant Professor, Department of Biomedical Science, College of Medicine, King Faisal University, Saudi Arabia. 3. Nurjahan Begam, Assistant Professor, Department of Clinical Neurosciences, College of Medicine, King Faisal University, Saudi Arabia. 4. Saleh Al Sumaih, Medical Student, College of Medicine, King Faisal University, Saudi Arabia. 5. Hamad Waleed Albuali, Medical Student, College of Medicine, King Faisal University, Saudi Arabia. Correspondence to: Mohammad Ayoob, Assistant Professor, Department of Clinical Neurosciences, College of Medicine, King Faisal University, Saudi Arabia, Email: ayooblone08@gmail.com; 250 Impact of Socio-demographic factors on Quality of life in Medical Students of Eastern Saudi Arabia Several studies have examined quality of life among medical students. For instance, in a cross-sectional study conducted among Saudi medical students, the students experienced environmental domain as their highest QOL, whereas physical domain and social relations domain were experiences as the lowest.9 Mahmoud and Fareed10 reported that final year medical students score higher in environmental domain than students in initial years. Another study reported that first year and final year medical students experienced better quality of life in terms of psychological and social domain.11 There is tremendous research works on the quality of life among medical students. However, few studies have examined the impact of demographic factors on quality of life among medical students12. There is presently dearth of psychological research on impact of different demographic factors on quality of life among medical students especially in Saudi Arabia. Therefore, the current study aimed to assess the effect of various socio-demographic factors on the quality of life of medical students, with the intention of improving learning and retention at medical college. Materials and Methods Setting and participants The present study was conducted in college of medicine, King Faisal University, Saudi Arabia. King Faisal University is one of the leading University in the Kingdom of Saudi Arabia with more than 42227 students. Among the total student body, 1070 students are enrolled at the college of medicine, with nearly 300 new medical students enrolling each semester. The bachelor of medicine and surgery in Saudi Arabia is six-year programme in which first three years are preclinical, and years 4–6 are clinical. College of Medicine utilizes an innovative teaching methodology to achieve their vision and mission of preparing future physicians. Participants of the present study were 223 medical students (Male =125, Females = 98) studying in college of medicine, King Faisal University, Saudi Arabia. Ages of these participants were ranging from18 to 25 years. Study Design A cross sectional study was performed among undergraduate medical students attending College of Medicine, King Faisal University between February and September 2019. Medical students in their Ist to 5th year in College of Medicine voluntarily participated in the study. In the beginning of the third semester of the academic year, students were asked to complete a self-reported questionnaire on demographic factors and quality of life. Measures In order to achieve the goals of the present study, the different measures were used. Quality of life was assessed by World Health Organization Quality of Life-Biomedical Research and Education Facility (WHOQOL-BREF). Demographic questionnaire prepared by the researcher was also include in the study. Demographic Questionnaire: The information about demographic profile of the participants was collected with the help of questions related to their age, sex, marital status, and academic year. In addition, information about their family were include area of residence, family type, education level of parents, family occupation, income, housing status etc. World Health Organization Quality of Life Assessment –Abbreviated Version (WHOQOL-BREF): Quality of life was measured by using WHOQOL-BREF.13 The scale comprises of twenty six items, covering four domains: physical health, psychological health, social relationships, and environment. The first two questions separately assess the overall perception of quality of life and general health. Participants were asked to rate their responses to each item using a 5-point Likert scale ranging from 1 (very dissatisfied/ very poor) to 5 (very satisfied/ very good). The total score is derived by adding the scores of the items belonging to different areas and mean scores are obtained. In this scale higher scores indicate better QOL14. Mahmoud and Fareed10 have reported internal reliability for the total scale as 0.83. Internal consistency reliability (Cronbach’s alpha) of this measure was found as 0.92. Procedure After seeking required permission from concerned college authorities, the participants were personally contacted. They were briefed about the purpose of research and questionnaire used in the study. After seeking consent of the student, a suitable time and date was fixed for data collection. Before administering the questionnaire, the purpose of the study was again explained to the participants and they were assured that their responses would be kept confidential and would be used for research and academic purpose only. A good rapport was built with the participants for getting correct responses. Necessary instruction and guidelines were provided to them for properly filling the questionnaire. After this, the questionnaires 251 Mohammad Ayoob, Abdulrehman Alsultan, Nurjahan Begam, Saleh Al Sumaih, Hamad Waleed Albuali were provided to them and they were requested to fill up the questionnaire as per the instructions given in the questionnaire. It took 20 minutes to complete the questionnaire. After completion of the questionnaire participants returned the questionnaire and they were thanked for their participation and cooperation. and socio-demographic factors, multiple regression models were performed. For this, separate analysis was run for each aspect of WHOQOL-BREF. Statistical Analysis Result The data were first exported to Microsoft Excel and then to Statistical Package for Social Sciences (SPSS version, 22) computer software used for quantitative statistical analyses. Prior to analysis, all variables were screened for possible code. In order to obtain an understanding of the socio-demographic characteristics of the participants, frequency, percentage, means, standard deviations and other descriptive statistics were conducted. To investigate a relationship between WHOQOL-BREF scores Ethical clearance: The ethical committee of human research at the College of Medicine approved the study protocol. Socio-demographic population characteristics of study The present study invited 250 students’ studying in colleges of medicine at King Faisal University. A total of 223 students answered the questionnaire. The response rate was 89.20%. Among the 223 students, 125 were males and 98 were females. The mean age of these students was 21.36 years. Table 1 shows that majority (83.4%) of participant were single. Only 30 (13.5%) participants were married. Most of students Table 1: Quality of life scores (Mean and Standard Deviation) according to Demographic characteristics N (%) (n=223) Physical Health (Mean and SD) Psychological Health (Mean and SD) Social Relationships (Mean and SD) Environment (Mean and SD) Male 125(57.0) 64.09±16.52* Female 98(43.0) 54.89 ± 15.74 29.31 ± 22.69 48.47 ± 16.33 63.47 ± 18.22 34.17 ± 17.27 52.38 ± 22.90* 56.36 ± 17.55 Gender Academic Year Ist 67(30.0) 63.59 ± 18.52 49.17 ± 21.82** 50.70 ± 16.47 56.11 ± 12.98 2nd 40(17.9) 59.45 ± 12.04 34.70 ± 17.72 41.95 ± 12.52 52.57 ± 16.11 3rd 41(18.4) 55.46 ± 13.79 19.75 ± 11.81 39.14 ± 18.80 51.53 ± 21.91 4th 50(22.4) 60.24 ± 14.25 17.16 ± 11.00 54.88 ± 21.33 65.36 ± 16.89 5th 25(11.2) 74.68± 14.65** 27.32 ± 7.35 75.00 ± 17.26** 75.44 ± 19.16** Marital Status Married 30(13.5) 64.93 ± 19.58** 32.37 ± 16.79 71.43 ± 19.18** 71.50 ± 13.27** Engaged 7(3.1) 61.57± 13.16 23.14 ± 7.88 60.17 ±21.76 69.86 ±14.54 Single 186(83.4) 53.33 ± 16.16 28.78 ± 22.23 48.22 ± 23.45 57.84 ± 19.07 Nuclear 90(40.4) 59.47 ±17.99* 29.44 ± 17.87 56.90 ± 24.26* 64.11 ±20.44* Joint 133(59.6) 51.71 ± 15.33 28.80 ± 23.37 46.26 ± 22.36 57.31 ± 17.31 <10000SAR 76(34.1) 49.03 ± 16.55 28.92 ± 22.01 44.76 ± 22.45 55.09 ± 19.99 10001-25000 107(48.0) 57.82 ± 6.76 28.64 ± 21.10 53.45 ± 24.04 61.83 ± 17.74 25000> 40(17.9) 57.90 ± 5.06** 30.60 ± 20.81*** 53.80 ± 23.51*** 64.72 ± 18.11 Own 198(88.8) 54.97 ± 17.09 29.12 ± 21.07 50.88 ± 24.12 60.11 ±18.77 Rented 25(11.2) 53.76 ± 15.11 28.84 ± 23.30 47.96 ± 20.05 Family Type Monthly Income Housing Status *p=.01 (t-test), **p=.01 (One-way ANOVA), ***p=.05 (One-way ANOVA), 252 59.64 ± 20.1 Impact of Socio-demographic factors on Quality of life in Medical Students of Eastern Saudi Arabia (30.0%) were from Ist year and 25(11.2%) were from 5th year. The percentages of participants belonging to nuclear and joint families were 40.4 and 59.6 respectively. With regard to different domains of quality of life presented in Table 1 showed that male participants scored higher in physical health domain of QoL than female participants, whereas, female participants scored better than male participants in social domain of QoL. In terms of academic year, fifth year student’s scored higher in physical, social and environmental domain of QoL, while as first year students score better in psychological domain of QoL. For marital status, married participants scored higher in physical, social and environmental domain of QoL. Participants belong to nuclear families showed higher score in physical and environmental domain of QoL. In terms of monthly income, participants having more income perceive higher score in physical, psychological and social domain of QoL. Quality of life according to socio-demographic factors Separate multiple linear regression analyses were performed to identify predictors variables for the physical, psychological, social and environmental quality of life, as measured by the WHOQOL-BREF. Result of multiple regression analysis presented in Table 2 revealed that five variables, gender (β= -.20), academic year (β= .25), marital status (β= -.12) monthly income (β= .11) and family type (β= -.15) are the strongest predictors in the physical domain of QoL. The obtained results clearly revealed that gender was negatively and significantly related to physical domain. This means that male participants showed good physical quality of life than female participants. Result also revealed that marital status was found negatively and significantly related to physical domain of quality of life. This indicates that married students showed good physical quality of life than unmarried students. Similarly, family type was negatively and significantly related to physical domain. This means that students belong to nuclear families showed good physical quality of life compared to students living in joint families. However, results clearly revealed that academic years were found positively and significantly related to physical component of quality of life. This indicates that students who are final stage of their degree showed good physical quality of life than students of initial years of their course. Also, study revealed that monthly income was found positively and significantly related to physical domain of quality of life. This means that students having high monthly income experience better physical quality of life than students having low monthly income. For the psychological domain, academic year (β= -.42) and monthly income (β= .13) were found strongest predictors. Results clearly revealed that academic years were found negatively and significantly related to psychological aspect of quality of life. This indicates that students who are initial stage of their degree showed good psychological quality of life Table 2: Association between Quality of life and Predictor variables Variables Physical QOL Psychological QOL β p β p Environment QOL Social QOL β p β p Gender -.20 .00 .01 .90 .16 .02 -.10 .14 Age .20 .17 .05 .77 -.14 .38 .13 .38 Academic Year .25 .05 .01 .54 .00 .35 .02 .01 -.42 Marital Status -.12 .05 -.11 .12 -.03 .67 -.18 Area of residence -.01 .85 -.04 .49 -.02 .80 .00 .96 Monthly income .11 .05 .13 .04 .12 .05 .10 .10 Family occupation -.05 .42 -.06 .32 -.03 .57 -.05 .38 Fathers education .06 .39 -.05 .44 -.07 .31 .02 .80 Mothers education -.01 .89 -.02 .74 -.03 .62 -.02 .81 House status -.05 .38 .04 .54 -.03 .66 -.03 .53 Family type -.15 .01 -.03 .63 -.14 .03 -.12 R2 38% 31% 26% .02 36% 253 Mohammad Ayoob, Abdulrehman Alsultan, Nurjahan Begam, Saleh Al Sumaih, Hamad Waleed Albuali than students of final years of their course. However, monthly income was positively and significantly related to psychological quality of life. This means that students having high monthly income experience better psychological quality of life than students having low monthly income. In regard to the social quality of life of participants results of multiple regression presented in Table 2 revealed that gender (β= .16), academic year (β= .54), monthly income (β= .12), and family type (β= -.14) were found significant predictors of social quality of life. The obtained results clearly revealed that gender was positively and significantly related to social domain of quality of life. This means that female participants showed good social quality of life than male participants. Similarly, academic years was positively and significantly related to social domain. This indicates that students were in final phase of their course showed good social quality of life as compared to students who are in initial years of their courses. Also, monthly income was positively and significantly related to social quality of life. This shows that students having high monthly income experience better social quality of life than students having low monthly income. However, family status was negatively and significantly related to social domain. This means that students belong to nuclear families showed good social quality of life compared to students living in joint families. Result of regression analysis predicting environmental domain from different demographic factors presented in Table 2 revealed academic year (β= .35) and marital status (β= -.18) as significant predictors of environmental quality of life. These results clearly indicate that academic years were positively and significantly related to environmental domain. This means that students were in final phase of their course showed good environmental quality of life as compared to students who are in initial years of their courses. However, result revealed that marital status was found negatively and significantly related to environmental component of quality of life. This indicates that married students showed good environmental quality of life than unmarried students. Discussion The present study was conducted to examine the role of demographic factors that potentially influence 254 the quality of life among undergraduate medical students attending college of medicine, King Faisal University, Saudi Arabia. It was hypothesized that demographic factors will significantly predict quality of life of medical students. The results showed a significant relationship between various socio-demographic variables and quality of life. The findings of the study revealed that gender, academic year, marital status, monthly income and family type were found significant predictors of quality of life among medical students. Gender effects Findings of the present study regarding gender differences in quality of life showed significant differences between male and female participants on the measure of WHOQOL-BREF. In the domain of physical health, male participant reported better quality of life in comparison to their counterpart female participants. These results regarding gender differences in quality of life of the participants extended support to the previous studies showing differences between males and females in quality of life.15-19 Better quality of life reported by the male participants in the present study may be because of a tendency to perceive their personal health as excellent and that they always feel energetic, as compared with females. This is in line with the cultural norms of the Saudi Arabia where men are generally considered to be the stronger and dominant sex, thus men perceive themselves to be so. These factors may be more responsible for better quality of life among male medical students. However, these issues could not appropriately be explained by our study and requires further investigation. In the present study, female students scored higher than males in social domain of quality of life, studies shown that females are better than men in dealing with interactions.20 Moreover, female medical students had handful of experiences and maturity to manage their social relations and the environment in comparison to male students. Academic years Regarding effect of academic years on quality of life among medical students, the present study observed significant relationship of academic years to different domains of quality of life. In the present study, it was observed that fifth year student’s scored higher in physical, social and environmental domain of QoL. A similar kind of research among Chinese medical Impact of Socio-demographic factors on Quality of life in Medical Students of Eastern Saudi Arabia students reported higher scores in psychological domain and social domain of QoL in clinical years as compared to third year students.21 Another study conducted on Brazilian medical students indicated significant differences in the quality of life according to academic year, particularly with respect to the psychological and physical domains.22 The reasons for better quality of life during later years in medical course are that the students had less theoretical content, more experience and maturity to improve their quality of life in comparison to students of preclinical years. Marital Status Several researches have suggested the effect of marital status on the quality of life in university students.23 In the present study, the married students have better physical and environmental domain of quality of life as compare to the unmarried student. The results are in line with previous studies examined marital status and quality of life.24 Previous findings reported that married people have strong mental health than those who are single due to the social support received from their spouse.25 Another research examined the relationship between marital status and mortality. The results indicated that people who were single showed higher mortality in particular diseases compared with those who were married.26 In the present study, good quality of life among married students may be because of care and support provided by their spouses especially during the tough times of their study. Monthly income To the best of our knowledge, this is the first study to examine the relationship between socio-economic status and quality of life among undergraduate medical students. The findings of the present study indicates that students with high monthly income showed better physical, psychological and social quality of life as compared to the students with low monthly income. These results regarding relationship between monthly income and quality of life of the participants received extended support from previous studies.27 Previous findings suggests that low income badly affects self-esteem, blocked aspirations, increase frustrations, reduce efficacy, fatalism and lower mastery and personal control.28 Family type Regarding the role of family type on quality of life among medical students, the present study indicated, the quality of life is better among the students belongs to nuclear families as compared to joint family subjects. This result is consistent with the findings from a study conducted in India, which suggested the quality of life is higher in nuclear family participants.29 A nuclear family is a very small family having only a few members, less engagements, more freedom, more attention to children and less responsibilities. However, nuclear family faces lot of problems of lower tolerance among individuals, lower maturity, more dependency, adjustment issues and egoism. Other studies also indicates that living in nuclear family unit is related to better health among males as compared to females, than other family structures.30 Several limitations of the present study must be noted. First, the data of the present study were collected from only one medical college from eastern region of Saudi Arabia. Data gathered in this context may therefore be unique, and it is entirely possible that a replication of this study in a different parts of the country might yield different results. Second, the convenience sampling method of medical students in eastern region is not likely to be representative of all medical students studying in other regions of the Saudi Arabia. Therefore, further study needs representative samples in order to establish the generalizability of findings on medical students studying in other parts of the country. Third, the cross-sectional design used in the present study does not allow drawing conclusions regarding causality. Longitudinal research will be needed to support such conclusions. Lastly, sample size of the present study was relatively small and homogeneous which also limits generalization. The present study demonstrated the differences in scores of different domains for the quality of life related to various demographic factors. Gender, academic year, marital status, monthly income and family type are found strongest predictors of quality of life. Betterment in the quality of life of medical students is very important to ensure the good medical education system. It is also important to maintain a well-balanced academic environment for improved learning experience. A focus on student needs and problems can help prevent the harmful effects of stress on quality of life. In addition, It is also suggested that all required facilities should be provided to 255 Mohammad Ayoob, Abdulrehman Alsultan, Nurjahan Begam, Saleh Al Sumaih, Hamad Waleed Albuali the medical students and programmes like career counseling and stress management training should be executed for the students to reduce their mental stress and improve quality of life during medical education. Conflict of Interest Statement There are no conflicts of interest or any financial or personal relationships with other people or organizations. Role of funding sources and author’s contribution: The funding sources did not play a role in the study conceptualization or design; collection, analysis, or interpretation of data; writing of the manuscript; or the decision to submit the article for publication. Acknowledgment I would like to thank everybody who was important to the successful realization of this study, especially the participants who took this work seriously enough to complete the study and pass the message on. Also, our special thanks to the college administration for allowing us to conduct this study. References: 1. Group WH. Development of the WHOQOL: rationale and current status. International Journal of Mental Health. 1994; 23: 24-56 https://doi.org/10.1080/00207411.1994.11449286 2. Pagnin D, De Queiroz V. Comparison of quality of life between medical students and young general populations. Education for Health 2015;28:209-12. https://doi.org/10.4103/1357-6283.178599 3. Bowling A. Concepts of functioning, health, well-being and quality of life. In: Measuring health 2005; 3rd edition. McGraw-Hill, New York 4. 256 Dyrbye LN, Thomas MR, Shanafelt TD. Systematic review of depression, anxiety, and other indicators of psychological distress among US and Canadian medical students. Academic Medicine 2006; 81(4):354-373 https://doi.org/10.1097/00001888-200604000-00009 5. Henning MA, Krägeloh CU, Hawken SJ, Zhao Y, Doherty, I. The quality of life of medical students studying in New Zealand: a comparison with nonmedical students and a general population reference group. Teaching and Learning in Medicine 2012; 24 (4): 334-340 https://doi.org/10.1080/10401334.2012.715261 6. Dyrbye LN, West CP, Satele D, Boone S, Tan L, Sloan J, Shanafelt TD. Burnout among US medical students, residents, and early career physicians relative to the general US population. Academic Medicine 2014; 89 (3): 443-451 Impact of Socio-demographic factors on Quality of life in Medical Students of Eastern Saudi Arabia https://doi.org/10.1097/ACM.0000000000000134 7. Gupta S, Choudhury S, Das M, Mondol A, Pradhan R. Factors causing stress among students of a medical college in Kolkata, India. Education for Health (Abingdon) 2015; 28: 92-95 https://doi.org/10.4103/1357-6283.161924 8. O’Rourke M, Hammond S, O’Flynn S, Boylan G. The medical student stress profile: A tool for stress audit in medical training. Medical Education 2010; 44: 1027-37. https://doi.org/10.1111/j.1365-2923.2010.03734.x 9. Malibary H, Zagzoog MM, Banjari MA, Bamashmous RO, Omer AR. Quality of Life (QoL) among medical students in Saudi Arabia: a study using the WHOQOLBREF instrument. BMC Medical Education 2019; 19(1): 344. doi: 10.1186/s12909-019-1775-8. https://doi.org/10.1186/s12909-019-1775-8 10. Mahmoud A, Fareed, M. Assessment of Quality of Life among Medical Students in Saudi Arabia: A Study Based on WHO-QOL-BREF Protocol. International Journal of Medical Research & Health Sciences 2018; 7 (10): 1-11 11. Zhang Y1, Qu B, Lun S, Wang D, Guo Y, Liu J. Quality of life of medical students in China: a study using the WHOQOL-BREF. PLoS One 2012; 7(11): e49714. doi: 10.1371/journal.pone.0049714 https://doi.org/10.1371/journal.pone.0049714 12. Serinolli MI, Novaretti MC. A cross-sectional study of sociodemographic factors and their influence on quality of life in medical students at Sao Paulo, Brazil. PLOS One. 2017; 12(7): e0180009. https://doi.org/10.1371/journal.pone.0180009 13. The World Health Organization Quality of Life assessment (WHOQOL). Position paper from the World Health Organization. Social Science Medicinal Journal 1995; 41: 1403-09. https://doi.org/10.1016/0277-9536(95)00112-K 14. WHOQOL Group. Development of the World Health Organization WHOQOL-BREF quality of life assessment. Psychological Medicine 1998; 28: 551-558. https://doi.org/10.1017/S0033291798006667 15. Pagnin D, Queiroz V. Influence of burnout and sleep difficulties on the quality of life among medical students. Springer Plus 2015; 4: 676. https://doi.org/10.1186/s40064-015-1477-6 16. Shareef MA, AlAmodi AA, Al-Khateeb AA, Abudan Z, Alkhani MA, Zebian SI, Qannita AS, Tabrizi MJ. The interplay between academic performance and quality of life among preclinical students. BMC Medical Education 2015; 15:193. https://doi.org/10.1186/s12909-015-0476-1 17. Naseem S, Orooj F, Ghazanfar H, Ghazanfar A. Quality of life of Pakistani medical students studying in a private institution. Journal of Pakistan Medical Association 2016; 66: 579-83. 18. Chazan AC, Campos MR, Portugal FB. Quality of life of medical students at the State University of Rio de Janeiro (UERJ), measured using WHOQOLBREF: a multivariate analysis. Cien Saude Colet. 2015; 20: 547-56. https://doi.org/10.1590/1413-81232015202.05182014 19. Paro HB, Morales NM, Silva CH, Rezende CH, Pinto RM, Morales RR, Mendonça TM, Prado MM. Health-related quality of life of medical students. Medical Education 2010; 44(3): 227-235. https://doi.org/10.1111/j.1365-2923.2009.03587.x 20. Palchykov V, Kaski K, Kertesz J, Barabasi AL, Dunbar RI. Sex differences in intimate relationships. Scientific Report 2012; 2:370. https://doi.org/10.1038/srep00370 21. Zhang Y, Qu B, Lun S, Wang D, Guo Y, Liu J. Quality of life of medical students in China: a study using the WHOQOL-BREF. PLoS One. 2012; 7:11: e49714. https://doi.org/10.1371/journal.pone.0049714 22. Paro CA, de C Bittencourt ZL. Qualidade de vida de graduandos da aÂrea da sauÂde. Brazilian Journal of Medical Education 2013; 37: 365-375. https://doi.org/10.1590/S0100-55022013000300009 23. Heidari M, Reza M, Pasalar P, Nedjat S. Quality of life of medical students in Tehran University of Medical Sciences. Acta Medical Iranica. 2014; 52(5):390-399. 24. Bierman A. Marital status as contingency for the effects of neighborhood disorder on older adults’ mental health. Journal of Gerontology 2009; 64: 425-434. https://doi.org/10.1093/geronb/gbp010 25. Bierman A. A multifaceted approach to the mental health advantage of the married assessing how explanations vary by outcome measure and unmarried group. Journal of Family Issues. 2006; 27: 554-582. https://doi.org/10.1177/0192513X05284111 26. Hu Y. Mortality differentials by marital status: an international comparison. Demography 1990; 27: 233-250. https://doi.org/10.2307/2061451 27. Fan AP, Chen CH, Su TP, Shih WJ, Lee CH, Hou SM. The association between parental socioeconomic status (ses) and medical students’ personal and professional development. Annals of Academy of Medicine Singapore 2007; 36: 735-42 28. Eaton WW. The Sociology of Mental Disorders. New York: Praeger, 1980. 29. Kumar, M, Tiwari, P. Structural Influence of Family on Religious Orientation, Quality of Life and Happiness. International Journal of Applied Psychology 2016; 6 (4): 94-99 30. Denton M, Prus S, Walters V. Gender differences in health: A Canadian study of the psychosocial, structural, and behavioral determinants of health. Social Science & Medicine 2004; 58; 2585-2600. https://doi.org/10.1016/j.socscimed.2003.09.008 257 

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150 POLÍTICAS PÚBLICAS E DIREITO À SAÚDE: A TENSÃO ENTRE OS PODERES – A NECESSIDADE DE ESTABELECER CRITÉRIOS JUDICIAIS PARA INTERVENÇÃO Leonardo Grison1 Têmis Limberger2 RESUMO O direito a saúde é um direito social e deve ser implementado por meio de políticas públicas. A experiência de Estado Social de outros países é diferente do Brasil. As políticas públicas são mandatos formulados pelo legislador e realizados pelo administrador. A discricionariedade administrativa encontra limites constitucionais. Nos caso de ineficiência ou omissão na execução das políticas públicas, cabe a intervenção judicial. As ações coletivas são o foro mais adequado para esta discussão. As ações individuais são um mecanismo de pressão. A cultura democrática implica no reconhecimento dos limites e possibilidades do controle judicial para efetividade do direito á saúde. Palavras­Chave: Controle Judicial. Direito à saúde. Estado Social. Políticas públicas. Resposta adequada. INTRODUÇÃO Os direitos não são um dado, mas um construído. Pode­se formular esta idéia a partir do pensamento de Hannah Arendt, que concebe os direitos humanos a partir da construção da igualdade e a cidadania como o direito a ter direitos. O primeiro direito humano é o direito da ter direitos. Isto significa pertencer pelo vínculo da cidadania, a algum tipo de comunidade juridicamente organizada e viver numa estrutura onde vige o princípio da legalidade, evitando­se o surgimento de um novo estado totalitário de natureza3. Dentro desta perspectiva, quando se comemoram 20 anos da promulgação da Constituição Cidadã, a questão principal não é mais a declaração de novos direitos, mas a busca pela efetividade dos mesmos. Aí se insere o direito à saúde, que por ser um direito social, requer a disponibilidade orçamentária para sua implementação. O direito à saúde teve a sua trajetória de construção nestas duas décadas, em que pese muito ainda ter de ser feito. No Brasil, devido à falta de planejamento, são despendidas elevadas quantias quando se trata da implementação dos direitos sociais. Ao invés de haver uma atitude de prevenção, gasta­se muito mais na reparação. Pode­se pensar na questão da educação, moradia, segurança, transporte, trabalho, previdência social, lazer, saúde, etc. Todos estes direitos sociais previstos no art. 6º, “caput”, da CF se prestam para investigação, mas devido a um corte metodológico necessário, é imprescindível delimitar a questão. Dentro desta perspectivas, os dois direitos sociais que têm por parte dos poderes públicos as chamadas “verbas carimbadas”, no sentido de que o 151 percentual de recursos já se encontra previamente definido são: educação (art. 212, da CF) e saúde (art. 198, § 2º, da CF), daí se constata a importância de investimento para estes setores tão prioritários para que o Brasil seja no futuro, um país sem tantas desigualdades sociais. A relação que se pode estabelecer entre estes dois direitos sociais: educação e saúde, é no sentido de que quanto maior o nível de instrução mais saudável é a população, via de regra. Estudos4 demonstram que a educação é uma ferramenta importante, quando se está a mencionar algumas problemáticas, tais como: mortalidade infantil, gravidez precoce e número de filhos por núcleo familiar. O trabalho optou por abordar o direito à saúde, especialmente nestes últimos anos da recente história constitucional democrática brasileira. O desafio que se impõe é como fazer com que o direito à saúde não seja um comando meramente programático e se torne efetivo. Diante desse quadro, impõe­se a seguinte indagação: quais as possibilidades e limites do controle judicial, atinente às políticas públicas que visem à implementação do direito à saúde? Para responder a esta questão, algumas tomadas de posição são necessárias, tais como: por que vigora(ou) no direito administrativo brasileiro o dogma da impossibilidade de revisão dos atos administrativos, que se albergavam sob o manto da discricionariedade administrativa; qual o conceito de política pública; quais os critérios para se estabelecer a intervenção do poder judiciário nesta seara. O trabalho se propõe a uma abordagem das políticas públicas como determinações constitucionais, ou seja, mandatos a serem realizados pelo administrador, sujeitos, portanto, ao controle judicial. Assim, tarefas que são previstas na Constituição e que devem ser realizadas de acordo com a capacidade orçamentária, mas que não se constituem em uma discricionariedade administrativa, não ficando, portanto, imunes à questionamento judicial. Pretende­se, então, revisar o conceito de discricionariedade da administração que foi transposto equivocamente do direito francês ao direito brasileiro e também, a forma de realização dos direitos sociais, aonde a doutrina alemã, não se adapta totalmente à realidade brasileira. Por isso, a necessidade de desenvolver uma teoria, voltada às especificidades de nosso país, desde a ótica de uma hermenêutica comprometida com uma nova crítica do direito e, ao final, a busca de uma solução adequada constitucionalmente. O direito administrativo é o direito constitucional concretizado, em uma síntese, formulada pela doutrina alemã5. Assim, o administrador na realização das políticas públicas deve observar os preceitos constitucionais. O mandato político não deve ser desempenhado conforme critérios subjetivos do governante, mas representam políticas públicas a serem desenvolvida em conformidade com os ditames constitucionais. Direitos sociais importantes foram consagrados no art. 6º da CF, visando a execução pelo administrador. Porém, estes preceitos não têm sido cumpridos, opera­se uma situação de crise, pois o Estado brasileiro não foi capaz de atender às demandas do liberalismo clássico, com os direitos de cunho individual e tampouco conseguiu realizar as demandas do Estado Social. O Estado imiscuiu­se em atividades que não eram 152 próprias do poder público e deixou de realizar atividades que eram prioritárias. Atualmente, não se afigura um projeto político claro com realização às demandas prestacionais sociais. Como conseqüência, os serviços públicos não são prestados ou o são de forma deficiente. Assim, no Brasil os direitos sociais para que sejam efetivos demandam um caminho diferente dos países europeus, onde estes são uma determinação da Constituição ao legislador ordinário ou ao administrador. Em nosso país, diante da particularidade própria em que estes comandos não são atendidos por seus destinatários, é importante a intervenção do Poder Judiciário. O desafio ocorre, no sentido de verificar os limites em que ocorre a atuação do Poder Judiciário, que não pode se substituir em atividades destes outros dois poderes, mas que tem de propiciar a efetividade dos direitos fundamentais sociais, quando decorrentes de políticas públicas insuficientes ou inexistentes. A realização dos direitos sociais é de suma importância, principalmente em países de modernidade tardia6 como o Brasil. Na época em que foi realizada a Constituinte, vivia­se uma euforia como se a partir da colocação das garantias na Constituição fosse haver uma transformação em nossa realidade. Pairava na sociedade a idéia de pensamento mágico, como se da previsão na Constituição fosse advir uma modificação na situação fática. É claro que a garantia representa um compromisso importante em termos de desenvolvimento de políticas públicas, isto é, que país queremos ser no terceiro milênio. Tiveram que se passar quase 20 anos para que se começasse a amadurecer no sentido de que os direitos sociais fossem relacionados com os dispositivos orçamentários. É o que Canotilho denomina de “Constituição Orçamental”7. As medidas de gestão orçamentária são importantes quando se pretende a realização dos serviços públicos. Questões vitais como saúde, educação, segurança e moradia, reclamam para sua implementação dispêndios por parte do poder público, que precisa contar com disposições orçamentárias. Assim, o administrador quando concretiza uma política pública encontra no orçamento o limite objetivo da reserva do possível. Da mesma forma, diante da omissão ou precariedade da implementação da política pública, o Poder Judiciário fica também adstrito ao orçamento, mas pode pronunciar­se quando provocado, para efetivar os direitos sociais. Durante muitos anos havia a errônea concepção de discricionariedade administrativa que servia para agasalhar todos os desmandos ou ineficácia do executivo. Sob o manto do ato discricionário não poderia o judiciário se imiscuir na atividade da administração. As ações judiciais interpostas e algumas decisões judiciais indicam que houve uma mudança de posição, é claro que isto não significa suprimir ou substituir a atividade administrativa, mas buscar a efetividade dos direitos sociais. Da análise das decisões jurisprudenciais proferidas pelo Supremo Tribunal Federal (preponderantemente) em matéria de políticas públicas, especialmente as que visam à implementação do direito social à saúde é que se terá um perfil dos limites e 153 critérios de atuação do Poder Judiciário, tomando­se como referência a promulgação da atual Constituição Federal. Deste modo, o trabalho a ser desenvolvido, utilizará o pensamento de Hannah Arendt, partindo­se da idéia de que os direitos não são um dado, mas um construído, neste aspecto o direito à saúde representa um marco importante de desenvolvimento, no sentido de que sua evolução vem sendo construída pelos operadores do direito, de modo a promover condição de dignidade para o cidadão. Com estes balizadores será possível travar uma discussão da eficácia e limites de implementação das políticas públicas de concretização dos direitos sociais, quando são submetidas ao crivo do poder judiciário, especialmente no que diz respeito ao direito à saúde, não no sentido de sentenças formuladas com critérios eminentemente subjetivos, mas a decisão adequada8, desde o ponto de vista constitucional. 1 O DOGMA DA IMPOSSIBILIDADE DE REVISÃO JUDICIAL DOS ATOS DA ADMINISTRAÇÃO, SOB O MANTO DA DISCRICIONARIEDADE ADMININISTRATIVA Durante muitos anos no direito brasileiro vigorava o entendimento da impossibilidade de revisão judicial da atividade administrativa. O controle judicial dos atos da administração não era realizado, fundado na justificativa da discricionariedade administrativa. Esta posição que vigorou em nosso país de forma inconteste, ainda encontra seguidores, embora se encontre superada, por no mínimo, três motivos: a impossibilidade de revisão dos atos administrativos por parte do judiciário, que é doutrina transposta do direito francês, onde existe uma especificidade que é o contencioso administrativo, que no direito brasileiro não encontra similar. O segundo motivo, reside na teoria transposta do direito alemão aonde os direitos sociais são comandos endereçados ao legislativo e administrativo, não cabendo ao judiciário este controle. Ocorre que naquele país estes poderes realmente levam a sério a implementação destas políticas públicas, sem que seja necessário a interferência do judiciário. Por fim, o terceiro aspecto a ser considerado, é que no Estado Democrático de Direito, somente existe escolha do administrador vinculada aos preceitos constitucionais, em especial pela pauta dos direitos fundamentais. O Poder Judiciário quando efetua esta decisão não pode cair na casuística, autorizando ou negando todas as pretensões que lhe vêm a julgamento, sem considerar os dispositivos orçamentários, mas deve buscar a solução adequada constitucionalmente, e aí reside a questão. Com esta perspectiva, pretende­se desenvolver o trabalho para investigar os limites em que são controladas as políticas públicas judicialmente. Assim, o questionamento judicial destes atos da administração vêm ocorrendo, mas o importante é apontar os critérios de como este vem se implementando, para que o judiciário não se substitua à atividade do administrador e tampouco o administrador fique livre dos controles judiciais. Esta sindicabilidade tem de ocorrer de forma a concretizar os preceitos estabelecidos pela Constituição Federal. As grandes questões do direito público neste terceiro milênio dizem respeito à possibilidade de controle judicial dos atos da administração referente às políticas públicas. 154 Em um país como o Brasil em que os direitos sociais ainda não estão assegurados para grande parte da população, a pergunta que se faz é a seguinte: fica ao alvedrio a implementação destas políticas públicas por parte do Executivo ou o Poder Judiciário pode ser um importante mecanismo de pressão, na implementação destes direitos e em que limites? Assim, exemplificativamente, referente ao direito à saúde, o que a Constituição objetiva assegurar: um tratamento caro no exterior para uma só pessoa ou o tratamento de doença epidemiológica relevante para uma parcela expressiva da sociedade? Por isso, quando está ausente ou insuficiente uma política pública, o judiciário deve agir, mas com critérios e limites, sob pena de se cair no subjetivismo judicial. Daí advém a necessidade desta investigação, pois em casos de omissão ou atuação precária do administrador, por vezes o judiciário manifesta­se de forma tímida ou sem critérios. Visa­se com este estudo contribuir para esta questão, no sentido de apontar critérios formulados a partir dos preceitos constitucionais, sem que se caia em um casuísmo destituído de fundamento legal. Com relação ao primeiro aspecto, a inadequação do modelo francês transposto à realidade brasileira, faz­se um retrospecto a respeito dos controles da administração. Pode­se afirmar que existem dois sistemas: o da unidade e o da dualidade da jurisdição. O sistema da unidade ou da dualidade da jurisdição decorrem da interpretação do Princípio da Separação dos Poderes. É importante salientar que a doutrina a que se convencionou a denominação “Separação dos Poderes”9, não foi assim conceituada por seu autor Montesquieu. Da leitura de sua obra, extraem­se duas idéias principais, quais sejam: a distinção tripartite das funções do Estado e a idéia de controle recíproco entre estas atividades. Em momento algum escreveu o autor a palavra “separação dos poderes”, isto decorreu da interpretação principal de sua obra, mormente a apropriação da doutrina que se constituiu em um dos paradigmas apregoados pela Revolução Francesa. A interpretação e crítica de Louis Althusser10, não teve fortes ecos, no sentido de que Montesquieu era de origem nobre, conhecido como Barão de La Brède e formulou a teoria apenas para fortalecer o poder monárquico, na época em que este se encontrava em crise. Na França, berço do princípio da Separação dos Poderes devido à doutrina de Montesquieu, este foi interpretado de forma drástica como a impossibilidade de um poder exercer o controle sobre outro. A origem histórica desta interpretação encontra­se na desconfiança que os legisladores da Revolução Francesa tinham para com o Poder Judiciário, pois este tinha se mostrado resistente às conquistas populares. Waline11 assevera que a Lei nº 16, de 24 de agosto de 1790, dispunha sobre a organização judiciária e proclamava a separação das funções administrativas e judiciais, que foi reafirmada com o período que se seguiu à Revolução Francesa. Houve proibição legal expressa aos juízes do conhecimento da matéria administrativa. As reclamações com conteúdo administrativo não poderiam ser, em nenhum dos casos, encaminhadas aos tribunais. Deveriam ser submetidas ao rei, então chefe da administração geral12 . Por contencioso administrativo, entende­se o conjunto de regras relativas aos litígios organizados que questionam a atividade dos administradores públicos. O progresso do direito administrativo francês deveu­se à existência de um contencioso. Essa instituição garantiu o desenvolvimento do direito propriamente dito, por meio das 155 garantias de legalidade criadas, pela importância do aspecto moral da conduta dos administradores, dando respaldo às teorias subjetivas. O recurso por excesso de poder é uma criação jurisprudencial devido ao próprio Conselho de Estado, constituindo­se o principal instrumento de controle da legalidade administrativa13. Assim, o Conselho de Estado14 é um órgão de fiscalização dos atos da administração, situado dentro da próprio Executivo e que desempenha suas funções decisórias com imparcialidade e em consonância com a legalidade. A justificativa é no sentido de que o legislativo é o poder constituído a partir da vontade popular e não seria democrático, a substituição da representação da soberania do povo pelo poder judiciário, que não é eleito democraticamente. Esta questão é bem enfrentada por Sérgio Cademartori15, que analisa a questão da legitimidade. Assim, o poder legislativo e poder judiciário possuem intervenção calcados em pressupostos de legitimidade distintos. A legitimidade do poder legislativo decorre da idéia de soberania popular. O poder legislativo é o representante da população, em que pese poderem ser feitas críticas ao modelo representativo16. Com pressuposto diferente de legitimidade se encontra o poder judiciário, que possui seu fundamento no Estado de Direito. Basicamente, são estas as razões de ordem histórica pelo qual não se permite a revisão dos atos administrativos no direito francês, que consagra a dualidade de jurisdição. Esta doutrina é inaplicável ao direito brasileiro em virtude do art. 5º , XXXV, da CF que consagra a inafastabilidade do controle judicial de todos os atos. Logo, os atos da administração que traduzem opções de políticas públicas não podem ficar à margem do questionamento judicial, o desafio está em estabelecer estes critérios. 2 DIREITOS SOCIAIS NO BRASIL E EM PAÍSES QUE REALIZARAM O ESTADO SOCIAL­ TRAJETÓRIAS DISTINTAS O segundo motivo de análise, são as especificidades do modelo alemão que foram equivocadamente transpostas para o Brasil, sem as necessárias adequações. Assim, no dizer de Hesse17: “ derechos sociales fundamentales como por ejemplo (...) el derecho a una vivienda adecuada (...) no se hacen ya efectivos por el hecho de que se respeten y amparen, sino que requieren de antemano, y em cualquier caso más que en los derechos fundamentales tradicionales, acciones del Estado tendentes a realizar el programa contenido en ellos. No sólo exige esto regularmente un actuar del legislador, sino también el de la Administración; y puede afectar a los derechos­libertad ajenos. Por ello, los derechos fundamentales no alcanzan a justificar pretensiones de los ciudadanos invocables judicialmente en forma directa, como cuadraría a la doctrina de los derechos fundamentales”. É claro que os direitos sociais têm uma estrutura distinta dos direitos individuais, do liberalismo clássico. São comandos voltados prioritariamente para o administrador e legislador, e para a sua implementação dependem de possibilidade orçamentária. Porém, no contexto brasileiro, diante da inoperabilidade destes poderes, por vezes, o judiciário representa um fator importante para pressionar a realização das políticas 156 públicas, visando assegurar a dignidade da pessoa humana, composto pelo mínimo existencial18. Um exemplo frutífero que pode ser apontado é o tratamento da AIDS em nosso país. Diante da inicial omissão dos poderes legislativo e executivo, no sentido de promover políticas públicas, no tocante à prevenção e tratamento do HIV, ações reiteradas no âmbito judicial pedindo medicamentos, incentivaram a que o Brasil se tornasse um país referência nesta área. Deste modo, apropriadas as considerações de Andreas Krell19, conhecedor das realidades alemã e brasileira: “ Face aos problemas sociais candentes de um país periférico como o Brasil, o princípio tradicional da Separação dos Poderes deve ser entendido sob os parâmetros e dimensões novas e diferentes dos das nações centrais. Ainda não foram aproveitadas as potencialidades dos modernos instrumentos processuais do direito brasileiro para correição judicial das omissões dos Poderes Executivo e Legislativo na área das políticas públicas, como a ação civil pública e a ação de inconstitucionalidade por omissão”. Alguns países como a França e Alemanha, em que o Estado Providência20 cumpriu seu papel, assegurando os direitos sociais à população é adequado que se discuta a permanência ou a diminuição de algumas garantias, devido à proporção robusta que estas alcançaram. Diferentemente, no Brasil, em que a população não conseguiu atingir níveis homogêneos em termos de acesso aos direitos sociais. A “reserva do possível”21 tem sido outro conceito mal transposto ao direito comparado. Esta teoria é adaptação de uma expressão cunhada no direito alemão, que entende que a construção de direitos subjetivos à prestação material de serviços públicos pelo Estado está sujeita à condição da disponibilidade dos respectivos recursos, ou seja, no campo orçamentário. Assim, é necessário para avaliar em que limites está ocorrendo o provimento judicial a respeito das políticas públicas, em um país como o Brasil, com especificidades próprias, principalmente no que diz respeito às grandes desigualdades econômicas e culturais. É perigoso importar diretamente conceitos cunhados em outros países com contexto cultural e sócio­econômico diferentes. O Brasil, que é um país em desenvolvimento não pode transportar diretamente teorias de países ricos. O debate europeu sobre a redução dos direitos conquistados durante o Estado Social não pode ser transferido, porque o Estado Providência nunca foi efetivado plenamente. Ainda, concernente à eficácia, importante a posição de Ingo Sarlet22, que posiciona os direitos de cunho prestacional como autênticos direitos fundamentais constituindo­se, por isso, direito imediatamente aplicável, nos termos do art. 5º, § 1º, da CF. Por isto, no embate entre o Estado Social intervencionista e altamente regulador e a nefasta tentativa de implementar um Estado minimalista desenhado a partir da globalização e da ideologia neoliberal, o correto manejo da proibição de retrocesso na esfera dos direitos fundamentais sociais, poderá se constituir uma ferramenta útil para a construção do perfil do Estado brasileiro, com dignidade para cada indivíduo e, portanto, uma vida saudável para todos os integrantes do corpo social23. 157 Deste modo, o trabalho abordará a da importância dos poderes executivo e legislativo na implementação de políticas públicas e o papel do judiciário na busca de efetividade do direito social à saúde, fixando os seus limites e possibilidades de atuação. 3 O CONTROLE JUDICIAL E SUA PERSPECTIVA CONSTITUCIONAL No Brasil, os poderes executivo e legislativo não têm implementado as políticas públicas a contento, por isso, a discussão se insere na órbita judicial. Neste contexto, outro aspecto a ser considerado é a sindicabilidade das políticas públicas como decorrência do Estado Democrático de Direito. Atualmente não existe um poder discricionário livre, mas somente um poder discricionário vinculado24. Vinculação esta, que se pode relacionar aos ditames constitucionais. Desta maneira, o conteúdo das políticas públicas não pode ficar ao alvedrio do administrador, mas tem de buscar efetivar os preceitos contidos na Constituição. O direito administrativo se estruturou no século XIX, a partir do Princípio da Legalidade, este foi o primeiro grande freio que foi imposto ao administrador, porém atualmente com a denominada fuga do direito administrativo ou a tendência neoliberalizante do Estado, ocasionando a chamada eficácia com relação a terceiros “drittwirkung”25, novos pressupostos devem ser erigidos. Para García de Enterría26, o aspecto subjetivo da Justiça Administrativa, que rompeu o mito histórico de sua suposta objetividade, atrás da qual de escondia uma superioridade formal da Administração sobre o cidadão, considerado ainda como súdito, que deveria ceder ante a suposta superioridade dos “interesses gerais”, gestionados pela administração, é uma conquista irrenunciável do nosso tempo. Por isso, o grande desafio que é posto aos estudiosos do direito da administrativo na atualidade é esta possibilidade de controle judicial. Afirma, Moreira Neto27, que há atuações administrativas constitucionalmente mandatórias, como é o caso das políticas públicas nacionais e não apenas governamentais, da educação, da saúde e da segurança pública. Assim, o Estado Democrático de Direito reclama a implementação dos direitos fundamentais, por meio das políticas públicas. Deste modo, a atividade administrativa no terceiro milênio deve ser pautada dentro dos balizadores que compõem o Estado Democrático de Direito: os direitos do homem e a democracia substantiva. A partir destes dois vetores são estatuídos os quatro paradigmas atuais do direito administrativo28: legitimidade, finalidade, eficiência e resultados. Devem ser analisados de forma conjunta e não isoladamente, tendo em vista a realização do serviço público. Estes critérios serão necessários quando são exercidos os controles com relação à atividade administrativa quer no âmbito preventivo ou repressivo. Assim legitimidade e finalidade, que eram requisitos intrínsicos dos atos administrativos, demandam um novo olhar. No tocante à legitimidade serão constitucionais as prestação públicas que respeitem os direitos fundamentais. Não basta mais somente a legitimidade fundada no que se denominava pacto social, mas deve­se perquirir a respeito do atendimento dos valores que visam implementar a pauta dos direitos humanos, principalmente quanto estes completam 60 anos29. Assim a 158 legitimação é, sobretudo, um fenômeno antropológico indissociável do consenso alcançado pelos grupos sociais, que consiste na verificação da concordância com os fenômenos políticos e valorativos relacionados ao poder, ou seja, à sua concentração, à sua distribuição, ao seu acesso, ao seu exercício e ao seu controle30. Concernente à finalidade, estamos sob a época da redescoberta dos valores e das finalidades, deve­se buscar identificar a finalidade nos princípios. A eficiência é um princípio trazido pela EC 19/98, que traduz um aspecto axiológico, no sentido de que o direito administrativo deve e pode ser um instrumento para a realização dos direitos fundamentais e facilitador do desenvolvimento econômico e social. Após o aspecto valorativo ocorre o último paradigma que é o do resultado. Cuida­ se de mensurador prático, em que são buscadas técnicas de controle da gestão31. Deve­se atentar para a prestação do serviço público, que possui um espectro distinto da seara privada. Já foi mencionado o fenômeno da eficácia horizontal dos direitos fundamentais, onde os conflitos migraram da seara pública à privada. É a denominada fuga do direito administrativo em direção ao direito privado32. Isto aconteceu com a questão da saúde, também. Há porém especificidades próprias, pois a iniciativa privada somente se interessa por setores aonde há a possibilidade de lucro. As áreas deficitárias são suportadas pelo poder público. Assim, a prestação de serviço pelo SUS responde por aproximadamente 75% população brasileira33. Isto demonstra que os 25% restantes incumbem à iniciativa privada porque são setores com a potencialidade de lucro. Desta forma, o princípio da eficiência deve ser aferido com características próprias e não com os mesmos balizadores privados. 4 CONCEITO DE POLÍTICA PÚBLICA, DESDE O PRISMA JURÍDICO, COM ÊNFASE NO DIREITO À SAÚDE O conceito de política pública é um conceito que partiu da Ciência Política e da Administração Pública, mas que hoje encontra importante espaço de reflexão no campo jurídico. Assim, a definição apresentada na ciência política é a dimensão normativa ou moral do Estado, que perpassa a filosofia política ocidental e objetiva garantir uma vida feliz ao cidadão34. A questão da política pública apresenta um núcleo com interseccção na organização do sistema internacional, de organização na sociedade e da organização do Estado. Não se apresenta como algo isolado, mas que deve dialogar tendo em conta os três vértices: internacional, estatal e social. Assim, já se percebe porque o tema das políticas públicas entra na pauta jurídica, nos últimos tempos. Da passagem do Estado Liberal com sua feição de abstenção35, chega­se ao Estado Social com seu conteúdo de intervenção, a partir das demandas concretas formuladas pelo cidadão. É o constitucionalismo social inaugurado no início do século XX, depois da Revolução Industrial e do Socialismo36, que tem sua marca na Constituição Mexicana de 1917, Constituição de Weimar de 1919 e em nosso país, na Constituição de 1934, da era Vargas. Passado o movimento pendular no sentido de constituições antidemocráticas e democráticas, chega­se à Constituição de 1988, onde 159 a democracia começa a se estabilizar e confere margem para a efetividade dos direitos. Deste modo, a discussão das políticas públicas ganha espaço no cenário jurídico. Um delineamento a respeito do tema das políticas públicas é trazido por Ana Paula Bucci, como programas de ação destinados a realizar, sejam os direitos a prestações, diretamente, sejam a organizações, normas e procedimentos necessários para tanto. As políticas públicas não são, portanto, categoria definida e instituída pelo direito, mas arranjos complexos, típicos da atividade político­administrativa, que a ciência do direito deve estar apta a descrever, compreender e analisar, de modo a integrar à atividade política os valores e métodos próprios do universo jurídico37. Uma questão que surge é no sentido de delimitar quais as ações que conduzem à implementação das políticas públicas, considerando­se as de cunho individual e coletivo. Estas últimas não suscitam dúvidas. A indagação diz respeito às ações individuais, prestam­se estas para estatuir políticas públicas? É sabido que o art. 5º, XXXV, CF consagra o acesso à justiça de forma ampla e irrestrita. Assim, não se pode impedir o acesso, mas a concessão de medicamentos não previstos como essenciais, demanda uma atenção para a sua decisão. O art. 196 da CF conjuga o direito à saúde a políticas sociais e econômicas, para que seja possível assegurar a universalidade das prestações e preservar a autonomia dos cidadãos, independente do seu acesso maior ou menor do Poder Judiciário. “Presume­se que Legislativo e Executivo, ao elaborarem as listas referidas, avaliaram, em primeiro lugar, as necessidades prioritárias a serem supridas e os recursos disponíveis, (...)”38. Este pressuposto, é corolário de um argumento democrático. Os recursos obtidos para o fornecimento dos medicamentos são obtidos pelos tributos suportados pela população. Desta forma, os representantes eleitos pelo processo democrático devem estabelecer quais são as prioridades na atual conjuntura. Pode ser simpático o argumento de ampla concessão de qualquer medicamento, porém isto é falacioso, pois os recursos orçamentários são limitados em qualquer país, não é possível pretender fazer tudo a qualquer gasto. O argumento de que os recursos públicos são mal utilizados e, por vezes, desviados, não é cabível nesta discussão, embora seja necessária a devida responsabilização na órbita da improbidade administrativa. Como os recursos são limitados não se pode pretender o pagamento de um medicamento, por vezes com valor altíssimo, não testado suficientemente, em prol de uma lista estatuida a partir dos critérios legais e de implementação do executivo. Por isso, importante a decisão proferida pela Ministra Ellen Gracie na SS 39 3073/RN que considerou inadequado fornecer medicamento que não constava da lista do Programa de Dispensação em Caráter Excepcional do Ministério da Saúde. A Ministra enfatizou que o Estado do RN, não estava se negando à prestação dos serviços à saúde e que decisões casuísticas, ao desconsiderarem as políticas públicas definidas pelo Poder Executivo, tendem a desorganizar a atuação administrativa, comprometendo as políticas de saúde ainda incipientes. O caráter de importância nas ações individuais somente se pode vislumbrar, quando se tornam mecanismo de pressão para implementação das políticas públicas, 160 como aconteceu nos casos dos portadores de vírus HIV40, em que o Brasil se constitui em um país com tratamento que é uma referência. Nas primeiras demandas, a contestação do Estado era no sentido de isentar­se de responsabilidade e dizer que se o demandante havia contraído a enfermidade era porque tinha dado causa a isso, numa atitude nitidamente preconceituosa. O foro adequado é o das ações coletivas, aí se pode discutir o arrolamento de algum outro remédio ou tratamento na lista dos essenciais, veja­se a questão atinente à mudança de sexo41. Houve ajuizamento de ação civil pública pedindo a inclusão na tabela do SUS da transgenitalização, que foi julgada procedente. Tal redundou em alteração da posição administrativa, ocasionando a portaria do Ministério da Saúde de nº 1.707/2008, que incorpora a alteração de sexo, como procedimento oferecido pela rede pública. Três ponderações para que a discussão se trave em âmbito coletivo são apresentadas por Barroso42. Por primeiro, a discussão no âmbito coletivo exigirá a análise do contexto em que se situam as política públicas; por segundo, evita­se a questão da micro­justiça, ou seja, a preocupação do juiz com o deslinde daquela ação esquecendo­se da macro­justiça, atendimento com recursos limitados a demandas ilimitadas; por terceiro, a decisão proferida em decisão coletiva terá efeitos “erga omnes”, preservando a igualdade e a universalidade do atendimento à população. Assim, a política pública visa a implementação pelo poder executivo de um comando constitucional. Especificamente, atinente ao direito à saúde, constata­se que com o constitucionalismo social inaugurado no Brasil em 1934, o direito à saúde aparece por primeira vez em sede constitucional. Os textos seguintes limitaram­se a atribuir competência à União para planejar sistemas nacionais de saúde, conferindo­lhe a exclusividade da legislação sobre normas gerais de proteção e defesa da saúde e mantiveram a necessidade de obediência ao princípio que garantia aos trabalhadores assistência médica sanitária. Na Constituição atual, o direito à saúde (art. 6º, CF) é reconhecido como direito social e um dever do Estado (art. 196, CF) que a Constituição institui obrigações para todos o entes federados. Constitui­se em competência comum (art. 23,II, CF) à União, aos Estados, ao Distrito Federal e aos Municípios, competência concorrente sobre a proteção e defesa da saúde, competindo à União o estabelecimento de normas gerais e aos Municípios as suplementares ( art. 24, §§ 1º e 2º, c/c art. 30,II, CF) e cooperação técnica e financeira com o auxílio da União e dos Estados (art. 30, VII, CF). Nenhum dos entes federativos está isento de atribuições, isto apresenta dupla crítica no sentido de que a todos incumbem tarefas, mas por outro lado, quando a responsabilidade é tão partilhada entre todos, fica mais difícil cobrar a atribuição de cada um. Porém, o escopo deve ser no sentido de construir a responsabilidade no âmbito individual, familiar e do poder público, em todas as áreas de atuação, conforme apregoa Rogério Gesta Leal43. Este balizador de atuação deve ser um objetivo para os juristas. O Sistema Único de Saúde (SUS) se propõe a ser uma estrutura descentralizada, com direção única em cada esfera do governo, oferece atendimento integral e conta com a participação da comunidade (art. 198, CF). Em atenção aos preceitos constitucionais foram elaboradas as Leis nºs 8.080/90 e 8.142/90, conhecidas como Lei 161 Orgânica da Saúde ­ LOS, que são leis nacionais, com o caráter de norma geral, onde contém as diretrizes e os limites que devem ser respeitados pela União, pelos Estados e pelos Municípios ao elaborarem suas próprias normas para garantir o direito à saúde para a população brasileira. Deste modo, a LOS colabora na definição do direito à saúde, estrutura o SUS, tornando claro seus objetivos e suas atribuições, as diretrizes que devem orientar sua organização, direção e gestão, a forma como estão distribuídas as tarefas entre as três esferas de poder e a forma de participação na comunidade na gestão do sistema em cada uma dessas esferas, além de organizar o seu financiamento, tratando expressamente dos recursos, da gestão financeira, do processo de planejamento e do orçamento, inclusive das transferências intergovernamentais de recursos financeiros. A questão dos medicamentos, de responsabilidade do SUS, encontra previsão legal na Lei Federal nº 8.080/90, art. 6º, VI, sendo um dos pontos de difícil operacionalização quando se trata da política de Estado para a saúde pública. Desde 197544 a Organização Mundial da Saúde – OMS aponta para a necessidade de os Estados formularem uma política que permita o acesso e favoreça o uso racional dos medicamentos por todas as pessoas, pois se verificou que o mercado mundial era composto, por aproximadamente 70% de substâncias não essenciais, desnecessárias e até perigosas, e que 1/3 da população não tem acesso a medicamentos essenciais45. Para a OMS, a formulação de uma política de medicamentos implica a definição de um conjunto de diretrizes com a finalidade de assegurar para toda a população uma utilização racional dos medicamentos. Isto significa diversos momentos que vão desde a produção até a prescrição dos remédios46. No Brasil, foi editada a Portaria do Ministério da Saúde nº 3.916/9847 contendo a Política Nacional dos Medicamentos, baseada nas recomendações da OMS, visando fornecer os medicamentos necessários à população mais desprovida de recursos econômicos. Importante passo para a acessibilidade dos genéricos foi conseguido por meio dos medicamentos genéricos, disciplinados pela Lei nº 9.787/99. A partir de então, a Agência Nacional de Vigilância Sanitária – ANVISA, tem estabelecido por meio de resoluções como deve ser feito o registro, a fabricação, o controle de qualidade, a prescrição dos medicamentos genéricos. 5 A BUSCA DE CRITÉRIOS CONSTITUCIONALMENTE DENTRO DA ESCOLHA ADEQUADA Feitas as considerações iniciais, no sentido de que é cabível a intervenção judicial dentro da perspectiva da busca da escolha adequada, passa­se à parte mais instigante, que é a busca dos critérios para nortear a interpretação jurisprudencial, em um estudo preliminar. Por primeiro, a formulação de políticas públicas cabe aos poderes legislativo e executivo, uma vez que estas opções se constituem em uma decorrência da 162 democracia representativa e, por vezes, da participativa. Não cabe aí uma manifestação jurisprudencial, portanto. Por segundo, nos casos de ineficiência ou omissão na execução das políticas públicas, cabe a intervenção judicial. Aí podem se apresentar algumas situações. O Judiciário está autorizado a intervir, quando o poder público não fizer o aporte exigido constitucionalmente à área da saúde, em conformidade com o art. 198, § 2º, da CF. O foro mais adequado para discutir a inclusão ou exclusão de algum medicamento é por via das ações coletivas, com legitimados ativos representativos e que permitem um amplo debate a respeito das prioridades, que são estabelecidas a partir das determinações da Organização Mundial da Saúde, levando em conta as peculiaridades de cada país e implementadas pelo Ministério respectivo, após debate junto ao Congresso Nacional. Atinente aos medicamentos em experimentação, que não têm a sua eficácia comprovada não podem ser objeto da inclusão em lista, pelo Poder Judiciário. Deve­se, ainda, optar pelo genérico de menor custo, quando houver esta possibilidade. Nos casos de demanda individual, compreende­se a agonia do cidadão que requer o remédio, da família que passa por uma situação difícil e do magistrado que muitas vezes se encontra em uma decisão entre a vida e a morte, iminentemente, mas se trava um embate entre as deliberações públicas e privadas e situações de risco em curto e médio prazo. Por exemplo, às vezes se gasta mais na parte curativa do que preventiva. Vejam­ se os números que são investidos em saneamento básico e construção de redes potável, comparativamente à saúde48. Os primeiros são muito tímidos se comparados aos segundos. E a ausência de investimento em saneamento básico é um foco para disseminar doenças, posteriormente. Então, na premência do momento, descuida­se de uma perspectiva de futuro. Deste modo, a concessão dos medicamentos não deve se pautar por uma abordagem individual dos problemas sociais, mas pela busca de uma gestão eficiente dos escassos recursos públicos, analisando­se os custos e benefícios, desde o prisma das políticas públicas. Aí se pode estabelecer mais uma reflexão a partir do pensamento de Hannah Arendt, quando trata da dicotomia público e privado49. As atividades humanas são condicionadas pelo fato de que os homens vivem juntos. O termo público denota dois fenômenos intimamente correlatos, mas não perfeitamente idênticos. Significa, em primeiro lugar, que tudo o que vem a público pode ser visto e ouvido por todos e tem a maior divulgação possível. Torna­se aparente, aquilo que é visualizado e ouvido pelos outros e nós mesmos, constituindo­se na realidade. Por segundo, público significa o próprio mundo, na medida em que é comum a todos nós e diferente do lugar que nos cabe dentro dele. Se o mundo deve conter um espaço público, não pode ser construído apenas para uma geração e 163 planejado somente para os que estão vivos: deve transcender a vida de homens mortais. Dentro desta perspectiva, as decisões de saúde não devem se pautar pelo casuísmo, ou no dizer de STRECK50, de modo solipsista, mas dentro de uma perspectiva de cumprimento do texto constitucional, em busca da resposta adequada dentro dos parâmetros preceituados pela Constituição Federal, sob pena de não haver avanços para as próximas gerações. Preocupado com a legitimidade social das decisões judiciais, e com a intenção de se evitar a discricionariedade (vale dizer, a arbitrariedade), Lênio Streck faz importante construção teórica, tendo como parâmetros a Hermenêutica Filosófica de Hans Georg Gadamer e a teoria integrativa de Ronald Dworkin. Deste, toma para si a importância dos princípios no processo interpretativo, que trazem para o Direito a faticidade, e daquele relembra como o sentido, no processo de interpretação/aplicação se constrói socialmente, e historicamente, através da tradição. De Dworkin vem a lição de que por trás de cada regra há um princípio instituidor da norma. Há na verdade uma co­originariedade entre princípio e norma. “Não há primeiro uma formação subjetiva de princípios e, depois, sua aplicação compartilhada no mundo da convivência, mas essa formação principiológica é formada pelo mundo e, ao mesmo tempo, forma mundo, na medida em que pode articular um significado novo que exsurge da interpretação do próprio mundo”.51 De plano impõe­se a apreciação dos princípios no plano caso concreto, eis que eles não existem em abstrato. No plano fático é que se verifica como o princípio é a história institucional do direito. Assim, a legitimidade de uma decisão advém do fato que um princípio institui aquela regra/norma. Sem um princípio, não há legitimidade para a aplicação da regra. Não se trata, portanto, de, como no caso do direito à saúde, simplesmente citar o direito à vida e alçá­lo a condição de princípio. De plano percebe­ se que não se trata de um princípio, como é o caso da igualdade, do devido processo legal, da presunção de inocência, da publicidade dos atos estatais, etc. Princípios possuem força normativa, e encontram sua realização em uma regra. A esse passo, cabe abordar outro aspecto importante: o sentido da tradição na interpretação/aplicação do direito. Gadamer52 mostrou como a interpretação autêntica, fundada nos pré­conceitos autênticos, é aquela fundada na tradição. Assim, os princípios adquirem sentidos porque ancorados na jurisprudência (tal qual o precedente na tradição anglo­saxã, a decisão institui um princípio, que aponta para a mesma aplicação da regra aos casos subseqüentes), na doutrina, e na própria opinião pública. No caso do direito à saúde, em especial, costuma se vislumbrar uma afronta à Lei 8.080/90. Esta legislação estabelece como se dará a aplicação do direito fundamental à saúde, estabelecendo­se os medicamentos de maior relevância, e a atribuição da competência de cada ente federativo. Ora, tal lei se presume constitucional, e insere­se naquilo que se pode chamar de tradição. É produzida democraticamente, e a elaboração da lista dos medicamentos é acompanhada por uma série de instituições 164 sociais (como já abordado no capítulo anterior). Portanto, possui uma história institucional que precisa ser levada em conta quando da sua interpretação/aplicação. Isso é uma questão de princípio, tal qual trata Dworkin. Esse sentido dado pela tradição é que faz com que não haja um esvaziamento hermenêutico do direito constitucional à saúde, que como cabe lembrar, se faz por meio de políticas públicas, portanto não se traduz em um direito subjetivo, de matriz liberal­individualista, que autorize o cidadão a postular para si um “direito à saúde” (o que importaria na exclusão de outros referente a esse direito, pela própria escassez). Dessa maneira, fica evidente a postura adequada pelos tribunais ao exercerem o controle que lhes é próprio relativo à implementação do direito fundamental à saúde. Deve sempre buscar a resposta correta (nem a única, nem a melhor, como bem salienta STRECK), vale dizer, adequada à Constituição, o que se faz recorrendo­se a tradição, que é quem confere sentido à norma. Portanto, impossível não aplicar a lei orgânica da saúde, e suas determinações em termos de políticas públicas, a menos que, em sede de jurisdição constitucional se faça: a) controle difuso da constitucionalidade, declarando a norma inconstitucional; b) deixe­se de aplicá­la em função de antinomia de normas; c) faça­se uma interpretação conforma à Constituição, o que implica em alteração do sentido da lei; d) aplicação da nulidade parcial sem redução de texto, por via da qual há a exclusão de determinada hipótese de aplicação; ou e) a declaração de inconstitucionalidade com redução de texto, hipótese em que excluindo­se uma palavra, mantém­se a constitucionalidade do dispositivo.53 Lembrando­se que para isso há de existir profunda fundamentação, no sentido de desvelar adequadamente os princípios constitucionais. Portanto, é possível dizer (com STRECK) que existe um direito fundamental à obtenção de uma resposta correta, e um dever fundamental de uma adequada fundamentação das decisões judiciais que não de azo à discricionariedade. Somente recorrendo­se a tradição, e buscando­se substrato nos princípios que orientam a resolução do conflito sob judice que se dará efetividade ao controle judicial das políticas públicas. Assim como o fundamento do controle é a Constituição, não pode ser ele quem a viole. Em vez de um sujeito solipsista que tem na interpretação um ato de vontade54, é preciso que o magistrado, em tempos de Estado Democrático de Direito, esteja atento para a necessidade de legitimação das decisões judiciais. É atentando para o caso concreto e tudo que dele exsurge que é possível controlar a discricionariedade. Pelo fortalecimento das instâncias democráticas de controle da saúde é que se dá a efetividade das políticas públicas de saúde, e não pela mera judicialização da política. CONCLUSÕES Na perspectiva de novos temas referentes à administração, o controle judicial das políticas públicas, é o tema pautado neste terceiro milênio, que coincide com o processo de redemocratização e reconstitucionalização do país, que é fruto da mudança de paradigma: a busca de efetividade dos direitos fundamentais. Assim, pretende­se contribuir ao debate, a fim de traçar os limites e possibilidades de atuação judicial, no tocante às políticas públicas, e construir um horizonte, com perspectiva 165 favorável, para milhares de brasileiros que necessitam da eficácia dos direitos fundamentais sociais. No final do século XX, produziram­se importantes mudanças no âmbito econômico, social e cultural, devidos à revolução tecnológica, à globalização, à circulação rápida da informação e das comunicações, aos processos de integração econômica e política, ao término da guerra fria, a dissolução da URSS, a queda do muro de Berlim, e o ataque de movimentos fundamentalistas. Todos estes fatores contribuíram para uma mudança no direito administrativo que até então havia se construído com o aporte iluminista e se estruturado a partir do princípio da legalidade. Com a mudança do aspecto territorial, como um dos elementos do Estado, devido à globalização, um novo contexto de Estado vem se definindo, conferindo ao direito administrativo um novo horizonte como a efetividade das garantias dos cidadãos, que fazem superar a concepção tradicional de busca de equilíbrio das prerrogativas da administração e as garantias dos cidadãos, para almejar o cumprimento do preceituado pela Constituição por parte dos poderes públicos, em especial, no que diz respeito aos direitos fundamentais. O Estado Social produziu a superação do conceito de interesse público, calcado exclusivamente a partir dos objetivos da administração e foi substituído pelo interesse coletivo, aberto à participação popular. Voltando ao pensamento de Hannah Arendt, percebe­se que o direito à saúde está sendo construído, por meio das políticas públicas de implementação de tratamentos e de medicamentos ditos essenciais, constituindo­se o Poder Judiciário um instrumento de realização dos direitos dos cidadãos, desde que se apliquem critérios adequados com os preceitos da Constituição, deixando­se o casuísmo e o subjetivismo, nesta seara. Outro importante aspecto a ser considerado quando da implementação das políticas públicas dos direitos sociais que devem atentar aos dispositivos orçamentários. Os últimos vinte anos de constitucionalismo democrático brasileiro demonstram esta busca de efetividade dos direitos fundamentais sociais. Deste modo, a crise do direito administrativo é uma mutação para adequar­se às transformações e mudanças sociais vividas no início do século XXI. É necessário voltar­ se à pauta dos direitos fundamentais, que até hoje não foram efetivados de maneira universal, considerando, especialmente, o direito à saúde no Brasil. Somente assim, poderemos acenar com a construção de um país realmente democrático com sua população mais saudável, nas próximas gerações.Tal tarefa não é fácil, e passa por uma transformação de imaginário. Somente por meio da produção de uma cultura democrática (que implica no reconhecimento dos limites e possibilidades do controle jurisdicional) é que se pode vislumbrar avanços nesta seara. 166 PUBLIC POLICIES AND RIGHT TO HEALTH: the tension between the powers – the need to establish judicial criteria to intervention ABSTRACT The right to health is a constitutional right, and its meant to be implemented by public policies. The experience of the Social State of other countries is different of the Brazilian one. The public policies are mandates created by the legislator and achieved by the administrator. The administrative discretion find constitutional limits. In cases of inefficiency or omission in execution of public policies, can occur judicial intervention. The collective actions are the most adequate way for this discussion. The individual actions are a mechanism of pressing. The democratic culture implies in the recognition of the limits and possibilities of the judicial control for the effectivity of the right to health. Keywords: Adequate answer. Judicial control. Public policies. Right to health. Social state. NOTAS 1 2 3 4 5 6 7 Bacharel em Direito pela UNISINOS, Mestrando em Direito Público na UNISINOS. Doutora em Direito pela Universidade Pompeu Fabra de Barcelona/Espanha, Mestra em Direito pela UFRS, Profa. do PPG em Direito da UNISINOS, Promotora de Justiça/RS. ARENDT, Hannah. Los orígenes del totalitarismo. Madrid: Taurus, 1998, p. 368­ 382. Vide também The rights of man, what are they? In Hannah Arendt’s Centenary: Political na Philosophie, vol. 74, number 3, Part I (fall 2007), guest editor: Jerome Kohn. SCLIAR, Moacyr. Do mágico ao social: trajetória da saúde pública, São Paulo: SENAC, 2002, p. 95. Assim, o médico sanitarista escreve: “(...) Sabe­se que a morbimortalidade materna, por exemplo, correlaciona­se inversamente com o grau de educação formal, independente das condições socioeconômicas. Para que o processo educativo em saúde atinja a plenitude de seus objetivos é preciso percorrer várias etapas: a partir de um diálogo com a população, procura­se informar as pessoas, para que, informadas adotem uma atitude positiva, que deve gerar um comportamento; esse comportamento, idealmente, deveria ser introjetado sob a forma de hábito, ou seja, comportamento automatizado. Disseminado, o hábito evolui para o costume, que é um prática generalizada, incorporada à própria cultura” (grifos do autor). Vide SZWARCWALD, Célia L. Mortalidade infantil no Brasil: Belíndia ou Bulgária? Cadernos de Saúde Pública, vol. 13, nº3, Rio de Janeiro, 1997. Disponível também em: http://www.scielo.br/pdf/csp/v13n3/0175.pdf Acesso em: 9/10/2009. COUTO e SILVA, Almiro in apresentação do livro Elementos de Direito Administrativo Alemão, MAURER, Hart, Porto Alegre: Fabris, 2000, p. 14. STRECK, Lênio. Jurisdição Constitucional e Hermenêutica: uma nova crítica do direito. 2. ed. Rio de Janeiro: Forense, 2004, p. 122. Para Streck: “o que houve (há) é um simulacro de modernidade. (...) Ou seja, em nosso país as promessas de modernidade ainda não se realizaram. E, já que tais promessas não se realizaram, a solução que o establishment apresenta, por paradoxal que possa parecer, é o retorno ao Estado (neo)liberal. Daí que a pós­modernidade é vista com a visão neoliberal. Só que existe um imenso deficit social em nosso país, e, por isso, temos que defender as instituições da modernidade contra esse neoliberalismo pós moderno”. CANOTILHO, J.J. Gomes e MOREIRA Vital. Fundamentos da Constituição. Coimbra: Coimbra Ed., 1991, p.173. Na Constituição orçamental estariam os preceitos relativos ao orçamento do Estado, principalmente a aprovação parlamentar do orçamento, incluindo a autorização parlamentar anual dos impostos e a autorização e controle parlamentar das despesas públicas. 167 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 STRECK, Lênio. Decisionismo e discricionariedade judicial em tempos pós­positivistas: o solipsismo hermenêutico e os obstáculos à concretização da Constituição no Brasil. Separata: o Direito e o Futuro do Direito, Almedina: Coimbra, 2008, p. 99. MOTESQUIEU, Charles Louis de Secondat. Do espírito das leis. 2. ed., São Paulo: Abril Cultural, 1979, pp.148/54. ALTHUSSER, Louis. Montesquieu – A política e a história. 2. ed., Lisboa: Editorial Presença. WALINE, Marcel. Traité Élémentaire de Droit Administratif, 6e édition, Recueil Sirey, Paris, 1952, p. 45. LAUBADÈRE, André. Manuel de Droi Administratif, 15e édition, L.G.D.J., Paris, 1995, p. 107. LAUBADÈRE, op. cit., pp. 105/7. A respeito da evolução do recurso de excesso de poder, o autor faz uma análise de sua evolução até o final do século XX. Da mesma forma, o Conselho Constitucional é o órgão a quem incumbe o controle da constitucionalidade das leis, que é realizado de uma maneira preventiva, não se permitindo ao judiciário o controle destas questões. CADERMATORI, Sergio. Estado de Direito e Legitimidade: uma abordagem garantista. 2. ed., Campinas: Millennium, 2006. A respeito das insuficiências do modelo representativo e o movimento que ocorre em prol da democracia participativa, não se tratará desta questão, por não ser objeto do presente trabalho. HESSE, Conrado. Significado de los derechos fundamentales. In Manual de Derecho Constitucional, BENDA et als. , Madrid: Instituto Vasco de Administración Pública, 1996, p. 98, g.n. BARROSO, Luis Roberto. Fundamentos teóricos e filosóficos do novo direito constitucional brasileiro (pós­modernidade, teoria crítica e pós­positivismo). Revista do Ministério Público, Porto Alegre, nº 46, jan/mar,2002, p. 59. KRELL, Andreas J. Direitos Sociais e controle judicial no Brasil e na Alemanha: os (des)caminhos de um direito constitucional comparado. Porto Alegre: Fabris, 2002, p. 109, g.n. EWALD, François. L’Etat Providence. Paris: Bernard Grasset, 1986. KRELL, Andreas J. op. cit., p. 51/2. Segundo o Tribunal Constitucional Federal da Alemanha, esses direitos a prestações positivas “estão sujeitos à reserva do possível no sentido daquilo que o indivíduo, de maneira racional pode esperar da sociedade”. Essa teoria impossibilita exigências acima de um certo limite básico social; a Corte recusou a tese de que o Estado seria obrigado a criar quantidade suficiente de vagas nas universidades públicas para atender todos os candidatos. SARLET, Ingo.W . A eficácia dos direitos fundamentais, 7. ed. Porto Alegre: Livraria do Advogado, 2007, p. 297. SARLET, Ingo.W . A eficácia do direito fundamental à segurança jurídica: dignidade da pessoa humana, direitos fundamentais e proibição de retrocesso social no direito constitucional brasileiro, in Constituição e Segurança Jurídica, ROCHA, Cármen Lúcia Antunes Coord., Belo Horizonte: Fórum, 2004, p.128. MAURER, Hart. Elementos de Direito Administrativo Alemão. Porto Alegre: Fabris, 2007, p. 50. Os conflitos não mais se situam na polaridade Estado x cidadão, mas nas relações grupo x indivíduo. Isso ocorre porque alguns grupos econômicos possuem poder paralelo ao Estado, decorrente, em alguns casos, das privatizações, em que muitos serviços públicos são prestados por empresas privadas. O desenvolvimento da dtittwirkung, também conhecida por eficácia diante de terceiros ou eficácia horizontal, é hoje questão principal nos direitos do indivíduo. Os litígios foram se deslocando do plano das relações públicas para o das privadas. A propósito vide BILBAO UBILLOS, Juan Maria. La eficacia de los derechos fundamentales frente a particulares. Madrid: CEC, 1997, p. 256. GARCÍA DE ENTERRÍA, Eduardo. Las Transformaciones de La Justicia Administrativa: de excepción singular a La plenitud jurisdiccional. ¿Um cambio de paradigma? Pamplona: Thomson Civitas, 2007, p. 147. MOREIRA NETO, Diogo de Figueiredo. Novos horizontes para o direito administrativo: pelo controle das políticas públicas. Revista de Direito do Estado, Rio de Janeiro, nº 4: 403­412, out/dez, 2006, p. 406. Vide também ALLI ARANGUREN, Juan­Cruz. Derecho Administrativo y Globalización, Madrid: Thomson Arandazi, 2004, p. 377. MOREIRA NETO, Diogo de Figueiredo. Quatro paradigmas do direito administrativo pós­moderno: legitimidade, finalidade, eficiência, resultados. Belo Horizonte: Fórum, 2008. MOREIRA NETO, Diogo de Figueiredo. op. cit., p. 33. 168 30 31 32 33 34 35 36 37 38 39 MOREIRA NETO, Diogo de Figueiredo. op. cit., p. 63. MOREIRA NETO, Diogo de Figueiredo. op. cit., p. 139. São apresentadas técnicas destinadas a realizar as seguintes funções: a) a definição de objetivos de políticas públicas; b) a identificação das estruturas administrativas responsáveis por sua consecução; c) definição das normas de desempenho que traduzam um compromisso quanto ao grau de realização dos objetivos demarcados em função dos meios a eles destinados; d) os resultados comensurados e sua comunicação; e) outorga de facilidades de gestão aos responsáveis proporcionalmente a seu desempenho (conforme convênios de gestão); f) análise dos dados de desempenho integrados com vista a informar as subseqüentes decisões sobre recursos e orçamento; g) fixação de acompanhamentos físicos e contábeis necessários para os controles, correntes e posteriores. FLEINER, Fritz. Institutionem des Verwaltungsrechts, 8. ed., 1928, p. 326, apud MIR PUIGELAT, Globalización, Estado y Derecho. Las transformaciones recientes del Derecho administrativo. Madrid: Civitas, 2004, p.158. De acordo com a pesquisa mundial sobre saúde, desenvolvida pela OMS e coordenada no país pelo Centro de Informação Científica e Tecnológica (CICT) da Fiocruz, em estudo que avalia os sistemas de saúde de 71 nações, consigna que os brasileiros dispensam em média 19% da renda familiar com saúde, da parcela da população de 25,8% que têm acesso aos planos de saúde privados, enquanto a maioria 74,2% conta com os serviços do Sistema Único de Saúde – SUS. Disponível em: http://www. ministeriodasaude.gov.br. Acesso em 20/8/2008. HOWLETT, Michael e RAMESH, M. Come studiare le poliche pubbliche. Bologna: Il Mulino, 1995, p. 5. Tradução do original Studying Public Policy: Policy cycles and policy subsystems, Oxford, Oxford University Press, 1995. A propósito a Constituição dos Estados Unidos de 1787 e a Constituição Francesa de 1791. SIGMANN, Jean. 1848­ Las revoluciones románticas y democráticas de Europa. 3. ed., Madrid: Siglo veintiuno editorial, 1985. BUCCI. O conceito de política pública em direito, p. 31, in BUCCI, Maria Paula Dallari (org). Política públicas: reflexões sobre o conceito jurídico. São Paulo: Saraiva, 2006. BARROSO, Luís Roberto. Da falta de efetividade à judicialização excessiva: direito à saúde, fornecimento gratuito de medicamentos e parâmetros para a atuação judicial, p. 28 Disponível em: http: www. lrbarroso.com.br/pt/noticias.medicamentos. pdf. Acesso em: 29/8/2008. STF, DJU 14/2/2007, SS 3073/ RN, Rel. Min. Ellen Gracie. Verifico estar devidamente configurada a lesão à ordem pública, considerada em termos de ordem administrativa, porquanto a execução de decisões como a ora impugnada afeta o já abalado sistema público de saúde. Com efeito, a gestão da política nacional de saúde, que é feita de forma regionalizada, busca uma maior racionalização entre o custo e o benefício dos tratamentos que devem ser fornecidos gratuitamente, a fim de atingir o maior número possível de beneficiários. Entendo que a norma do art. 196 da Constituição da República, que assegura o direito à saúde, refere­se, em princípio, à efetivação de políticas públicas que alcancem a população como um todo, assegurando­lhe acesso universal e igualitário, e não a situações individualizadas. A responsabilidade do Estado em fornecer os recursos necessários à reabilitação da saúde de seus cidadãos não pode vir a inviabilizar o sistema público de saúde. No presente caso, ao se deferir o custeio do medicamento em questão em prol do impetrante, está­se diminuindo a possibilidade de serem oferecidos serviços de saúde básicos ao restante da coletividade. Ademais, o medicamento solicitado pelo impetrante, além de ser de custo elevado, não consta da lista do Programa de Dispensação de Medicamentos em Caráter Excepcional do Ministério da Saúde, certo, ainda, que o mesmo se encontra em fase de estudos e pesquisas. Constato, também, que o Estado do Rio Grande do Norte não está se recusando a fornecer tratamento ao impetrante. É que, conforme asseverou em suas razões, “o medicamento requerido é um plus ao tratamento que a parte impetrante já está recebendo” (fl. 14). Finalmente, no presente caso, poderá haver o denominado “efeito multiplicador” (SS 1.836­AgR/RJ, rel. Min. Carlos Velloso, Plenário, unânime, DJ 11.10.2001), diante da existência de milhares de pessoas em situação potencialmente idêntica àquela do impetrante. Ante o exposto, defiro o pedido para suspender a execução da liminar concedida nos autos do Mandado de Segurança nº 2006.006795­0 (fls. 31­35), em trâmite no Tribunal de Justiça do Estado do Rio Grande do Norte. 169 40 41 42 43 http://www.ioc.fiocruz.br/aids20anos/linhadotempo.html Acesso em: 9/10/2009. Em 1991, O Ministério da Saúde dá início à distribuição gratuita de antirretrovirais. A OMS anuncia que 10 milhões de pessoas estão infectadas pelo HIV no mundo. No Brasil, 11.805 casos são notificados. O antirretroviral Videx (ddl) é aprovado nos Estados Unidos e a fita vermelha torna­se o símbolo mundial de luta contra a Aids. A Fiocruz foi convidada pelo Programa Mundial de Aids das Nações Unidas e Organização Mundial da Saúde (Unaids/OMS) para participar da Rede Internacional de Laboratórios para Isolamento e Caracterização do HIV­1. Em 1992, pesquisadores franceses e norte­americanos estabelecem consenso sobre a descoberta conjunta do HIV. A Aids passa a integrar o código internacional de doenças e os procedimentos necessários ao tratamento da infecção são incluídos na tabela do SUS. Combinação entre AZT e Videx inaugura o coquetel anti­aids. O início foi uma ação civil pública proposta pelo MPF/ RS, que ocasionou a decisão de 2007 do TRF­ 4 (Tribunal Regional Federal da 4ª Região) em que obrigava o SUS a fazer esse tipo de cirurgia. A decisão do TRF determinava que o governo federal tomasse todas as medidas que possibilitem aos transexuais a realização da cirurgia de mudança de sexo pelo SUS. “Direito Constitucional. Transexualismo. Inclusão na tabela do SUS. Procedimentos médicos de transgenitalização. Princípio da proibição de discriminação por motivo de sexo. Discriminação de gênero. Direitos fundamentais de liberdade, livre desenvolvimento da personalidade, privacidade e à dignidade humana e direito à saúde. Força normativa da Constituição.” Rel. Roger Raupp Rios, Processo nº 2001.71.00.026279­9. D.E. 22/08/2007. A União recorreu ao STF (Supremo Tribunal Federal), alegando que as operações gratuitas trariam prejuízo aos cofres públicos. Em 12 de dezembro de 2007, a então presidente do STF, Ministra Ellen Gracie, suspendeu os efeitos da decisão do TRF­4, em um pedido de suspensão de tutela antecipada. Agora, o próprio Ministério da Saúde decidiu incluir a cirurgia na tabela do SUS. A cirurgia para mudança de sexo fará parte da lista de procedimentos do Sistema Único de Saúde (SUS). A Portaria n.º 1.707 do Ministério da Saúde, publicada na edição desta terça­feira, no Diário Oficial da União, prevê a realização do processo nos hospitais públicos. A regulamentação dos procedimentos caberá à Secretaria de Atenção à Saúde do Ministério da Saúde. Segundo o texto da portaria publicada nesta terça­feira, a secretaria vai adotar as providências necessárias à estruturação e implantação do processo transexualizador no SUS, definindo os critérios mínimos para o funcionamento, o monitoramento e a avaliação dos serviços. O anúncio de que o SUS faria este tipo de cirurgia foi feito no início de junho, durante a Conferência Nacional de Gays Lésbicas, Bissexuais Travestis e Transexuais, a primeira, no mundo, organizada por um governo federal. Na ocasião, o ministro da Saúde, José Gomes Temporão, lembrou que essa cirurgia é um processo extremamente complexo e delicado, e serão selecionados pouquíssimos centros no Brasil, possivelmente hospitais universitários para o procedimento, que atualmente é feito em hospitais particulares, custa caro e muitas vezes envolve riscos para o paciente. O processo cirúrgico transexualizador vai além da cirurgia e envolve uma equipe multidisciplinar composta por psicólogos, assistente social e médicos endocrinologista, psiquiatras, urologistas e ginecologistas. O Ministério promete que todo o atendimento será livre de discriminação. Disponível em: http://www.agenciabrasil.gov.br/noticias/2008/08/19/materia.2008­08­ 19.1741052107/view. Acesso em: 9/10/2009. Cirurgia para mudança de sexo passa a fazer parte de procedimentos do SUS. BARROSO, Luís Roberto. Da falta de efetividade à judicialização excessiva: direito à saúde, fornecimento gratuito de medicamentos e parâmetros para a atuação judicial, p. 31. Disponível em: http://www.lrbarroso.com.br/pt/noticias/medicamentos.pdf Acesso em: 9/10/2009. LEAL, Rogério Gesta. A quem compete o dever de saúde no direito brasileiro? Esgotamento de um modelo institucional. Revista de Direito Sanitário, v. 9, p. 50­69, 2008. O autor assevera que o Direito Civil pátrio estatui dever de solidariedade entre as pessoas que compõem a família. A solidariedade impõe um dever de cuidado, que se exprime, dentre outros, nas prestações alimentícias. Ocorre que tais prestações não englobam apenas a subsistência, devendo ela ser adequada, do que se depreende que inclusive a assistência à saúde está incluída, não sendo apenas uma verba para alimentação. Isso é reflexo do dever de cuidado que os familiares tem para si, posição que os colocam como devedores solidários da promoção à saúde. Prova disso é que nossos tribunais ao fixar o valor dos alimentos já incluem o valor das despesas médicas, com plano 170 44 45 46 47 48 49 50 51 52 53 54 de saúde inclusive, não estando apartados os valores. A lei 9.908/93­RS, que determina o fornecimento por parte do Estado de medicamentos, desde que se comprove a condição de pobre, demonstrando a incapacidade da família em arcar com os custos. A construção de Gesta Leal vai no sentido de preocupar­se com a efetividade da Constituição, rejeitando­se os discursos que apregoam que a simples referência à ela promoveria milagres no plano fático. Cabe lembrar que é pressuposto de eficácia do Estado Social a solidariedade, razão pela qual há que se impedir o avanço das práticas clientelistas, que mantém os cidadãos infantilizados. Resolução WHA 28.66, da 28ª Assembléia Nacional da Saúde. SOBRAVIME, O que é o uso racional de medicamentos, São Paulo: SOBRAVIME, 2001, p.13. Organização Mundial da Saúde, Pautas para establecer políticas farmacéuticas nacionales, Ginebra: OMS, 1988. Publicada no DOU nº 215­E, Seção I, de 10/11/98, p. 18­22. No Estado do Rio de Janeiro foram gastos com os programas de assistência farmacêutica R$ 240.621.568,00, enquanto com o saneamento básico foram investidos R$ 102.960.276,00. Comparando­se as duas quantias, o gasto com saneamento é menos da metade. Disponível em: http://www.planejamento.rj.gov.br/orcamentoRJ/2007_LOA.pdf. Acesso em: 9/10/2009. ARENDT, Hannah, A Condição Humana, 10. ed., 2ª reimp., Rio: Forense, 2002, capítulo II – as esferas pública e privada, pp. 31/88. STRECK, Lenio Luiz. Decisionismo e discricionariedade judicial em tempos pós­positivistas: o solipsismo hermenêutico e os obstáculos à concretização da Constituição no Brasil. Separata: o Direito e o Futuro do Direito, Almedina: Coimbra, 2008, p. 99. STRECK, Lenio Luiz. Verdade e Consenso. Constituição, Hermenêutica e Teorias Discursivas Da possibilidade à necessidade de respostas corretas em Direito. 3..ed Rio de Janeiro: Lumen Juris. 2009 p. 499 Nesse sentido ver GADAMER, Hans Georg. Verdade e Método: Traços Fundamentais de uma Hermenêutica Filosófica. 4. ed. Petrópolis: Vozes, 2002. Nesse sentido ver STRECK, Lenio Luiz. Op cit. p. 561­562. Tal qual Kelsen explicita em seu cap. 8°, no qual trata da interpretação. KELSEN, Hans. Teoria pura do direito. 7. Ed. São Paulo: Martins Fontes. 2006. REFERÊNCIAS ALTHUSSER, Louis. Montesquieu – A política e a história. 2. ed., Lisboa: Editorial Presença. ARENDT, Hannah. A Condição Humana, 10. ed., 2ª reimp., Rio: Forense, 2002. _______, Hannah. Los orígenes del totalitarismo. Madrid: Taurus, 1998, p. 368­ 382. BARROSO, Luís Roberto. Da falta de efetividade à judicialização excessiva: direito à saúde, fornecimento gratuito de medicamentos e parâmetros para a atuação judicial. Disponível em: http://www.lrbarroso.com.br/pt/noticias/medicamentos.pdf Acesso em: 9/10/2009 _________, Luis Roberto. Fundamentos teóricos e filosóficos do novo direito constitucional brasileiro (pós­modernidade, teoria crítica e pós­positivismo). Revista do Ministério Público, Porto Alegre, nº 46, jan/mar, 2002. BILBAO UBILLOS, Juan Maria. La eficacia de los derechos fundamentales frente a particulares. Madrid: CEC, 1997. 171 BUCCI. O conceito de política pública em direito, p. 31, in BUCCI, Maria Paula Dallari (org). Política públicas: reflexões sobre o conceito jurídico. São Paulo: Saraiva, 2006. CADERMATORI, Sergio. Estado de Direito e Legitimidade: uma abordagem garantista. 2. ed., Campinas: Millennium, 2006. CANOTILHO, J.J. Gomes e MOREIRA Vital. Fundamentos da Constituição. Coimbra: Coimbra Ed., 1991. COUTO e SILVA, Almiro in apresentação do livro Elementos de Direito Administrativo Alemão, MAURER, Hart, Porto Alegre: Fabris, 2000. EWALD, François. L’Etat Providence. Paris: Bernard Grasset, 1986. GADAMER, Hans Georg. Verdade e Método: Traços Fundamentais de uma Hermenêutica Filosófica. 4. ed. Petrópolis: Vozes, 2002. GARCÍA DE ENTERRÍA, Eduardo. Las Transformaciones de La Justicia Administrativa: de excepción singular a La plenitud jurisdiccional. ¿Um cambio de paradigma? Pamplona: Thomson Civitas, 2007. HESSE, Conrado. Significado de los derechos fundamentales. In Manual de Derecho Constitucional, BENDA et als. , Madrid: Instituto Vasco de Administración Pública, 1996. HESSE, Konrad; MENDES, Gilmar Ferreira (trad.). A forca normativa da constituição. Porto Alegre: Sergio Antonio Fabris, 1991. HOWLETT, Michael e RAMESH, M. Come studiare le poliche pubbliche. Bologna: Il Mulino, 1995. KELSEN, Hans. Teoria pura do direito. 7. ed. São Paulo: Martins Fontes, 2006. KRELL, Andreas J. Direitos Sociais e controle judicial no Brasil e na Alemanha: os (des)caminhos de um direito constitucional comparado. Porto Alegre: Fabris, 2002. LAUBADÈRE, André. Manuel de Droi Administratif, 15e édition, L.G.D.J., Paris, 1995. LEAL, Rogério Gesta. A quem compete o dever de saúde no direito brasileiro? Esgotamento de um modelo institucional. Revista de Direito Sanitário, v. 9, p. 50­69, 2008. MAURER, Hart. Elementos de Direito Administrativo Alemão. Porto Alegre: Fabris, 2007 MIR PUIGELAT, Globalización, Estado y Derecho. Las transformaciones recientes del Derecho administrativo. Madrid: Civitas, 2004. MOREIRA NETO, Diogo de Figueiredo. Novos horizontes para o direito administrativo: pelo controle das políticas públicas. Revista de Direito do Estado, Rio de Janeiro, nº 4: 403­412, out/dez, 2006. 172 ______, Diogo de Figueiredo. Quatro paradigmas do direito administrativo pós­ moderno: legitimidade, finalidade, eficiência, resultados. Belo Horizonte: Fórum, 2008. MOTESQUIEU, Charles Louis de Secondat. Do espírito das leis. 2. ed., São Paulo: Abril Cultural, 1979. Organização Mundial da Saúde, Pautas para establecer políticas farmacéuticas nacionales, Ginebra: OMS, 1988. SARLET, Ingo.Wolfgang . A eficácia dos direitos fundamentais, 7. ed. Porto Alegre: Livraria do Advogado, 2007. SARLET, Ingo.Wolfgang . A eficácia do direito fundamental à segurança jurídica: dignidade da pessoa humana, direitos fundamentais e proibição de retrocesso social no direito constitucional brasileiro, in Constituição e Segurança Jurídica, ROCHA, Cármen Lúcia Antunes Coord., Belo Horizonte: Fórum, 2004. SOBRAVIME, O que é o uso racional de medicamentos, São Paulo: SOBRAVIME, 2001. SIGMANN, Jean. 1848­ Las revoluciones románticas y democráticas de Europa. 3. ed., Madrid: Siglo veintiuno editorial, 1985. SCLIAR, Moacyr. Do mágico ao social: trajetória da saúde pública, São Paulo: SENAC, 2002. STRECK, Lênio Luiz. Decisionismo e discricionariedade judicial em tempos pós­ positivistas: o solipsismo hermenêutico e os obstáculos à concretização da Constituição no Brasil. Separata: o Direito e o Futuro do Direito, Almedina: Coimbra, 2008. _______, Lênio. Jurisdição Constitucional e Hermenêutica: uma nova crítica do direito. 2. ed. Rio de Janeiro: Forense, 2004. _______, Lênio Luiz. Verdade e Consenso. Constituição, Hermenêutica e Teorias Discursivas Da possibilidade à necessidade de respostas corretas em Direito. 3. ed. Rio de Janeiro: Lumen Juris. 2009. SZWARCWALD, Célia L. Mortalidade infantil no Brasil: Belíndia ou Bulgária? Cadernos de Saúde Pública, vol. 13, nº 3, Rio de Janeiro, 1997. Disponível também em: http://www.scielo.br/pdf/csp/v13n3/0175.pdf. Acesso em: 9/10/2009. WALINE, Marcel. Traité Élémentaire de Droit Administratif, 6e édition, Recueil Sirey, Paris, 1952. Recebido para publicação 23/10/2009 Aceito para publicação 12/12/2009 

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RESEARCH ARTICLE Human Tregs at the materno-fetal interface show site-specific adaptation reminiscent of tumor Tregs Judith Wienke,1 Laura Brouwers,2 Leone M. van der Burg,1 Michal Mokry,3,4 Rianne C. Scholman,1 Peter G.J. Nikkels,5 Bas B. van Rijn,2,6 and Femke van Wijk1 Center for Translational Immunology, 2Wilhelmina Children’s Hospital Birth Center, 3Regenerative Medicine Utrecht, 1 Laboratory of Clinical Chemistry and Hematology, and 5Department of Pathology, Wilhelmina Children’s Hospital, 4 University Medical Center Utrecht, Utrecht University, Netherlands. 6Obstetrics and Fetal Medicine, Erasmus MC University Medical Center Rotterdam, Rotterdam, Netherlands. Tregs are crucial for maintaining maternal immunotolerance against the semiallogeneic fetus. We investigated the elusive transcriptional profile and functional adaptation of human uterine Tregs (uTregs) during pregnancy. Uterine biopsies, from placental bed (materno-fetal interface) and incision site (control) and blood were obtained from women with uncomplicated pregnancies undergoing cesarean section. Tregs and CD4+ non-Tregs were isolated for transcriptomic profiling by Cel-Seq2. Results were validated on protein and single cell levels by flow cytometry. Placental bed uTregs showed elevated expression of Treg signature markers, including FOXP3, CTLA-4, and TIGIT. Their transcriptional profile was indicative of late-stage effector Treg differentiation and chronic activation, with increased expression of immune checkpoints GITR, TNFR2, OX-40, and 4-1BB; genes associated with suppressive capacity (HAVCR2, IL10, LAYN, and PDCD1); and transcription factors MAF, PRDM1, BATF, and VDR. uTregs mirrored non-Treg Th1 polarization and tissue residency. The particular transcriptional signature of placental bed uTregs overlapped strongly with that of tumor-infiltrating Tregs and was remarkably pronounced at the placental bed compared with uterine control site. In conclusion, human uTregs acquire a differentiated effector Treg profile similar to tumor-infiltrating Tregs, specifically at the materno-fetal interface. This introduces the concept of site-specific transcriptional adaptation of Tregs within 1 organ. Introduction Conflict of interest: The authors have declared that no conflict of interest exists Copyright: © 2020, Wienke et al. This is an open access article published under the terms of the Creative Commons Attribution 4.0 International License. Submitted: April 1, 2020 Accepted: August 12, 2020 Published: September 17, 2020. Reference information: JCI Insight. 2020;5(18):e137926. https://doi.org/10.1172/jci. insight.137926. In the past decade, T cells have been identified in various human and murine nonlymphoid tissues (1, 2). These tissue-resident memory T cells (TRM) do not recirculate, serve as first-line responders to infections, and are characterized by expression of signature molecules such as CD69, which prevents their tissue egress (1, 3–8). TRM adapt to tissue environments by acquiring a specialized functional phenotype that depends on microenvironmental cues (9, 10). Also Tregs, critical gatekeepers of immune homeostasis (11), have been recently identified in murine and human tissues (12–16). Like TRM, Tregs can become resident and gain a polarized phenotype, with functional specialization depending on the tissue or organ, which is controlled on a transcriptional level (14–22). Although increasing evidence in mice supports functional adaptation of Tregs to nonlymphoid tissue environments (23), studies in humans are still scarce (14, 17). However, transcriptional adaptation of Tregs has gained special interest in the tumor environment, due to the important therapeutic implications (24). Tumor-infiltrating Tregs (TITR) display a unique and specialized transcriptional signature (25), associated with activation and functional specialization, including increased suppressive capacity (25–27). Tissue and tumor Tregs undergo differentiation reminiscent of effector Tregs, with potent suppressive capacity, and are characterized by expression of CD45RO and increased CD25, CTLA-4, and HLA-DR (28–31). Furthermore, effector Tregs (in tissues and tumors) express high levels of immune checkpoint molecules OX-40, 4-1BB, GITR, TIGIT, and ICOS and transcription factors such as BLIMP-1 (encoded by PRDM1) and BATF (19, 25–27, 31–34). Effector Tregs can mirror effector Th cell polarization by acquiring coexpression of FOXP3 with chemokine receptors and transcription factors associated with Th1 (CXCR3, T-bet), Th2 (GATA3, IRF4), or Th17 (RORγt, STAT3) differentiation (19, 35–38). insight.jci.org   https://doi.org/10.1172/jci.insight.137926 1 RESEARCH ARTICLE This specific polarization is associated with an enhanced suppressive efficacy toward the matching T effector response (31, 36–45). Since most of these insights have been generated in mice, it is still largely unknown whether these principles also apply to human tissue Tregs. As recently highlighted (46), one of the most interesting yet elusive tissue sites for Treg function in humans is the materno-fetal interface. Pregnancy is a mystifying biological process when viewed from an immunological perspective, posing a unique challenge to the maternal immune system (47, 48). While peripheral immunity against pathogens needs to remain intact, the semiallogeneic fetus and placenta, which may harbor foreign paternal antigens, need to be tolerated (49). The maternal immune response is therefore delicately balanced and requires tight regulation especially locally at the materno-fetal interface, which is underlined by the fact that human decidual T cells can recognize and actively respond to fetal cord blood cells (47–51). Maternal Tregs are consequently indispensable for successful embryo implantation and pregnancy outcome, and they contribute to materno-fetal tolerance on multiple levels (47, 52, 53). Depletion of murine maternal Tregs causes pregnancy loss due to immunological rejection of the fetus (53, 54). In humans, maternal Tregs are abundantly present in the gravid uterus (55–62), and normal human pregnancy is characterized by increased numbers of Tregs in the periphery and at the materno-fetal interface (56, 61, 63, 64). In patients with preeclampsia, a severe hypertensive pregnancy disorder, and patients with recurrent miscarriages, Treg numbers are reduced both at the materno-fetal interface and in the periphery (57, 65–70), implying that — also in humans — local presence of Tregs in the pregnant uterus is required for successful pregnancy outcome. Previous studies investigating the maternal, uterine immune system in humans have been limited by the practical challenge of acquiring biopsy material of the uterine wall and have made use of the thin superficial decidual layer attached to the delivered placenta, which is heavily contaminated by fetal immune cells and may not be representative of the maternal Treg status during pregnancy. Moreover, the functional and transcriptomic profile of human uterine Tregs (uTregs) from the materno-fetal interface and its relation to Tregs from other human tissues remain to be elucidated. Here, we investigated functional adaptation and specialization of highly purified human, exclusively maternal, resident uTregs in myometrial biopsies from the materno-fetal interface. We performed transcriptomic profiling and functional in vitro assays, as well as flow cytometry, to study their phenotypic heterogeneity on protein level in single cell resolution. To identify tissue site–specific functional adaptation, we compared these uTregs with uTregs from a distant uterine control site and maternal peripheral bTregs, in addition to tissue- and site-matched resident CD4+ non-Tregs. Last, we observed the specific profile of functional adaptation of uTregs compared with known Treg signatures from other human and murine tissue sites, including tumors. Results uTregs are bona fide suppressive Tregs. The frequency of CD25hiFOXP3+ Tregs within the CD4+ T cell population was similar between blood and uterine tissue and ranged from 2.5% to 13.5% (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci. insight.137926DS1). For transcriptional profiling, the CD3+CD4+CD25hiCD127– population (Tregs) and CD3+CD4+CD25–CD45RA– memory T cells (Tconv) were FACS sorted from peripheral blood and myometrial biopsies from 5 women with uncomplicated pregnancies undergoing cesarean section. In myometrium, Tconv were selected for CD69 positivity. The sorting strategy is shown in Supplemental Figure 1B. Confirming the maternal origin of the sorted cells, the female-specific gene XIST was highly expressed in all samples, whereas transcripts of the male-specific gene SRY were undetectable in all samples, including pregnancies with male offspring (Supplemental Figure 1C). Principal component analysis (PCA) of transcriptomic profiles showed that uTregs from the materno-fetal interface are clearly distinct from blood-derived Tregs (bTregs), and that also uterine T conv (uTconv) and blood-derived Tconv (bTconv) clearly cluster apart (Figure 1A). Notably, PC1, mounting the difference between the cell sources, accounted for > 60% of the variance, whereas PC2, explaining variance between Treg and Tconv populations, accounted for only 11% of the variance. To assess whether the sorted population of uTregs consisted of bona fide Tregs, we analyzed enrichment of a published core Treg gene signature (71) in uTregs compared with uTconv and bTregs by gene set enrichment analysis (GSEA). Expression of Treg core signature genes was not only enriched compared with uTconv, but, remarkably, also more pronounced in uTregs than in bTregs, indicating that uTregs are bona fide Tregs with enhanced expression of Treg core signature genes (Figure 1, B and C). Indeed, expression of many of the published Treg markers (71) was higher in uTreg than bTreg (Figure 1D). insight.jci.org   https://doi.org/10.1172/jci.insight.137926 2 RESEARCH ARTICLE Figure 1. Tregs at the materno-fetal interface are bona fide Tregs. (A) Principal component analysis of bTregs, bTconv, uTregs, and uTconv (all n = 5). (B and C) GSEA with published Treg signature gene set (71) comparing uTreg and uTconv (B) and uTreg and bTreg (C) (all n = 5). NES, normalized enrichment score; FDR, FDR adjusted P value. (D) Heatmap of genes in leading edge of GSEA comparing enrichment of published Treg signature genes in uTregs and bTregs. Expression values were mean centered and scaled per gene. (E) Representative gating strategy of bTregs, uTregs, and uTconv out of 5 experiments. (F) Representative expression of CTLA-4 in uTregs out of 6 experiments. (G) Ex vivo protein expression of core Treg molecules FOXP3 (n = 10) and CTLA4 (n = 6) measured by flow cytometry. (H) Ex vivo protein expression of Treg signature molecule TIGIT (n = 6) measured by flow cytometry. (G and H) Multiplicity adjusted P value of 2-way ANOVA with Tukey’s post hoc test. (I) GSEA of TIGIT+ Treg signature (n = 5) (72). Higher expression of the Treg-identifying molecules FOXP3 and CTLA-4 in uTregs than bTregs was confirmed on protein level (Figure 1, E–G). Also TIGIT, a key checkpoint molecule associated with specialized suppressive function (72), was highly expressed in uTregs, with the majority of uTregs being positive for TIGIT (Figure 1H). Consistently, GSEA showed significant enrichment of a previously identified TIGIT+ Treg signature (Figure 1I) (72). Suppression assays, although technically challenging due to low cell numbers, confirmed the suppressive potential of uTregs on proliferation and cytokine production of healthy donor peripheral blood–derived CD4+ T cells (Figure 2 and Supplemental Figure 1, D and E). Two of 4 uTreg donors showed particularly high suppressive capacity of uTregs on cytokine production of IL-2, IL-10, IFN-γ, and TNF-α, already at a 1:8 (Treg/Tconv) ratio, compared with bTregs. These results confirm that the sorted uTregs are bona fide functional Tregs, with enhanced expression of Treg signature genes. The uTreg signature indicates an activated and effector Treg profile. To investigate the functional adaptation of uTregs to the specific environment of the materno-fetal interface, we determined both their functional differentiation and Th polarization, both of which may be influenced by the tissue environment (12, 13, 20, 33, 73–75). insight.jci.org   https://doi.org/10.1172/jci.insight.137926 3 RESEARCH ARTICLE To identify the uTreg-specific transcriptional signature, we assessed their differential gene expression with both bTregs and uTconv. Many genes were differentially expressed between uTregs and bTregs (Figure 3A), with significant upregulation of 1966 genes and downregulation of 1997 genes in uTregs (adjusted P value [Padj] < 0.05 and |log2FC| > 0.5). To isolate the uTreg-specific signature, we also compared gene expression between uTreg and uTconv, yielding 465 upregulated genes — including the Treg-identifying genes FOXP3, IL2RA, CTLA4, TIGIT, and IKZF2 — and 103 downregulated genes in uTregs (Padj < 0.05 and |log2FC| > 0.5; Figure 3B); 236 genes were specifically upregulated (225 after removal of duplicate genes) and 23 genes specifically downregulated in uTregs compared with both bTregs and uTconv (Figure 3C, Supplemental Table 4, and Supplemental Table 5). Among the downregulated genes were ITGA6, IL7R, CCR7, TTC39C, PLAC8, ATF7IP2, ABLIM1, MGAT4A, PRKCB, and GIMAPs, as well as transcription factors TCF7, LEF1, and SATB1, indicating late-stage differentiation of Tregs (76, 77). The 225 upregulated genes were involved in cytokine signaling, TNF receptor signaling, and glycolysis (Figure 3D). Selected genes from the top 5 pathways included those related to Treg activation and effector differentiation, such as immune checkpoints of the TNF receptor superfamily (TNFRSF13B [TACI], TNFRSF18 [GITR], TNFRSF1B [TNFR2], TNFRSF4 [OX-40], TNFRSF8 [CD30], TNFRSF9 [4-1BB]) and HLA-DR, CD80, and LRRC32 (GARP). Furthermore, genes associated with suppressive capacity (CTLA4, ENTPD1, HAVCR2, IL10, IL2RA, LAG3, LAYN, LGALS1, PDCD1, and TOX2) were highly expressed in uTregs (Figure 3E) (19, 31), and cytokine receptors of the IL-1 and IL-2 family (IL1R1, IL1R2, IL1RAP, IL1RN, IL2RA, and IL2RB) and specific chemokine receptors (CCR1, CXCR6) showed increased and specific expression in uTregs (Figure 3E). Transcription factors specifically upregulated in uTregs included BATF, CEBPB, ETS2, ETV7, HES1, IKZF4, MAF, NFIL3, PRDM1, VDR, and ZBTB32 among others (Figure 3E). This transcriptomic profile, and especially high expression of BATF, PRDM1, and immune checkpoint molecules, reflects previously identified crucial signatures of effector Treg differentiation and function, especially in tissues (29, 32, 33, 78–80). We confirmed upregulation of immune checkpoints associated with effector Treg differentiation/ chronic stimulation GITR, OX-40, 4-1BB, PD-1, HLA-DR, and ICOS in uTregs, even compared with uTconv, on the protein level (Figure 3F). Since increased expression of many of these genes pointed toward an activated phenotype, we confirmed this by demonstrating significant enrichment of published gene sets of in vitro–activated Tregs in uTregs (Supplemental Figure 2 and Supplemental Table 3) (81–85). Taken together, uTregs at the materno-fetal interface have a highly differentiated transcriptional signature suggestive of a specialized function with high suppressive capacity and high responsiveness to environmental cues, which is reflective of late-stage effector differentiation and chronic activation. uTregs have a tissue-resident phenotype and share transcriptional specialization with uTconv. To examine whether uTregs at the materno-fetal interface represent a resident population or rather transiently infiltrating cells, we assessed the expression of tissue residency–related markers and gene signatures. uTregs had a significantly higher gene and protein expression of key residency molecule CD69 than bTregs and bTconv, similar to uTconv (Figure 4A). Expression analysis and GSEA with published human TRM signatures showed a pattern of upregulated and downregulated genes as previously described for human TRM in general (Figure 4B) and specifically in CD4+ (and CD8+) TRM from lung and skin (Figure 4C) (4, 14, 86), confirming the tissue-resident profile in uTregs as compared with bTregs. Next, we identified the shared tissue-specific adaptation of uTregs and uTconv to the materno-fetal interface. A large proportion of upregulated and downregulated genes was shared between uTregs and uTconv compared with their counterparts from blood (Supplemental Figure 3A; 1032 genes up and 1348 down; Padj < 0.05 and |log2FC| > 0.5), which again suggests that the specific tissue environment at the materno-fetal interface accounts for a significant part of their adapted transcriptional profile. Shared upregulated genes were involved in cytokine signaling (Supplemental Figure 3B), highlighting the integration of a spectrum of microenvironmental cues, while shared downregulated genes were reflective of ribosomal processes involved in RNA translation (Supplemental Figure 3C). Taken together, uTregs have a TRM signature that reflects a shared adaptation to the tissue environment of the materno-fetal interface between uTregs and uTconv. uTregs mirror uTconv Th1 polarization with a predominance of T-bet+CXCR3+ Tregs. Effector Tregs can acquire different Th phenotypes with coexpression of FOXP3 and lineage-defining transcription factors T-bet (TBX21, Th1), GATA3 (Th2), and RORγt (RORC, Th17), as well as lineage-associated cytokine and chemokine receptors (35). We investigated whether uTregs and uTconv underwent a, possibly shared, Th polarization. uTregs showed significantly increased expression of Th1-related TBX21 compared with bTreg, which mirrored the increased expression of TBX21 in uTconv (Figure 5A). Th2-related GATA3 and Th17-related RORC were not significantly differentially expressed between uTreg and insight.jci.org   https://doi.org/10.1172/jci.insight.137926 4 RESEARCH ARTICLE Figure 2. Tregs at the materno-fetal interface are bona fide Tregs with suppressive capacity. (A–D) Suppression assay assessing cytokine production of IL-2 (A), IFN-γ (B), TNF-α (C), and IL-10 (D) by anti-CD3–stimulated (or unstimulated) healthy CD4+ T cells in the supernatant by multiplex immunoassay after 4 days of coculture with healthy donor bTregs, maternal bTregs, or uTregs at a 1:8, 1:4, and 1:2 ratios. Data represent median with interquartile range. n = 4 donors, but not every condition could be measured for each donor due to limited availability of material. Therefore, some conditions contain data from 3 donors. bTreg (and uTconv and bTconv), although RORC showed a trend toward downregulation, which was confirmed on the protein level (Figure 5, A and B). Increased expression of T-bet was also confirmed on the protein level, with 6%–87% (median 22%) of uTregs showing positivity for T-bet (Figure 5, C and D). Also, the Th1-related cytokine receptor IL-18R1 was increased in both uTregs and uTconv compared with blood T cells on the gene and protein levels (Figure 5E). Investigation of chemokine receptor expression, related to both Th polarization and tissue-specific homing (87, 88), showed that chemokine receptors associated with naive Tregs and lymphoid tissue environments CCR7 and CXCR5 were downregulated in uTregs compared with bTregs, on the gene and protein levels (Figure 5, F and G). Chemokine receptors upregulated in uTregs included CCR2, CCR5, CXCR3, CXCR4, CCR1, and CXCR6 (Figure 5, F and H), which largely mirrored expression by uTconv. CCR1 and CXCR6 were specifically upregulated in uTregs, both previously identified as part of the conserved murine tissue Treg signature (18). The Th1-associated CXCR3 (36, 89) and Th1/inflammation-associated CCR5 (89–92) had significantly higher gene and protein expression in uTregs and uTconv compared with their counterparts from blood (Figure 5, F and H). Although the variable percentage of T-bet+ Tregs suggests heterogeneity in uTreg subspecialization, virtually all uTregs (and uTconv) were positive for CXCR3 (84%–100%, median 93%), and the majority expressed CCR5 (22%–83%, median 62%) (Figure 5I). Consistent with these findings, a previously published gene signature of T-bet+CXCR3+ Tregs from the pancreas of prediabetic mice was highly enriched in uTregs compared with bTregs (Figure 5J) (38). In conclusion, uTregs at the materno-fetal interface show Th1 polarization mirroring uTconv, with high expression of Th1-related markers T-bet and CXCR3. Furthermore, uTregs express an array of chemokine receptors with which they can integrate a variety of locally produced signals. While some of these receptors are uTreg specific, others are shared with uTconv. uTreg and uTconv cells may therefore rely on both unique and shared cues to guide their migration to and retention at the uterine materno-fetal interface. The uTreg signature at the materno-fetal interface overlaps with TITR signatures. We questioned whether the highly differentiated uTregs from the materno-fetal interface would resemble Tregs from other human and murine tissue sites or would show a uniquely adapted profile. Well-studied murine tissue Treg populations include Tregs from visceral adipose tissue (VAT), muscle, and intestines (12, 16, 18, 73, 93). Each population displays a tissue-specific phenotype with expression of certain (a) transcription factors, (b) chemokine receptors, and (c) preference toward a Th lineage differentiation when compared with spleen Tregs (12, 13, 16, 18, 73). insight.jci.org   https://doi.org/10.1172/jci.insight.137926 5 RESEARCH ARTICLE Figure 3. The uTreg core signature. (A and B) Volcano plot of differential gene expression between uTregs and bTregs (A) or uTregs and uTconv (B) (all n = 5). (C) Venn diagrams yielding genes specifically upregulated (Padj < 0.05 and log2FC > 0.5, left panel) or downregulated (Padj < 0.05 and log2FC < –0.5, right panel) in uTreg compared with bTreg and uTconv. (D) Pathway analysis (ToppGene pathways) of 236 genes specifically upregulated in uTregs. P < 0.05 after Bonferroni’s correction were considered significant. (E) Heatmaps showing gene expression of genes in top 5 pathways and selected downregulated genes in the uTreg core signature, related to Treg activation or effector differentiation (left panel), (cytokine) signaling (middle panel; including downregulated CCR7 and IL7R), and transcription factors (right panel). Expression values were mean centered and scaled per gene. (F) Protein expression of GITR (TNFRSF18), OX-40 (TNFRSF4), 4-1BB (TNFRSF9), PD-1 (PDCD1), HLA-DR, and ICOS. uTregs were gated as CD3+CD4+CD25hiFOXP3+ cells. Multiplicity adjusted P value of 2-way ANOVA with Tukey’s post hoc test. n = 6 each. A murine PAN-tissue signature, shared by VAT, muscle, and intestinal Tregs, was also identified (18). GSEA in Figure 6A shows that the shared murine PAN-tissue Treg signature was also strongly enriched in uTregs, again highlighting its generalized expression in tissue Tregs, apparently even conserved across species. Overlaying significantly upregulated genes in uTreg (versus bTreg) with murine tissue–specific or murine tissue–shared Treg signatures (18), yielded a large amount of shared genes between uTregs and murine VAT–, colon–, and muscle–derived Tregs (Figure 6B, numbers in each field represent overlap of the specific field with significantly upregulated genes in uTreg). Fifty-nine genes were shared among all 3 murine tissues and uTregs, including IL1RL1 (receptor for IL-33, ST2), AREG, IL10, IRF4, GZMB, TNFRSF9, BHLHE40, NR4A1, NR4A3, and CCR2, many of which have been described as crucial regulators insight.jci.org   https://doi.org/10.1172/jci.insight.137926 6 RESEARCH ARTICLE Figure 4. Tregs at the materno-fetal interface have a tissue-resident profile. (A) Gene and protein expression of CD69 in sorted T cell populations. DE, differentially expressed genes with Padj < 0.05. Box plots with median: box indicates 25th to 75th percentiles, whiskers indicate minimum and maximum values (n = 5). FACS data: representative plot of 5 experiments. uTregs were gated as CD3+CD4+CD25hiCD127–. MFI, median fluorescence intensity. Two-way ANOVA with Tukey’s post hoc test. (B) Heatmap with a published human core tissue–resident signature (4) in uTreg compared with bTreg. Expression values were mean centered and scaled per gene. (C) GSEA with published genes identifying human lung CD4+ and CD8+ TRM compared with blood memory cells; ref. 86) and genes upregulated/downregulated in skin CD4+ TRM compared with blood CD4+ T cells (14), in uTregs versus bTregs. NES, normalized enrichment score; FDR, FDR adjusted P value. for effector and/or tissue Treg function (Figure 6C) (12, 32, 40, 78–80, 94). Twelve of the 59 genes were even part of the uTreg-specific core signature as defined in Figure 3 (CCR1, CXCR6, ELL2, FGL2, GEM, IL10, LAPTM4B, SNX9, TNFRSF8, NFIL3, NR4A3, and PRDM1). This indicates that uTregs display features of tissue adaptation, which are highly conserved across tissues and species. The investigation of human tissue–derived Tregs has proven challenging, and only limited data are available. To assess how uTregs compare with other human tissue Tregs, we analyzed enrichment of 3 previously published gene sets of significantly upregulated genes in healthy skin, colon, and lung Tregs compared with bTregs (Supplemental Table 3) (14, 17). All 3 signatures were significantly enriched in uTregs compared with bTregs, indicating that the tissue profile of uTregs shows similarities with human Tregs from various tissue sites (Supplemental Figure 4). Human Tregs infiltrating the unique tissue-environment of tumors (TITR) have been studied more extensively. Comparison of genes significantly upregulated in uTregs versus bTregs with 7 recently published gene signatures of TITR infiltrating a variety of human tumors (Supplemental Table 3) (25, 95–100) yielded a remarkable overlap with each of the TITR signatures, with up to 65% of genes shared with uTregs (Supplemental insight.jci.org   https://doi.org/10.1172/jci.insight.137926 7 RESEARCH ARTICLE Figure 5. uTreg and uTconv polarization at the materno-fetal interface. (A) Gene expression of lineage-defining transcription factors TBX21 (T-bet), GATA3 (GATA-3), and RORC (RORγt). P values from differential gene expression analysis. DE, differentially expressed Padj < 0.05. Box plots with median; box indicates 25th to 75th percentiles, and whiskers indicate minimum and maximum values (all n = 5). (B–D) Protein expression of RORγt (B) and T-bet (C and D). uTregs were gated as CD3+CD4+CD25hiFOXP3+ cells. MFI, median fluorescence intensity. Multiplicity adjusted P values of 2-way ANOVA with Tukey’s post hoc test.(n = 5) (E) Gene and protein expression of IL18R1 (IL-18R1). Gene expression: box plots with median — box indicates 25th to 75th percentiles, and whiskers indicate minimum and maximum values (n = 5). DE, differentially expressed Padj < 0.05. Protein expression: uTregs were gated as CD3+CD4+CD25hiFOXP3+ cells. Multiplicity adjusted P values of 2-way ANOVA with Tukey’s post hoc test (n = 5). (F) Heatmap showing gene expression of chemokine receptors. Expression values were mean centered and scaled per gene. DE, differentially expressed Padj < 0.05. (G–I) Protein expression of chemokine receptors downregulated (G) and upregulated (H and I) in uTregs. uTregs were gated as CD3+CD4+CD25hiFOXP3+ cells. P values of 2-way ANOVA with Tukey’s post hoc test (n = 5). (J) GSEA with published gene set of CXCR3+T-bet+ Tregs from the pancreas of prediabetic mice (38), comparing uTregs and bTregs. NES, normalized enrichment score; FDR, FDR adjusted P value. Table 6). Of the 41 genes that were shared among ≥ 4 of the 7 TITR signatures (Supplemental Table 7), 31 were also part of the 225 genes in the uTreg core signature. Figure 6D shows the number of genes shared between the uTreg core signature, as well as each of the TITR signatures and healthy tissue–derived Treg signatures. Remarkably, 93 (41.3%) of the 225 core uTreg genes were overlapping with specifically upregulated genes from hepatocellular carcinoma–infiltrating (HCC-infiltrating) Tregs (98), 54 with the unique TITR signature identified by De Simone et al. (25), 49 with breast cancer TITR genes (95), and 40 with OX-40+ Treg from cirrhotic/ tumor liver tissue (Figure 6D) (99). Importantly, the 225 uTreg core signature genes showed less overlap with healthy tissue–derived Treg–specific signatures from human healthy colon, lung, and skin. The genes that were most often shared between uTregs and TITR were IL1R2 (7 of 7); TNFRSF1B, CTSC, DPYSL2, and LAPTM4B (6 of 7) and TNFRSF4, TNFRSF18, LAYN, IL2RA, ENTPD1, NCF4, SDC4, and CRADD (5 of 7) (Figure 6E), whereas — with healthy tissue-Treg signatures — PDGFA was most often shared (3 of 3) (Figure 6F). insight.jci.org   https://doi.org/10.1172/jci.insight.137926 8 RESEARCH ARTICLE Figure 6. uTregs share their transcriptional signature with tissue- and tumor-infiltrating Tregs. (A) GSEA with a published murine PAN-tissue gene signature (18), comparing uTregs and bTregs. NES, normalized enrichment score; FDR, FDR adjusted P value. (B) Venn diagram showing the numbers of genes upregulated in uTregs compared with bTregs (Padj < 0.05) (and genes in the uTreg core signature in parentheses), which are represented in tissue-specific and tissue-shared published murine gene signatures (18). VAT, visceral adipose tissue. (C) Heatmap showing the expression of the 59 genes that were part of the murine PAN-tissue signature and upregulated in uTregs compared with bTregs (Padj < 0.05) (18). Expression values were mean centered and scaled per gene. (D) The number of genes shared between the uTreg core signature and published human TITR signatures or healthy tissue Treg signatures (14, 17, 25, 95–100). Numbers behind bars indicate the number of shared genes out of the total number of genes in the specific signature. (E) The genes that were most often shared between the uTreg core signature and human TITR signatures (shared in ≥ 4 of 7 signatures). (F) The genes that were most often shared between the uTreg core signature and human healthy tissue Treg signatures (shared in ≥ 2 of 3 signatures). GSEA also showed that many of the nonoverlapping genes from the published TITR signatures were significantly enriched in uTregs compared with bTregs (Figure 7A). Genes in the leading edge of ≥ 3 of 7 tumor-specific GSEA that were highly expressed in uTregs compared with bTregs, but not part of the uTreg core (mostly because their high expression was shared with uTconv), are shown in Figure 7B. These included CREB3L2 (6 of 7); EBI3, GCNT1, and ICOS (5 of 7); ACTA2, ARHGEF12, BCL2L1, CCND2, PRDX3, and SLAMF1 (4 of 7); and CXCR3, CD7, CAECAM1, CD79B, and MICAL2 (3 of 7), among others. insight.jci.org   https://doi.org/10.1172/jci.insight.137926 9 RESEARCH ARTICLE Remarkably, genes specifically upregulated in breast cancer–infiltrating Tregs compared with Tregs from normal breast parenchyma or significantly upregulated in colon cancer Tregs compared with healthy colon Tregs showed a particularly high enrichment in uTregs, suggesting that uTregs are not just similar to Tregs from breast or colon tissue, but specifically to the highly differentiated/activated Tregs from the tumor environment (Figure 7C) (26, 100). By overlapping these cancer-versus-healthy tissue Treg signatures with significantly upregulated genes in uTregs (versus bTregs), we identified 12 cancer-specific genes expressed by uTregs (Figure 7D): CD80, IL1R2, LAYN, MYO7A, TNFRSF4, TNS3, TRAF3, VDR, DUSP4, HSPA1A, HSPA1B, and IFI6. The first 8 of these were also part of the uTreg-specific core signature, again highlighting the specificity c.q. importance of receptors IL1R2, LAYN, TNFRSF4, and CD80 and transcription factor VDR for human Tregs in a tumor-like microenvironment. Furthermore, tumor-specific downregulated genes were shared with the uTreg core signature: CCR7, PLAC8, and TCF7. In conclusion, these results indicate that uTregs from the materno-fetal interface have a transcriptional core signature that is shared specifically with the specialized transcriptional profile of TITR. uTregs show site-specific adaptation within the uterus. Next, we wondered whether uTregs would be merely adapted to the microenvironment in uterine tissue or specifically adapted to the tissue site at the maternal-interface. To investigate this site-specific adaption within 1 organ, we compared uTregs from the materno-fetal interface (i.e., placental bed; pbuTregs) with uTregs from a distant uterine site (i.e., the incision site made during cesarean section; incuTregs). Confirmation of Treg identity and TRM signature for incuTregs is shown in Supplemental Figure 5, A–F. The differentially expressed genes between inc uTregs and bTregs were similar to those between pbuTregs and bTregs (Figure 8A). Furthermore, PCA showed that gene expression profiles of pbuTregs and incuTregs were rather similar, compared with bTregs (Figure 8B). However, direct comparison of pbuTregs and incuTregs revealed a substantial difference between the 2 populations (Figure 8C). First, protein expression of the core Treg transcription factor FOXP3 was lower in incuTregs than pbuTregs, comparable with bTegs (Figure 8D). This was not due to incuTreg contamination with bTregs, since expression of CD69 was similar between pbuTregs and incuTregs (Supplemental Figure 5D). Protein expression of other core Treg markers CTLA-4 and TIGIT was also lower in incuTregs than pbuTregs (Figure 8D). This indicates that pbuTregs, derived from the materno-fetal interface, have a more pronounced expression of Treg signature markers, suggesting enhanced activation/differentiation in comparison with their uterine counterparts from the incision site. Differential gene expression analysis revealed 558 upregulated and 125 downregulated genes in pb uTregs versus incuTregs (Figure 8E). The heatmap in Figure 9A shows a selection of previously highlighted genes in this manuscript that proved to be differentially expressed between pbuTregs and incuTregs. These results suggest that Tregs cannot only adapt to the microenvironment within a certain tissue, but they will specifically adapt to the environmental cues at a certain tissue site. Pathway analysis showed that upregulated genes in pb uTregs versus incuTregs were related to PD-1 signaling, cytokine signaling, TCR signaling, and Th cell differentiation (Supplemental Figure 5G). Indeed, PD-1 was expressed higher in pbuTregs than inc uTregs on gene and protein levels (Figure 9, A and B), and GSEA showed enrichment of a TCR-activated Treg signature in pbuTregs compared with incuTregs (Supplemental Figure 5H). Furthermore, pb uTreg-specific core genes associated with effector Treg differentiation including TNFRSF4 (OX-40 protein, Figure 9C) and transcription factors BATF, MAF, PRDM1, and VDR, among others, were significantly expressed higher in pbuTreg than in incuTregs (Figure 9A), again suggesting that pbuTregs show more pronounced differentiation toward an effector Treg phenotype. Since pbuTregs appeared to be especially differentiated at the materno-fetal interface, we assessed whether the TITR-like profile of pb uTregs was also more pronounced than in incuTregs. Remarkably, 5 of 7 tested published TITR signatures were significantly enriched in pbuTregs compared with incuTregs (P < 0.05; Figure 9D). More specifically, GSEA with signatures differentiating between TITR and their counterparts from a matched healthy tissue site showed significant enrichment in pbuTregs compared with incuTregs (Figure 9E). CCR8 and ICOS, which were present in 6 of 7 TITR signatures, as well as TNFRSF18 (GITR), were expressed significantly higher in pbuTregs than in incuTregs and bTregs on the protein level (Figure 9F). CCR8 has been shown to be highly enriched in tumor Tregs and associated with a poor prognosis in several cancers (25, 26, 74). Thus, pbuTregs at the materno-fetal interface specifically acquire a highly differentiated effector profile similar to TITR, which is more pronounced even compared with a uterine tissue site distant from the materno-fetal interface. insight.jci.org   https://doi.org/10.1172/jci.insight.137926 10 RESEARCH ARTICLE Figure 7. uTregs have a functional profile similar to tumor-infiltrating Tregs. (A) GSEA with published TITR-specific signatures in uTregs versus bTregs (25, 95–100). NES, normalized enrichment score; FDR, FDR adjusted P value. (B) Heatmap showing expression of genes in the leading edge of ≥ 3 of 7 GSEA from Figure 7A, which were not represented in the uTreg core signature. Expression values were mean centered and scaled per gene. (C) GSEA with published gene signatures specific to Tregs from tumor tissue compared with the healthy tissue counterpart in uTregs versus bTregs(26, 100). NES, normalized enrichment score; FDR, FDR adjusted P value. (D) Venn diagrams showing shared genes between uTregs and genes specifically upregulated in Tregs from tumor tissue compared with the healthy tissue counterpart (26, 100). Discussion Here, we demonstrate for the first time to our knowledge that, in pregnancy, human uTregs have a highly differentiated transcriptional profile, which is specifically enriched at the materno-fetal interface and is reminiscent of the specialized profile of TITR. With these findings, we answer a long-standing question on how Tregs are functionally specialized at the materno-fetal interface to modulate local effector T cell responses, preventing an allo-reaction against the fetus. Moreover, we introduce the potentially novel concept of site-specific adaptation of Tregs within 1 organ or tissue. This again substantiates the notion that Tregs are capable of adapting their transcriptional program driven by microenvironmental cues (15–19). We have demonstrated that uTregs at the materno-fetal interface display a highly activated and late-stage differentiated effector profile (32, 34, 76, 77, 79), with high BATF and PRDM1, low SATB1, increased expression of molecules associated with suppressive capacity, and abundant expression of TNFR superfamily members. insight.jci.org   https://doi.org/10.1172/jci.insight.137926 11 RESEARCH ARTICLE Figure 8. uTregs show site-specific adaptation to the materno-fetal interface. (A) Venn diagrams of genes upregulated (left panel) and downregulated (right panel) in both incuTregs and pbuTregs compared with bTregs. (B) PCA of bTregs (n = 5), pbuTregs (n = 5), and incuTregs (n = 4). (C) PCA of pbuTregs (n = 5) and incuTregs (n = 4). (D) Protein expression of FOXP3, CTLA-4, and TIGIT. uTregs were gated as CD3+CD4+CD25hiFOXP3+ cells. Multiplicity adjusted P value of 2-way ANOVA with Tukey’s post hoc test. Left upper P value, blood versus placental bed; right upper P value, placental bed versus incision site; lower P value, blood versus incision site. MFI, median fluorescence intensity. (E) Volcano plot of differentially expressed genes between pbuTregs and incuTregs. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Also, others have reported that nonlymphoid tissue Tregs display an activated phenotype compared with lymphoid organ and circulating Tregs (12, 20, 73). Both BATF and the TNFRSF/NF-κB signaling axis are crucial in the survival of Tregs and maintenance of a stable effector Treg phenotype, especially in tissues (15, 28, 32, 33, 78, 79, 101). It is now recognized that Tregs adapt to their tissue environments, with, on the one hand, common adaptations across many tissues — such as increased expression of IL10, IL1RL1 (encoding ST2, an IL-33 receptor subunit), AREG (encoding amphiregulin), CTLA4, TIGIT, BATF, and IRF4 and decreased expression of LEF1, TCF7 — but, on the other hand, importantly, tissue-specific signatures (12, 13, 15, 16, 18). These tissue-specific signatures counter the notion that tissue Tregs merely have a more activated effector or memory state. Rather, they have a specialized adapted program (93), likely matching the specific requirements of a certain tissue site (10, 13, 18, 102, 103). To our knowledge, the concept of site-specific transcriptional adaptation of Tregs within 1 tissue or organ is novel, taking into account that tumors represent a completely altered tissue and not a different site within the same organ. We show that uTregs display features suggestive of a high responsiveness to microenvironmental cues, such as a range of TNF receptor superfamily members and chemokine receptors. With such a matrix of options to detect signals from the microenvironment, Tregs are likely able to adjust not only to the tissue or organ of their residence, but even to specific sites within that tissue, based on microenvironmental cues. Most likely, implantation of the placenta (i.e., the multitude of signals produced by myometrium-invading trophoblast; ref. 104) are the primary cues effectuating microenvironmental changes at the materno-fetal interface. Trophoblast attracts Tregs to the materno-fetal interface by production of hCG and CXCL16, the ligand for CXCR6.(105, 106). Moreover, in vitro coculture of HLA-G+ extravillous trophoblast with CD4+ T cells increased Treg numbers and FOXP3 expression level (107, 108), indicating insight.jci.org   https://doi.org/10.1172/jci.insight.137926 12 RESEARCH ARTICLE Figure 9. uTregs show site-specific adaptation to the materno-fetal interface. (A) Heatmap with previously highlighted genes in this manuscript that were differentially expressed between pbuTregs and incuTregs. Expression values were mean centered and scaled per gene. (B and C) Protein expression of PD-1 (B) and OX-40 (C). uTregs were gated as CD3+CD4+CD25hiFOXP3+ cells. Multiplicity adjusted P value of 2-way ANOVA with Tukey’s post hoc test. Left upper P value, blood versus placental bed; right upper P value, placental bed versus incision site; lower P value, blood versus incision site. MFI, median fluorescence intensity (n = 6). (D) GSEA with published TITR-specific signatures in pbuTregs versus incuTregs (25, 95–100). NES, normalized enrichment score; FDR, FDR adjusted P value. (E) GSEA with published gene signatures specific to Tregs from tumor tissue compared with the healthy tissue counterpart in pbuTregs versus incuTregs (26, 100). (F) Protein expression of CCR8 (n = 5), ICOS (n = 6), and GITR (n = 6). uTregs were gated as CD3+CD4+CD25hiFOXP3+ cells. Multiplicity adjusted P value of 2-way ANOVA with Tukey’s post hoc test for protein. Left upper P value, blood versus placental bed; right upper P value, placental bed versus incision site; lower P value, blood versus incision site. MFI, median fluorescence intensity. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. that Tregs may also be locally induced or expanded by trophoblast. Thus, it is likely that signals produced by invading trophoblasts at the materno-fetal interface account for at least some of the site-specific transcriptional adaptations in uTregs. The Th response at the materno-fetal interface was previously suggested to be skewed away from a proinflammatory Th1 response, to prevent a pathogenic allo-reaction against the fetus, resulting in a Th2-dominant response during the second trimester. However, during the third trimester, a proinflammatory Th1 response insight.jci.org   https://doi.org/10.1172/jci.insight.137926 13 RESEARCH ARTICLE may be essential for initiation of labor (reviewed in ref. 48). In line with this, our findings indicate that the Th response in the uterus at term is dominated by Th1 polarization, although well controlled. uTregs at the materno-fetal interface appear to be specifically equipped to effectively suppress Th1 responses. Although we observed heterogeneity of T-bet protein expression in uTreg, CXCR3 expression was remarkably homogeneous, with 84%–100% of uTregs being CXCR3+. CXCR3-expressing (and T-bet– expressing) Tregs are especially adept to suppress Th1 responses (36, 38, 45, 89). Furthermore, the majority of uTregs expressed TIGIT, OX-40, and/or CCR5. Tregs expressing TIGIT preferentially inhibit Th1 and Th17 responses (72), a subpopulation of OX-40–expressing Tregs can differentiate into Th1-suppressing Tregs (109), and also CCR5 expression on Tregs has been associated with more effective suppression of Th1 responses (90). Thus, the necessary proinflammatory Th1 response at the materno-fetal interface at term appears to be controlled by specifically differentiated and Th1-polarized Tregs. So far, Th1-like Tregs have been described mainly in inflammatory environments, such as infections, autoimmune diseases, and transplantation reactions (45, 89, 110–112), whereas tissue-resident Tregs were mostly characterized as being Th2 skewed (VAT, muscle) (13, 18) or Th17 skewed (intestines) (73). DiSpirito et al., however, recently also identified a subset of T-bet– expressing Tregs in muscle and colon (18), indicating that they can also be present in steady-state tissues. We are the first to our knowledge to study exclusively maternal, myometrial tissue–resident Tregs from the materno-fetal interface. Previous studies of uTregs had to resort to the use of more easily accessible decidua, due to the difficulty of acquiring human myometrium. Since decidual tissue is of fetal origin, it may not only be contaminated with fetal immune cells, but it also does not allow for studying the unique, and specifically maternal, uterine environment underlying the placenta, in which the complex process of spiral artery remodeling takes place. The only publications that we know of investigating FOXP3 expression in actual human placental bed biopsies demonstrated that the percentage of FOXP3+ T cells was significantly decreased in patients with preeclampsia, and FOXP3 mRNA expression was reduced in endometrial biopsies of infertile women, highlighting the importance of functional Tregs for a healthy pregnancy (57, 113). From human decidual data, it is known that the frequency of clonally expanded populations of effector Tregs is increased in decidua of third trimester cases compared with first trimester cases (114). Decidual Tregs display a more pronounced suppressive phenotype than in blood, with increased expression of FOXP3, CTLA-4, CD25, HLA-DR, ICOS, GITR, and OX-40, which recapitulates our findings (58, 59, 63, 115). Recently, 3 types of functional Tregs were identified at the human materno-fetal interface, of which the CD25hiFOXP3+ population matches the population investigated in this study (108). These Tregs effectively suppressed CD4+ and CD8+ T cell proliferation and IFN-γ and TNF-α production. Transcripts identified by qPCR array as specific for this subset were IL2RA, FOXP3, TIGIT, ENTPD1, LRRC32, IL1RL1, BATF, and CCR8, and increased expression of CCR5, IL10, and GITR compared with bTregs was also observed (108), which confirms our findings of an activated Treg phenotype at the materno-fetal interface. A study investigating chemokine receptor expression of CXCR3, CCR4, and CCR6 in decidual Tregs by flow cytometry showed that CCR6–CXCR3+ Th1 cells were increased and CCR6+CCR4+ Th17 cells were nearly absent, whereas CCR4+ Th2 frequencies were similar in blood and decidua (58), which is also in line with our findings. Taken together, this indicates that the here-identified activated phenotype of myometrial uTregs has overlapping characteristics with decidual Tregs. We observed that uTregs from the materno-fetal interface display a peculiar differentiated effector phenotype similar to TITR, defined by high expression of IL1R2, LAYN, CD80, VDR, and TNFRSF4, among others, with specific enrichment of TITR signatures compared with Treg signatures from matched, unaffected tissue sites. This observation may be explained by recent insights on the similarity of the immune environment at the materno-fetal interface and tumors (48). Both the receptivity of the myometrium toward implantation of the blastocyst and invasiveness of the trophoblast show striking similarities with implantation of tumor metastases in healthy tissues (116, 117). Tumor cells can modulate their immune environment into an antiinflammatory milieu and can recruit and/or induce suppressor cells, among which are high numbers of Tregs (118, 119). Just as in tumors, a tolerogenic mode of antigen presentation with indirect allorecognition of low levels of antigens predominates at the materno-fetal interface (120). Furthermore, others reported striking similarities between early Treg responses to embryo and tumor implantation (54). These findings imply that the microenvironment at the materno-fetal interface may be a unique mammalian tissue site that, under challenged but physiological conditions, resembles a tumor microenvironment: an actively remodeling tissue site distinct from a steady-state tissue, with low-grade inflammation and newly infiltrating/invading cells. These dynamic characteristics may account for the unique transcriptional adaptation of Tregs. insight.jci.org   https://doi.org/10.1172/jci.insight.137926 14 RESEARCH ARTICLE Although we observed global changes in gene expression patterns in uTregs, flow cytometry revealed an expression gradient of many markers across the uTreg population, suggesting that uTregs consist of a heterogenic population with various stages of differentiation and possibly subspecialization. Single cell sequencing techniques and mass cytometry are indeed starting to reveal the heterogeneity of Treg populations in tissues and tumors (15, 27, 97, 98, 121–123). A unique strength of our study is that we compared the transcriptomic profile of a highly specific and highly purified maternal Treg subset from myometrial biopsies, not only to their counterpart in blood, but also to a tissue-specific and site-specific Treg control population and matched Tconv population. We validated our key findings on the protein level in single cell resolution by flow cytometry. We only studied term pregnancies, due to the practical limitation of delivery of the infant and placenta. It would therefore be interesting to investigate term-dependent changes in uTreg profiles in future studies. Although protocols for tissue digestion may induce transcriptional changes (124), many of the uTreg-specific genes were previously found not to be affected by a tissue digestion protocol similar to but harsher than the one used here (17). Our findings have important implications. TITR are currently under heavy investigation as targets in cancer immunotherapy. However, we demonstrate that signatures identified in TITR are not as unique as previously assumed and that they may be shared by Tregs with specialized functions in other human tissues that may still be unknown. On the other hand, our results may lead to new targets for cancer immunotherapy, since profiling of Tregs in a variety of tissues under physiological, but not necessarily steady-state, conditions may help to identify truly TITR-specific expression patterns. Moreover, increased understanding of immunoregulatory mechanisms at the materno-fetal interface during healthy pregnancy gives unique insights into human immunobiology of pregnancy and also aids to elucidate the pathological changes in Tregs in pregnancy disorders such as preeclampsia, fetal growth restriction, or recurrent miscarriage, as many studies have pointed toward a role for Treg defects or deficiency in these disorders (65–67, 105, 114, 125, 126). Last, functional adaptation of human Tregs to different tissues and specific tissue sites is still largely unexplored. The receptivity of Tregs to their environmental stimuli and subsequent subspecialization may be exploited for therapeutic purposes. In conclusion, we have shown that human Tregs show functional adaptation with tumor-infiltrating– like features specifically at the materno-fetal interface, which introduces the concept of tissue site-specific transcriptional adaptation of human Tregs. Methods Participants and biopsies. This study is part of the Spiral Artery Remodeling (SPAR) cohort study, which is an ongoing effort to investigate the adaptation of the uterus to placental development by obtaining site-specific uterine biopsy samples in women undergoing cesarean section. A detailed description of the study set-up and protocol was previously published (127). For this analysis, we included 20 women who delivered by elective cesarean section (i.e., without any contractions or other signs of labor such as rupture of membranes) after an uncomplicated pregnancy and without any major underlying pathology, n = 5 of which were included for transcriptomics of T cell populations, n = 4 for suppression assays, and n = 11 for flow cytometry. Baseline characteristics are provided in Supplemental Table 1. One tube of sodium-heparin blood was taken from each donor before cesarean section. After delivery of the neonate and placenta, the placental bed was manually located, and 2 biopsies of the central placental bed from the inner uterine myometrial wall were obtained as previously described (127). Additionally, biopsies were taken from the incision site when the placenta was not situated on this part of the uterine wall. Lymphocyte isolation. Peripheral blood mononuclear cells (PBMC) were isolated from blood diluted 1:1 with basic medium (RPMI 1640 [Thermo Fisher Scientific] with penicillin/streptomycin [Thermo Fisher Scientific], L-glutamine [Thermo Fisher Scientific]), by ficoll-density centrifugation (at room temperature, 1100g for 20 minutes; GE Healthcare-Biosciences, AB). PBMC were washed in basic medium with 2% FCS (Biowest) and PBS or staining buffer consisting of cold PBS supplemented with 2% FCS and 0.1% sodium-azide (Severn Biotech Ltd.). The biopsy samples were collected in basic medium supplemented with 10% FCS and minced into pieces of 1 mm3 in PBS (Thermo Fisher Scientific). The biopsies were enzymatically digested with 1 mg/mL collagenase IV (MilliporeSigma) in medium for 60 minutes at 37°C in a tube shaker under constant agitation at 120 rpm. To dissolve the remaining biopsy pieces after digestion and remove any remaining lumps, the biopsies were pipetted up and down multiple times and poured over a 100 μm cell strainer (BD Falcon). Cells were subsequently washed in staining buffer and filtered through a 70 μm cell strainer, and they were prepared for flow cytometry or flow cytometry-assisted cell sorting. insight.jci.org   https://doi.org/10.1172/jci.insight.137926 15 RESEARCH ARTICLE Flow cytometry. For flow cytometric experiments without restimulation, PBMC and uterine cells were first incubated in fixable viability dye eFluor506 (eBioscience) in PBS (1:300) for 20 minutes at 4°C and washed in PBS. For surface staining, cells were incubated with the antibodies shown in Supplemental Table 2 for 20 minutes in staining buffer at 4°C and were subsequently washed in the same buffer. Cells were permeabilized with 1 part fixation/permeabilization concentrate and 3 parts fixation/permeabilization diluent (eBioscience) for 30 minutes at 4°C and subsequently incubated overnight with intracellular antibodies (Supplemental Table 2) in 10× diluted Permeabilization buffer (Perm; eBioscience) 4°C. The next day, cells were washed with Perm and measured on the LSR Fortessa (BD Biosciences). For intracellular cytokine measurement, PBMC and uterine cells were first incubated with surface staining, washed, and then restimulated with 20 ng/mL phorbol 12-myristate 13-acetate (PMA, MilliporeSigma) and 1 μg/mL ionomycin (MilliporeSigma) for 4 hours with addition of Monensin (Golgistop, BD Biosciences) during the last 3.5 hours at 37°C. Afterward, cells were stained with the viability dye, permeabilized, intracellularly stained, and measured as described above. Flow cytometry–assisted cell sorting. Cells were incubated with surface antibodies (Supplemental Table 2) for 20 minutes in staining buffer at 4°C, washed in the same buffer, and filtered through a 50 μm cell strainer (Filcon, BD Biosciences). For suppression assays, cells of the CD3+CD4+CD25+CD127– cell population (Tregs) and CD3+CD4+CD25– cell population (Tconv) were directly sorted into tubes with 500 μL FCS on a FACSAria III (BD Biosciences). For RNA sequencing (RNA-seq), 2000 cells of the CD3+CD4+CD25+CD127– cell population (Tregs) and CD3+CD4+CD25–CD45RA– (CD69+ from biopsies, CD69– from blood) cell population (Tconv) were sorted into Eppendorfs containing 125 μL PBS. After sorting, 375 μL Trizol LS (Thermo Fisher Scientific) was added to each vial, and vials were stored at –80°C until RNA isolation. Suppression assays and cytokine measurement. After sorting, peripheral blood, uTregs, and Tconv were washed in PBS and resuspended in basic medium with 10% human AB serum (Sanquin). Previously isolated and frozen healthy (HC) PBMC were labeled with 2 μM CellTrace Violet (Thermo Fisher Scientific) as described previously (128). Treg or Tconv populations were added to 15,000 HC PBMC at different ratios, and cells were coincubated for 4 days at 37°C. Supernatants were collected for cytokine measurement by multiplex assay before cells were stained with surface antibodies for CD3, CD4, and CD8 as described above and measured on a FACS Canto (BD Biosciences). Whole transcriptome sequencing. For RNA isolation, the vials were thawed at room temperature, and 100 μL chloroform was added to each vial. The vials were shaken well and spun down at 12,000g for 15 minutes at 4°C. The aqueous phase was transferred into a new tube, and RNA was mixed with 1 μL of GlycoBlue (Invitrogen) and precipitated with 250 μL isopropanol. Samples were incubated at –20°C for 1 hour and subsequently spun down at 12,000g for 10 minutes. The supernatant was carefully discarded, and the RNA pellet was washed twice with 375 μL 75% ethanol. Vials were stored at –80°C until library preparation. Low-input RNA-seq libraries from biological sorted cell population replicates were prepared using the Cel-Seq2 Sample Preparation Protocol (129) and sequenced as 2 × 75 bp paired-end on a NextSeq 500 (Utrecht Sequencing Facility). The reads were demultiplexed and aligned to human cDNA reference using the BWA (0.7.13) (130). Multiple reads mapping to the same gene with the same unique molecular identifier (UMI, 6bp long) were counted as a single read. Statistics. RNA-seq data were normalized per million reads per sample. Differentially expressed genes between the cell populations were identified using the DESeq2 package in R 3.5.1 (CRAN), with correction for donor batch (design = ~donor + cell population) and input of all genes. Genes with FDR Padj < 0.05 and |log2FC| > 0.5 were considered differentially expressed. PCA was performed in DESeq2 based on the constructed model, including donor correction. Pathway enrichment analysis was conducted in the Toppgene Suite publicly available online portal (https://toppgene.cchmc.org/enrichment.jsp), and pathways with Bonferroni-corrected P < 0.05 were considered statistically significant (131). For heatmap analysis, gene expression was mean centered and scaled per gene, and hierarchical clustering was performed with Ward’s method and Euclidian distance. GSEA (132) was conducted with Broad Institute software, by 1000 random permutations of the phenotypic subgroups to establish a null distribution of enrichment score, against which a normalized enrichment scores and multiple testing FDR-corrected q values were calculated. Gene sets with an FDR < 0.05 were considered significantly enriched. Gene sets were either obtained from provided data in publications or by analyzing raw data using GEO2R (NCBI tool) (133). An overview of used signatures is provided in Supplemental Table 3. For flow cytometric data, median fluorescence intensities (MFI) and percentages of positive cells were analyzed in FlowJo. For graphic representation, data were analyzed in GraphPad Prism. To assess significant differences on protein level between groups, 2-way ANOVA with Tukey’s post hoc test was used, and multiplicity-adjusted P < 0.05 were considered statistically significant. insight.jci.org   https://doi.org/10.1172/jci.insight.137926 16 RESEARCH ARTICLE The data sets generated for this study have been submitted to a public repository on GitHub ((https:// github.com/JudithWienke/Human-uterine-Tregs; branch name, master; Commit ID, f788ba7f8163d7f892ce4069a2c9c304cf922902) The raw data files could not be submitted due to GDPR constraints, but any additional required data can be requested with the corresponding author. Study approval. All patients received study information and signed informed consent before participation. This study was reviewed and approved by the local Institutional Ethical Review Board of the University Medical Center Utrecht (no. 16-198). Author contributions LB recruited and included patients, and collected clinical data. JW, LB, RCS, and LMVDB performed all wet-lab experiments. MM performed the RNA-seq and helped with data analysis. JW performed all data analyses and wrote the manuscript. PGJN consulted on biopsy preparation, tissue integrity, and uterine T cell distribution and phenotype. BBVR and FVW supervised JW, LB, LMVDB, and RCS and were closely involved in setting up the study protocol, collecting data, conducting data analysis, and writing the manuscript. All authors critically revised the manuscript. Acknowledgments We thank Michal Mokry, Noortje van Dunen, and Nico Lansu for their help with RNA-seq. 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Human HLA-G+ extravillous trophoblasts: Immune-activating cells that interact with decidual leukocytes. Proc Natl Acad Sci USA. 2015;112(23):7219–7224. 108. Salvany-Celades M, et al. Three Types of Functional Regulatory T Cells Control T Cell Responses at the Human Maternal-Fetal Interface. Cell Rep. 2019;27(9):2537–2547.e5. 109. Piconese S, et al. Human OX40 tunes the function of regulatory T cells in tumor and nontumor areas of hepatitis C virus-infected liver tissue. Hepatology. 2014;60(5):1494–1507. 110. Dominguez-Villar M, Baecher-Allan CM, Hafler DA. Identification of T helper type 1-like, Foxp3+ regulatory T cells in human autoimmune disease. Nat Med. 2011;17(6):673–675. 111. McClymont SA, et al. Plasticity of human regulatory T cells in healthy subjects and patients with type 1 diabetes. J Immunol. 2011;186(7):3918–3926. 112. Imanguli MM, et al. Comparative analysis of FoxP3(+) regulatory T cells in the target tissues and blood in chronic graft versus host disease. Leukemia. 2014;28(10):2016–2027. 113. Jasper MJ, Tremellen KP, Robertson SA. Primary unexplained infertility is associated with reduced expression of the T-regulatory cell transcription factor Foxp3 in endometrial tissue. Mol Hum Reprod. 2006;12(5):301–308. 114. Tsuda S, et al. Clonally Expanded Decidual Effector Regulatory T Cells Increase in Late Gestation of Normal Pregnancy, but Not in Preeclampsia, in Humans. Front Immunol. 2018;9:1934. 115. Nagamatsu T, Barrier BF, Schust DJ. The regulation of T-cell cytokine production by ICOS-B7H2 interactions at the human fetomaternal interface. Immunol Cell Biol. 2011;89(3):417–425. 116. Beaman KD, et al. Pregnancy is a model for tumors, not transplantation. Am J Reprod Immunol. 2016;76(1):3–7. 117. Holtan SG, Creedon DJ, Haluska P, Markovic SN. Cancer and pregnancy: parallels in growth, invasion, and immune modulation and implications for cancer therapeutic agents. Mayo Clin Proc. 2009;84(11):985–1000. 118. Lin WW, Karin M. 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Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations. Nat Methods. 2017;14(10):935–936. 125. Inada K, Shima T, Ito M, Ushijima A, Saito S. Helios-positive functional regulatory T cells are decreased in decidua of miscarriage cases with normal fetal chromosomal content. J Reprod Immunol. 2015;107:10–19. 126. Jianjun Z, Yali H, Zhiqun W, Mingming Z, Xia Z. Imbalance of T-cell transcription factors contributes to the Th1 type immunity predominant in pre-eclampsia. Am J Reprod Immunol. 2010;63(1):38–45. 127. Veerbeek JH, et al. Spiral artery remodeling and maternal cardiovascular risk: the spiral artery remodeling (SPAR) study. J Hypertens. 2016;34(8):1570–1577. 128. Petrelli A et al. Self-Sustained Resistance to Suppression of CD8+ Teff Cells at the Site of Autoimmune Inflammation Can Be Reversed by Tumor Necrosis Factor and Interferon-gamma Blockade. Arthritis Rheumatol. 2016;68(1):229–236. 129. Hashimshony T, et al. CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq. Genome Biol. 2016;17:77. insight.jci.org   https://doi.org/10.1172/jci.insight.137926 20 RESEARCH ARTICLE 130. Li H, Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics. 2010;26(5):589–595. 131. Chen J, Bardes EE, Aronow BJ, Jegga AG. ToppGene Suite for gene list enrichment analysis and candidate gene prioritization. Nucleic Acids Res. 2009;37(Web Server issue):W305–W311. 132. Subramanian A, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA. 2005;102(43):15545–15550. 133. Edgar R, Domrachev M, Lash AE. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 2002;30(1):207–210. insight.jci.org   https://doi.org/10.1172/jci.insight.137926 21 

W1999051990.txt

Document downloaded from: http://hdl.handle.net/10251/134138 This paper must be cited as: Chieregato, A.; Soriano Rodríguez, MD.; Basile, F.; Liosi, G.; Zamora Blanco, S.; Concepción Heydorn, P.; Cavani, F.... (2014). One-pot glycerol oxydehydration on multifunctional catalysts: influence of acid and redox properties on the catalytic performance. Applied Catalysis B Environmental. 150-151:37-46. https://doi.org/10.1016/j.apcatb.2013.11.045 The final publication is available at https://doi.org/10.1016/j.apcatb.2013.11.045 Copyright Elsevier Additional Information One-pot glycerol oxidehydration to acrylic acid on multifunctional catalysts: Focus on the influence of the reaction parameters in respect to the catalytic performance Alessandro Chieregato1, M. Dolores Soriano2, Francesco Basile1, Giuseppe Liosi1, Segundo Zamora2, Patricia Concepción2, Fabrizio Cavani*, José M. Lopez Nieto* 1. Dipartimento di Chimica Industriale e dei Materiali, ALMA MATER STUDIORUM Università di Bologna, Viale Risorgimento 4, 40136 Bologna, Italy. Also: INSTM, Research Unit of Bologna. 2. Instituto de Tecnología Química, UPV-CSIC, Campus de la Universidad Politécnica de Valencia, Avda. Los Naranjos s/n; 46022 Valencia (Spain) Abstract We report here about an investigation of the key catalyst features and reaction parameters in glycerol oxidehydration into acrylic acid over W-V-Nb mixed oxides with the hexagonal tungsten bronze structure. The incorporation of Nb allowed to considerably improve the catalytic behaviour not only because of the higher yield to acrylic acid and acrolein compared to W-V bronzes, but also to increase of more than order of magnitude the oxygenated productivity rate, because of the greater concentration of glycerol used in the inlet feed and the lower contact time needed. Experiments were carried out by reacting acrolein, and by oxidation of methanol, as a model molecule for the determination of surface acid and redox properties. These experiments demonstrated that with the W-V-Nb catalyst the optimal ratio between the two consecutive steps of acid-catalysed glycerol dehydration and acrolein oxidation, and between the two parallel reactions of acrolein oxidation and transformation into by-products (ketals and oligomers), was achieved in the presence of defined glycerol-to-oxygen inlet ratio. Indeed, oxygen played the fundamental role of accelerating the oxidation of the intermediately formed acrolein into acrylic acid, by allowing a greater concentration of the oxidizing V5+ sites. 1 Introduction With the aim of reducing the carbon footprint of fuels, bio-diesel represents one of the most important options, theoretically providing neutral CO2 balance and significant reduction of greenhouse gas emission.1-3 The increasing trend of biodiesel production goes hand-in-hand with the availability of large volumes of glycerol that is co-produced in the conventional transesterification reaction exploited to synthesize the bio-fuel.2,4 Since the late 90s, this abundance of glycerol has significantly impacted the glycerin market resulting in a decline of its price,5,6 and finally making glycerol a low-cost raw material desirable to be converted into different chemicals with higher economic value. On the other hand, it must be mentioned that starting from 1997 the glycerin market has faced very volatile conditions, increasing price and actual shortage of refined glycerol supply,7 that nowadays renders difficult any accurate economic evaluation of chemical processes designed for its transformation. However, since the over-supply of crude glycerol from biodiesel production (Bio-Crude) is actually one of the main reasons for price volatility, the transformation of glycerol toward more added-value products represents a key-point so as to develop a new bio-based chemical industry. Amongst the several options focused on glycerol conversion, a great deal of attention has been paid to the dehydration of glycerol into acrolein,1,4,7-31 and more recently also to the oxidehydration of glycerol into acrylic acid.32-42 From an economic and an engineering point of view, an interesting option to perform the latter reaction is the one-pot (single-step) transformation, by means of a multifunctional catalyst able to carry out both the dehydration of glycerol into acrolein and aldehyde oxidation into acrylic acid. Indeed, the use of a single catalyst can simplify the reactor engineering and level the heat balance between the endothermic dehydration of glycerol and the exothermic oxidation step. 2 Previously we reported that W-V oxides with hexagonal tungsten bronze (HTB) structure and V4+ incorporated in WO3 lattice are effective catalysts for the direct synthesis of acrylic acid from glycerol;38 under the best conditions these systems showed 25% acrylic acid yield plus 11% yield to residual acrolein. Later, we studied the influence of Nb addition to V-doped HTB structures, with the aim of improving the acidic features of the catalysts so as to enhance the glycerol dehydration step. Three-component catalysts (W-V-Nb) further increased the catalytic performance, finally leading to 33% acrylic acid yield plus 17% yield to residual acrolein;41 moreover, since the optimum working condition for W-V-Nb catalysts is located at a much lower contact time (from 0.38 s to 0.15 s) than for the W-V system, the catalyst productivity was also consistently increased. In order to fully investigate the catalyst activity of the best performing tri-component sample (reported as WVNb-1 sample)41, we report here a complete study of the catalyst behavior as a function of the inlet feed composition. Moreover, it is here reported about the methanol gas-phase transformation into formaldehyde and dimethyl ether (DME) on both W-V and W-V-Nb catalysts; indeed, methanol is known to be a suitable probe to explore the acid and redox properties of metal oxides.45,46 The results obtained allow to draw important and general conclusions on the catalyst features needed to convert oxygenated molecules by means of direct processes, using a single multifunctional catalyst. Finally, so as to support the conclusions depicted on HTB doped structures, the same reactions were carried out on Mo-V-(W) oxides with laminar structure. In order to obtain a complete picture of the catalysts behavior, the acrolein oxidation reaction was also studied on both HTBs and laminar-type oxides. 2. Experimental 2.1 Synthesis of W-V, W-V-Nb HTBs and Mo-V-(W) laminar oxide 3 W-V and W-V-Nb catalysts were prepared hydrothermally from gels obtained from aqueous solution of the corresponding salts. The hydrothermal synthesis was carried out at 175°C 48 h. Then the solid were washed, dried at 100°C overnight. Finally the solids were heat-treated in N2 at 600°C during 2 h. More details on the preparation have already been reported elsewhere.38,41 Mo-V-(W) catalyst was prepared by evaporation method; an aqueous solution of ammonium heptamolybdate, vanadyl sulphate and ammonium tungstate was evaporated in rotavapor. The solid was dried at 100°C overnight and then calcined in air at 350°C. Finally the solid was heattreated in N2 at 500°C for 2 h. (Josè: MORE DETAILS ON THE LATTER CATALYST ?). 2.2 Catalysts characterization Surface areas were obtained from N2 adsorption isotherms using the BET method; a Micromeritics ASAP 2000 instrument was used. The samples were degassed in-situ under vacuum at a temperature of 250°C. Metal atomic composition of both hydrothermal and heated samples was determined by inductive coupled plasma (ICP). Powder X-ray diffraction patterns (XRD) were collected using a PANalytical X’Pert PRO diffractometer with CuKα radiation and an X’Celerator detector in Bragg-Brentano geometry. Unit cell parameters were refined using the program FullProf.25 Termogravimetric analysis (TG) was performed on a Mettler Toledo (TGA/SDTA 851) instrument in the temperature range 20-700°C with a 0.02 g sample. The heating rate was 10°C min1 and the flow air 100 ml min-1. Infrared spectra were recorded at room temperature in the 300-4000 cm-1 region with a Nicolet 205xB spectrophotometer equipped with a Data station at a spectral resolution of 1 cm-1 and accumulations of 128 scans. Raman spectra were obtained with an “in via” Renishaw spectrometer, equipped with an Olympus microscope. The exciting wavelength was 785 nm from a Renishaw HPNIR laser with a power of approximately 15 mW on the sample. Dehydration of 4 catalysts was carried out using a home-designed microreactor for in situ Raman spectroscopy measurement. The samples were dehydrated under 20 ml min-1 argon flow at 150°C. Temperature-programmed reduction (TPR) experiments were carried out on 10–20 mg of catalyst with a N2:H2 flow (10% H2, total flow 50 ml min-1). The temperature range explored was from room temperature to 650°C. The heating rate was maintained at 10°C min-1. Temperatureprogrammed desorption of ammonia (TPD) experiments were carried out on a TPD/2900 apparatus from Micromeritics. 0.30 g of sample was pre-treated in a He stream at 450°C for 1 h. Ammonia was chemisorbed by pulses at 100°C until equilibrium was reached. Then, the sample was fluxed with He stream for 15 min, prior to increasing the temperature up to 500°C in a helium stream of 100 ml min-1 and using a heating rate of 10°C min-1. The NH3 desorption was monitored with a thermal conductivity detector (TCD) and a mass-spectrometer following the characteristic mass of ammonia at 15 a.m.u. X-ray photoelectron spectroscopy (XPS) measurements were performed on a SPECS spectrometer equipped with a Phoibos 150 MCD-9 detector using a monochromatic Al K-alpha (1486.6 eV) X-ray source. Spectra were recorded using analyzer pass energy of 50 V, an X-ray power of 200W, and an operating pressure of 10-9 mbar. Spectra treatment was performed using the CASA software. Binding energies (BE) were referenced to C1s at 284.5 eV. 2.3 Reactivity experiments Reactivity experiments for glycerol, acrolein and methanol transformation were carried out using a continuous flow reactor made of glass, operating at atmospheric pressure. An amount of catalyst ranging from 0.1 to 0.3 g of catalyst was loaded in the form of powder. Overall reactor gas residence time was varied. Inlet feed molar ratios were also varied according to the desired compositions. For each condition, all the reaction parameters are always specified in each figure. 5 For both glycerol oxidehydration and acrolein oxidation the effluent stream was bubbled through two in-series abatement devices, which were filled with water (but in some cases anhydrous acetone was used, for the identification of compounds which are less soluble in water) and maintained at a temperature of 0-2°C; a third refrigerated condenser was left unfilled with any solvent. After this abatement, the gaseous stream, still containing oxygen and carbon oxides, was fed to an automatic sampling system for gas-chromatography (GC-TCD) analysis. The water solution containing the unconverted glycerol and reaction products was analyzed by GC, using a HewlettPackard 5890 instrument equipped with a FID detector. A semi-capillary wide-bore OV 351 (polyethylenglycol treated with terephthalic acid) column was used for the separation of condensed compounds; oven temperature was set from 40°C to 190°C (heating rate 10°/min, isothermal step at 190°C, 3 min), then from 190°C to 225°C (heating rate 30°/min, final isothermal step at 225°C, 30 min). Two wide-bore columns were used for the separation of uncondensable products: a Molsieve 5A for oxygen and CO, and a Silica Plot for CO2 (oven temperature 80°C). Compounds were identified by means of both GC-MS and the injection of pure reference standards for the comparison of retention times in the GC column. A few unknown compounds were eluted in the GC column; we attributed to these compounds the same response factor of the corresponding known compound with the closest retention time. In the figures included with this paper, minor identified products and unknown compounds have been grouped together under the heading “Others”. Josè: SPECIFIC REACTION PARAMETERS FOR METHANOL REACTION 3. Result and discussion 3.1 Characterization of mixed-oxide catalysts Josè: you should write this part. The figures and Tables, those which you sent us, are at the end of the paper. 6 3.2 Reactivity experiments: oxidehydration of glycerol on W-V-Nb with HTB structure In order to examine the catalyst activity of W-V-Nb sample (WVNb-1 in ref [41]), the inlet feed composition was varied; for each value of oxygen-to-glycerol ratio, the catalytic performance was analyzed in the temperature range 260-410°C. Specifically, Figure N+1 reports the catalytic results obtained at 265°C, where the maximum acrylic acid yield was registered (however, analogous trends were obtained for the other temperatures examined). In this experiment both the glycerol molar content in feed (6%) and the water one (40%) were kept constant; oxygen molar and inert gas (He) molar contents were varied so as to satisfy both the desired feed composition and the pursued residence time. The latter was always set constant to 0.15 s (measured at room temperature). When a stoichiometric amount of oxygen and glycerol was fed (ratio 3:6), the glycerol conversion was close to 50% and very low selectivity to both acrolein and acrylic acid were registered. On the other hand, selectivity into heavy compounds was very high. Increasing the amount of oxygen, the heavy compounds selectivity dropped in favor of selectivity to the other products; the acrylic acid selectivity trend showed a maximum (39%) with the oxygen-to-glycerol molar ratio 12:6, finally decreasing in favor of carbon oxides. Figure N+2 shows the results of a set of experiments where the oxygen to glycerol molar ratio was kept constant equal to 2, which showed to be the best ratio in Figure N+1, but the inlet molar fraction of reactants was proportionally decreased compared to the ratio oxygen-to-glycerol 12:6. In spite of the constant optimal ratio between the reactants, the heavy compounds selectivity increased mainly in favor of acrylic acid when the molar fraction of reactants was decreased, which is the opposite one might expect based on the fact that higher reactants loading should favour condensation reaction leading to heavy compounds. This indicates that the most important 7 parameter affecting catalytic behavior is the oxygen partial pressure; higher oxygen contents enhance the oxidation of the intermediately formed acrolein into acrylic acid, so limiting the undesired formation of acrolein condensation into by-products. When instead lower oxygen molar fractions are used, irrespectively of glycerol partial pressure (i.e., at both low and high glycerol partial pressure), the kinetically preferred reaction involves the transformation of acrolein into byproducts (Scheme 1). This strongly support the hypothesis that the rate-determining step in the redox mechanism of acrolein transformation into acrylic acid is the oxidation of the reduced V sites into V5+, the latter being the species involved in the aldehyde oxidation, and that the surface concentration of the oxidizing species is thus affected by oxygen partial pressure. OH O O 1/2 O 2 - H 2O H OH OH OH H+ Oligomers Scheme 1. The overall reaction mechanism for glycerol oxidehydration. On the other hand, if the partial pressure of oxygen is too high, the selectivity to acrylic acid drops, with a concomitant increase of carbon oxides (Figure N+1). These findings allow to draw the important conclusion that in order to minimize the undesired competitive reaction of heavier compounds formation, likely catalyzed by the same acid sites which are also required for the glycerol dehydration step, the crucial point is to develop a steady-state showing a controlled amount of oxidizing surface sites on catalyst surface. Figure N+3 compares the productivity into acrolein + acrylic acid for the W-V catalyst41 and for the W-V-Nb catalyst under non optimized reaction conditions,38 with productivity achieved 8 under the conditions shown in the present work, and with results reported in the literature as well. It is shown that the productivity achieved with W-V-Nb is by far higher than that previously reported, and also much better than that reported for other catalytic systems and for the two-catalyst inseries reactor configuration, which the alternative approach proposed in the literature. Indeed, the best value achieved of ca 2 h-1 is quite of significance for industrial processes aimed at the synthesis of bulk chemicals or intermediates. 3.2 Reactivity experiments: oxidehydration of glycerol on W-V-Nb catalyst, time-on-stream test Figure N+4 (top) shows the catalytic behavior of the W-V-Nb catalyst plotted in function of time-on-stream (tos), using the experimental conditions that allowed to obtain the best acrylic acid yield (feed molar ratio O2/Gly/H20/He=12/6/40/40, temperature 265°C, residence time 0.15 s). The yields shown in figs. N+1 and N+2 were obtained after ca 90 min tos, and well fit the trends here shown; on the other hand, after 6 h tos an activation phenomenon was registered. Indeed, until ca 40 h tos the consecutive selective oxidation of acrolein to acrylic acid was clearly favored, since the acrolein yield decreased in favor of acrylic acid. At ca 40 h tos, the highest 50% acrylic acid yield was registered. However, afterwards the catalyst started to deactivate and the selectivity to acrolein and acrylic acid inverted their trends. Figure N+4 (bottom), plotting the yield to both acrolein and acrylic acid registered during experiments shown in Figure N+4 (top), demonstrates that a clear kinetic relationship between acrolein and acrylic acid exists; indeed, the sum of yields to both compounds was always close to 55%, even if the relative quantity of the two was consistently different during the period of the experiment. The XRD analysis on the spent W-V-Nb sample indicates that at the end of the lifetime experiment the catalyst structure was deeply changed with a loss of the HTB structure…… (Josè: 9 ANY SUGGESTION AND COMMENT IS HERE VERY WELCOMED! ANY COMMENT FROM XPS/RAMAN ANALYSIS ON SPENT SAMPLE?). More complex is the explanation for catalyst activation shown during the 6-to-20 h tos; this might be due to the formation of different crystalline or amorphous surface structures. Indeed, the formation of more active amorphous structures is well known to occur on Mo-V-(W) catalysts, typically used to selectively oxidize acrolein into acrylic acid44 and we cannot exclude that a similar phenomenon may occur on our W-V-Nb systems. Josè: here it should be necessary to characterise the catalyst downloaded after the first 20 h tos, that is after the activation has occurred; would it be possible to make TEM analysis of the freshly calcined, of the activated (after 20 h tos) catalyst and finally of deactivated catalysts ? We shall prepare samples and mail you. 3.3 Reactivity experiments: reaction with methanol on W-V and W-V-Nb catalysts Figures N+5 and N+6 report the results obtained when methanol was made react in the presence of oxygen on W-V-Nb and W-V catalysts; this latter sample was investigated in detail for glycerol oxidehydration in a previous work.38 As a general trend, at low temperature the formation of dimethyl ether (DME) was favored over its oxidation into formaldehyde (HCHO) (higher ratio DME/HCHO); when the temperature was increased the DME-to-formaldehyde ratio decreased. At high temperature the decomposition and total oxidation were also favored, thus increasing the overall carbon oxides selectivity, mainly carbon monoxide through formaldehyde decomposition. Minor amounts of dimethoxymethane and methylformiate also formed. Comparing the catalytic behavior of W-V and W-V-Nb, it is possible to notice that for the latter sample a much higher selectivity to DME was shown, in accordance with the higher acid strength of this sample. Increasing the temperature, the reaction of methanol oxidehydrogenation to formaldehyde kinetically prevailed over the etherification reaction; finally, at high methanol 10 conversion (90%) formaldehyde selectivity was almost identical for the two catalysts. Moreover, it is interesting to notice the different temperatures needed to reach similar conversions; for the WV-Nb catalyst, 90% methanol conversion was shown at 330-335°C, whereas for the W-V catalyst the same conversion was achieved at 380°C; this difference is attributable to higher surface specific surface area of the Nb-containing sample, which is one of the main features of this catalyst compared to the W-V; moreover, a contribution to the higher activity may also derive from the faster reaction of methanol etherification. The CO selectivity also was slightly different for the two catalysts, respectively 10% for W-V-Nb and 16% for W-V (at the same methanol conversion of 90%). 3.4 Reactivity experiments: acrolein conversion on W-V and W-V-Nb catalysts Figure N+7 compares the catalytic results obtained when acrolein was made react on W-V and W-V-Nb catalysts. Under the same working conditions, the two catalyst showed important differences: W-V sample exhibited both lower acrolein conversion than W-V-Nb, which again can be explained by taking into account the higher specific surface area of this latter sample, and lower selectivity into acrylic acid, with higher selectivity to heavy compounds. Therefore, It may be hypothesized that the improved acid features of this latter sample may help the desorption of acrylic acid,43 so limiting its combustion. On the other hand, the low selectivity to acrolein oligomers suggests that the aldehyde oxidation into acrylic acid is much faster than the side acid-catalysed reactions, which may also relate to the high concentration of V sites. The latter in turn is greatly affected by oxygen partial pressure (vide supra), but a role of Nb ions in facilitating the reoxidation of reduced V sites cannot be excluded. 3.4 Reactivity experiments: comparison with Mo-V-(W) laminar oxide 11 With the aim of demonstrating the general conclusions drawn on W-V-Nb catalysts, showing superior performance compared to W-V, we report here the catalytic results obtained on Mo-V-(W) laminar oxide, which is a catalyst for the selective oxidation of acrolein into acrylic acid [REF]. Figure N+8 reports the results obtained in the oxidehydration of glycerol using the same conditions found as optimal for W-V-Nb. As highlighted by the catalyst characterization (NH3 adsorption and NH3-TPD tests, fig. N) this mixed oxide shows very poor acid properties; this finding is very well reflected on the reactivity results of both glycerol (fig. N+8) and methanol (fig. N+9). Indeed, when glycerol was made react on this catalyst very high selectivity to heavier compounds was registered since the unconverted glycerol can react with the produced aldehyde so as to form ketals (cyclic ethers). In fact, if the catalyst does not hold the acid properties needed to quickly and selectively dehydrate glycerol into acrolein, the formation of other by-products is detected, eg, cyclic ethers such as 1,3-dioxan-5-ol and 1,3-dioxolan-4-yl-methanol, formed by reaction between glycerol and formaldehyde or acetaldehyde, the latter compounds being in turn formed by retroaldol condensation of 3-hydroxy-propanal, one product of glycerol mono-dehydration.35 The intermediately formed acrolein can react in a similar way with glycerol to form other cyclic ethers as reported in Scheme 2; indeed, the formation of these ethers was confirmed by means of ESI-MS analysis of the reaction mixture. 12 OH O O GLY O O O - H2O HO OH - H2O O H OH O H H OH GLY GLY OH HO O HO O O O O O HO O O Scheme 2. Ketals formation pathways as by-products of the glycerol oxidehydration reaction. When the temperature was raised the ketals selectivity decreased mainly in favor of COx and acrolein formation; on the other hand, the acrylic acid selectivity remained limited, even if the catalyst is specifically designed for acrolein oxidation into acrylic acid. Apparently, this was due to both the low selectivity to acrolein and the specific residence time used (optimal for glycerol dehydration on Nb-V-HTB structures), which is too low to efficiently convert acrolein into acrylic acid on Mo-V-(W) laminar oxides [REF]. This conclusion was confirmed by reacting acrolein: good acrylic acid selectivity was obtained on Mo-V-(W) oxide, but with low conversion (figure N+10). As already reported for glycerol oxidehydration, the lack of acid sites in laminar Mo-V-(W) is also evident from the catalytic behavior shown in methanol oxidation (fig. N+9); indeed the acid catalyzed route to DME was very low. On the other hand this system showed to be very selective in the oxidation of methanol to formaldehyde: a maximum aldehyde selectivity equal to 93% was registered at 87% methanol conversion (T 350°C). At low temperature, we also noticed the 13 formation of dimethoxymethane, which may derive from the acetalization of formaldehyde with the unconverted methanol. On the other hand, because of the low specific surface area of this catalyst, in order to obtain methanol conversions close to those obtained by both W-V and W-V-Nb catalysts, higher contact time (0.12 s) was needed as compared to that used with the latter systems (0.06 s). Josè: ARE THESE VALUES of contact time CORRECT ? Conclusions The studies carried out on both HTBs and laminar oxides have led to draw important conclusions on the features needed by multifunctional catalysts for the one-pot oxidehydration starting from oxygenated compounds. Indeed, the consecutive oxidation step must be fast compared to the dehydration one, in order to avoid the formation of heavy compounds generated by both ketals formation and oligomerisation reactions that take place from the intermediately formed aldehyde. Because of this, strongly oxidizing conditions, the latter being achieved by using oxygen partial pressures well above that needed for the stoichiometric oxidehydration of glycerol into acrylic acid, are necessary with the best performing W-V-Nb catalyst. The latter is characterized by higher surface area and higher concentration of stronger acid sites, properties which on one hand are important for an efficient dehydration of glycerol into acrolein, but on the other hand might favour the formation of undesired by-products. Under selected reaction conditions, an outstanding productivity to acrolein + acrylic acid close to 2 h-1 with an overall yield as high as 55% were achieved. Acknowledgements CIRI (Centro per la Ricerca Industriale), Università di Bologna, is acknowledged for the grant to A.C. The Valencia group also thanks the financial support of Dirección General de Investigación Científica y Técnica in Spain (Project CTQ2012-37925-C03-01). 14 References 1. B. Katryniok, S. Paul, V. Belliere-Baca, P. Rey, F. Dumeignil, Green Chem., 2010, 12, 2079– 2098. 2. P. Pin Oh, H. L. Nang Lau, J. Chen, M. Fong Chong, Y. May Choo, Renewable and Sustainable Energy Reviews, 2012, 16, 5131–5145. 3. E. Atabani, A.S. Silitonga, I. Anjum Badruddin, T.M.I. Mahlia, H.H. Masjuki, S. Mekhilef, Renewable and Sustainable Energy Reviews, 2012, 16, 2070–2093. 4. B. Katryniok, S. Paul,M. Capron, F. Dumeignil, ChemSusChem, 2009, 2, 719 – 730. 5. J. A. Kenar, Lipid Technology, November 2007, Vol. 19, No. 11. 6. James M. Clomburg, Ramon Gonzalez, Trends in Biotechnology January 2013, Vol. 31, No. 1 7. M. Ayoub, A.Z. 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Catal. A 413-414 (2012) 109-116. 41. A. Chieregato, F. Basile, P. Concepción, S. Guidetti, G. Liosi, M. D. Soriano, C. Trevisanut, F. Cavani, J. M. López Nieto, Catalysis Today 197 (2012) 58– 65. 42. C. F. M. Pestana, A. C. O. Guerra, Glaucio B. F., C. C. Turci C. J. A. Mota, J. Braz. Chem. Soc., 2013, 24, No. 1, 100-105. 43. Josef Tichy, Applied Catalysis A: General , 157 (1997) 363-385. 44. Philip Kampe et al., Phys. Chem. Chem. Phys., 2007, 9, 3577–3589. 45. J.M. Tatibouët , Applied Catalysis A: General 148 (1997) 213-252 46. Mohit Badlani, Israel E. Wachs, Catalysis Letters Vol. 75, No. 3–4, 2001 17 Fig. N. XRD spectra of the catalysts. A) Fresh samples; B) W-V-Nb sample, fresh and after 100 hours of time on stream test. 18 Fig. N. Spectra of fresh catalysts, Mo-V-W (a), W-V-Nb (b) and W-V (c). A) FTIR spectra, B) Raman spectra Fig. N. A) H2-TPR B) NH3-TPD 19 Figure N+1 100 80 Gly conversion 60 Acrylic Acid Acrolein 40 COx Heavy C. 20 0 Others 0 1 3:6 2 6:6 3 9:6 4 12:6 5 18:6 6 Fig. N+1. W-V-Nb catalyst. Variable oxygen to glycerol ratio. Others: mainly acetaldehyde and acetic acid (in minor amounts, < 4%: allylic alcohol, acetone, propionaldehyde, propionic acid and hydroxyacetone). 20 Figure N+2 50 100 45 90 40 80 35 70 30 60 25 50 20 40 15 30 10 20 5 10 0 0 0 1 2:1 2 4:2 38:4 4 12:6 5 Acrylic Acid Acrolein COx Heavy C. Others Gly conversi on Fig. N+2. W-V-Nb catalyst. Constant Oxygen:Glycerol molar ratio (equal to 2). Others: mainly acetaldehyde and acetic acid (in minor amounts, < 4%: allylic alcohol, acetone , propionaldehyde, propionic acid and hydroxyacetone). 21 Figure N+4 60 100 90 80 70 Yield (%) 40 60 50 30 40 20 30 20 10 0 Acrylic acid Acrolein Glycerol conversion (%) 50 Others COx Gly conv 10 0 10 20 30 40 50 60 70 80 90 100 0 Time on stream (h) 60 Acrylic acid yield (%) 55 50 45 40 35 30 25 20 0 5 10 15 20 25 30 Acrolein yield (%) Fig. N+4. Top: Time on stream test; feed condition Oxygen/Glycerol/H2O=12/6/40, tau 0.15 s. 22 Figure N+5 DO112d600_(W1V0,2Nb0,15) Selectivity (%) 70 60 50 40 30 20 10 0 20 40 60 80 100 350 340 330 320 310 300 290 280 270 260 250 Conversion (%) Fig. N+5. Methanol oxidation on W 23 HCHO temperature (°C) 80 DME FM DMM CO2 CO Treaction (º C) 

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153 A.HMED RASIM'İN İBRAHIM MÜTEFERRIKA YE MATBAACtLIK ÜZERINEY AZOIK.LAIUNININCELENMESİ ' 'Yaşar A. Tonta * GIRiş Bu çalı~manınamacı,VCirP\an bilgive düşüncelerinkjt1eseıı~şmesindc en önemli , , etmenlerd~nbirisi olandevingen (müteharrik) h,arflimatbaanınTürkiye'ye girişi konu9Jyla ilgili oJaliık, ö~eıdeAhmed Rasim'in ()Şmanlı Tarihi adlı eserindeyazmışol. duğu "ibrahim Müteferrika ve~atbaacdık"adl i makalesini değerlendinnek, genelde konuyla ilgili .basth kaynaklardanyararlanarakAhmed Rasim'in yazdıkları çerçeve. sinde Müteferri~vematbaa.cllıkkQpU$Unu iôc.lemektir. Bilindi~i gibi)yaıının bulunuşuyla.birlikte yazı malzemesi de hızla değişmiş ve kağltbulumıuştut.J.s. 1.yüzyJlcfaÇinlilet tarafından bulunduğu sanılan kağıt,ozamana değin elde edilen bilgi biri,kiminikaYda geçinnekte. kullandmıştır. Böylece, mevcutbilgi defontıasyonauğ'ramakstZınkijşaktan kuşağa aktarıtabUmiştir.. . Yazıda mekanizasyondevingen harfii ma1baanıhbulunuşuyla sağlarımış ,Müteferrika'nm dadeğindiğigibi,bir C'nty~azahmetiyıe b.inlerce.ciltdoğru yazılrmş eser eldeedebilmeolanağı doğmuştur.. . ÇalışmamlZ,da,matbaanın Türkiye'ye giı'işigemil hatlarıylain~lenmektedir. Ah. ıned Rasirn.'in ma:kal~sin.de ilk,basılaneserlece k,ısaca değiniJdiği,nden Müteferrika Ma1bclas.1arafındanbasllan eserlerkomı~Qdaay"ıntıh,biıgi verilmemiştir. Ycızıkaleme alınırken "kesin anlatım" yerine ';de~yimciantatım If. yeğlenmıştir. Bu dunim, Iı>-, rahim Müteferrika ve Matbaa,lhkkônu9Jndaaydmlatllmasl~eken birçok hU9JS ol- maSındMt ,kaYnaklanmakta~ır.Hatta, çok. kesin. bir 'yargıOlmakla. birlikte' konuyla . ilgili olarakNiyazi B~esşunlar. $Öytonektedir; ",..basılı Oımanlıkaynaklarıncfa ibrah im MÜ1eferrilf.t üzerine, Qrijinal kaynaklı hiçbir bilgi yoktur; arşlvlerdeki bilgile. rin hiçbiri de incelenmemiştir :,1' , , AJiMEDRAS~f,: . YAŞMlIVE Y APffLARI ıstanbul'da doğan yazar gazete,i ve1arihçLA.l:imed ~sim (1864-1932),ç~k.IuğUruakraba veyakınlarıom k()I'UyUc;ulJ.ıjualtında geçirim iştir. Darijşş afaka'y ibirinciHkle bitirdikten sonra bir$Üı:e Qevletmemu,",ı.ığu yapan Ahmed Rasiin, Ahmed Mid. hat Efendit'niı1 teşviklyle basın Yatarmııaatılrjtjşttr.Çeşitli gazetelerde makale, söyleşi,şiir ve çevlril~ri yaV,ttnlanınış~1927 ydlndula miUe~kili().Jarak Mec::ll$6ginniş- tır. (.) ., . ' Araştırma göıevlisi.N.Ü, KütiiiIDarteÇilikBö!iiinü (,1;) N.~rkea,Ti1rkiye;deÇ41dtif~tıf~;t~talt):Jtiı.19'NbJ.Sl AH~,,!ET RASi M 'tN,! DHP,r!i t'~'1~.1UTEFE r~Ri f{..,4 VE i\.lAT81,...,c..CI'L.i ,< Ahmed ,Rasim 'in toplanı 140 kadar yapltıva,.dır.İstanbuf yaşam 101anlatan ÖykÜ ve romanıarı, dtiy!}lsal aşk öyküleri,çeşitli kondlarda yazdığı fen kit.ıpları ve çevirileri de olan yazarın yapıtlarından bazılarışunlardır: JIh Sevgi (189'1), Güzel Eleııi (1892), Me;yl-i Ôil (Gönli! Kayması) (1 892);ıJc/~tL'p 1'1rhadaşıııı (1895), N ciJ;am (mera. mına Ererneyen) (1898), Askeroğlu (1899). Ahmed' Rasim Meşıutiyetten sonra tarihsel konuları da işleycreko.rtlokullarda (idadi) okutulmak üzer~ dört ciltlik Resimli ue Ha/ilah Osmanlı Tarihi'ni (19101912) yazdı. Bu yapıtın asıl değeri "faide" başlığı altında verilen dipnotla~dır. Say,. fanın yarısını kaplayan bu dipnot/arda ~ski devirlerin gelenek.gö.rencklerine, askeri ve yönetimsel örgütlenmesine vcta.rihscl terimIere jJJşkin b'jlgile( verilmektedi~. Nitekim'çalışmamıza tertı;çl olan "ibrahim Müteferrika veM;ıtbaacllık" adlımakale de ikinci cildin 811-819. sayfa hırında yeralan 18~numaralı "faide" de buluıunakt.dır. Adı geçen makale metinincelemesi yöntemiyle aşağıdaverilmektedir. iBRAaİM MÜTEFERRiI(A.VEi MATBAACILlK , f i' "Osmanı) hizmetine dahil olan Mac;ırlard;ın iki zatiki biiyük hizmetle saiderin. den serfıraz olmuşlardır (diğerleri nden üstiiıı tıı tuhnuş:lardır). Bunlardan biri topçu Urban, diğeri matbaacı ibrahim,Müteferrika'dır. '. "Urban kendi arzU!ıJ ile Türklerin y.anına gelip Fatih Sultan Mehmet zamanında Osınanlılara ilk bwü~ toplip'1 döktü. istanbul fethinde şehrin duvarlarında rahneler (büyük delikler) açan cesim toplar Urban Usta'nı~ malıaretiyle meydana gelmiştir. "ıbrahim! Miiteferrika kendi arzusıı iledeğilvukua'tın ilcasıylaOsl11anlı hizmetine gin~ıiştir. MiiialUnileyh (adıgeçen) H Iristi.yanlıkmczaJı ibinden Kaluinist mezhebinde fakir bir Mac~r aileye mensup olan 108S'teMacaristan'ın şark tarafında kiiin Kolojva.r §ehrinde ~oğmuştur. 1104 senesinde OrtaMacar Kralı Tököl~BeyOs'narılı ordusuyla müttefikan (birlikte) Nemçeaı~hine vuku bırtan muhareoode ıbrahim Mütefer>rika esir dÜşıüş. FidYe-i necat (kurtulmalık) veremediği için 1l06'da Istanbul/a getirilip burada esir olarak 5.1nldl. \-lin.i esaretinde (köleliği sırasında) kaba, z.'1limbir ada. mın elinedüşerek köleliğe ademJ tahammülüne binaen (köl'eliğe tıhammül edeme- yerek)islamolarakıbrahimte5miyeolundu(adlandırıldı). ' ..ibrah~ Efendi Risale,i Jslamiyye nam,l/altındabirkitap' te'lif etmiş.idi. Kitabın muhteviyatı dinJ Islamı müdafaaya niÜllhasır makalat..-ilmiyyeden (bilimsel makale- . lerden) olmağla bilahare sadrazam olan 'Damat ıbrahim Paşa'nın nazar-ı dikkatine . çarpmış ve mumalleyhi (a(fıgeçe,ni)himayesinealmıştır." 2 , ' Ahmed Rasim 'in makalesinın başınctıu'lyaptığ.m1Zbu alıntıdan sonra verilen (2) Ahmed Rasim, Asma/ılı Tarihi' eili Il, ısta~bul:~eıns ss. 811-812. l\1-atbaası,'1326.-1329 YAŞAR 155 A. TONTA bilgilere kısaca bir göz atalım; Ahmea--Rasim. Müteferrika'nı" kendi ısteği dışında müslünfdr'i.olduğunu. ~alvinci bir aileycmenSllp olup H. lO~5'te Kolojvar'da d~duğunu; Osmai'ılı ordu!U ile yapılan savaşta esjr düştüğünü ve kurtUlmalık veremediği için Jstanbul'a getirilip köle olarak s3.tl.ldığını. köleliğe tahammüledemediği için de islam.iyeti kaPu! ettiğini yazmaktadır. Yazar; Müteferrika'nm RisaLe-i lsliimi~'ye adlı Islam dinini savunan bilim.seı makaleler içeren bir kitap yazdığını ve yazdığı bu .de eklemektedir,Bu kitapla da sadrazamm dik!<;ıtini çekerek him.ayesi,oe.girdiğiıı,i koroda araHırma yapanlarR~ale-i İslamiyye'nin Islam dirıini savunmak için yaz'ılmış bir kitap olduğu konuSlInda birleşmektcdirler. OYsak:i. söz konu!U kitabın Islamiyeri savun.anW eser olmadığı Niyazi . ~fkes'in çalışmaJarısornıcu ortıya çıkarılmıştır. Berkes şunları söylem~ktedir: . "'Gerçektebue$er ne,lstanıhğın savunmaşıdır. nede islam biljmleri üzerine yazılmış bir eseidir.(...) Bu eser ı<atol;ildiğe.papahğa ve testis inancına hücüm edenbir polemiktir. Asıl önemli yanıbize ıbrahim'in aSlında sanıldığı gibi Kalvinist olmadığını göstermesindedif. Bunu. öğrendiğimiz zaman Ibrahi~ı'~n TÜrklere karş~ ~vusturya ordu!Undasavaştığına, kölelik yüzündenmüslümanlığa, geçtiğ ine inanmakgüçleşir.~" Herkes, Risale-i ISl6mıyyehakkirıda dahagen.iş bilgi vcrdiği:20 ıiumarah dipnot. ta yazmanın kendisinde bile Rısale-i lsldmi)')'eadının geçn1edlğini; bu adınbir katalogcu tarafından rastgele verilmiş bir ad olduğunu söylemektedir .Dahası. "Islamlığı bilmeyenbir kiş,i olmayan Ibi'ahim'in Katolik Habsburg'larııi yönetimi altında yaşam:ıktansaÖsriıanh!ığa geçerek.miisliJrıan olduğU apaçıktır"4 sözleri de Betkes'e ;ut~ . tir. Berkes'in verdiği bu bilgilere.dayanatak Ahmed R.asim'in değindiği (Mütefer, rika'nın Kalvincibir aileye memubiyeti, kurtulmahkveremediği. içinköle olarak ~. tl!ıŞI. köleliğe dayanamadığı için mUslümanoluşu. RisaleJ lsldmiyye'nin "din~ . isiSını müdafaaya münhasır" bir eser olduğu.yc yine aynıkitap d61ayısıyla müteferrika'nın Damat ıbrahim P3$a'nın him~yesinegirmesi vd. )ansikloı'edik bilgilerin gerçeklerlebağdaşıriadığt görülmektedir. Sait Maden'in deyişiyle. bu ansiklop~dik bilgiler şimdiye dek bu' konUda araştırma yapanlarınbirazeksik birazfaZla. küllana . geldikleri bir "ortak mal" duıurnundadtr.s , ' Tarihsel bilgTaçlsındal1bu d'önernlerdekldinsel gelişmelere . dekısacadeğinmek gerekmektedjr.. . ': . XVIII. yüzyılda Batı Avrupa Hıristıyanh~ı blrkaçm'eZhebe bö~nmliştü. (Katolik. (3): Berkes, a-.g.)'., s: 52 (4). a.)'. s:53. (5). Sait M aden , "İbrahim Müteferrika tçinBil'YenidenDeiteriendirme . . "Milliyet Sanat De rgisi Yeni Dizi No. 1, (Şubat 1980),8. 87 Çalışması, AHMET 156 RASIM'iN IBRAHIMMOTEFE;:RRiKA VE MATBAACtLlK luterd, Anglikan). Kalvincilik de Qu'mezhebierden birisidir.Hıristiyan dininde görülebuklikleşme o zamana'de~in herl<esçekaböledU1m"tek ve mutlak bir gerçekfik. ri" üzerinde olums.ız etki yaptı. Dinsel savaşımlarda birçok kan aktL Ama zıt inezhepterin biraradayaŞamasl$rektiği'düş~esiningetişmesıne de katkıda bulundu. Bu arada, tek ve mutlak bir ger'çek fikrinidevam ettirmek isteyenlerUnitarizmi kurdular. Müteferrika Katvinci d~il, terslne Unitariusçudur. Unitarizmin Islam dinine yakın bir doktrin oluşu, gene\ÇJetektanncılığa dayanması Müteferrika'nın zor yoluy. la müslümanolmadığına-da birjkariıtolabitir.ÇÜOkü; kili~ebağnazlığına karşı Çıkan, din ve inanç ayrımını, h~ türfüdüşünce~gürfüğünü $awnan, diğerbilim dallarıYla yakıml'anilgilenen, kısacasısöıP1ğün tırnan.lamıyla tam.birentelektüel olan Müteferrika'nın ıslamiyet korAls.ındada bilgisi olması beklenir. Ayrıca, Unitariiminkunıcusu olan Michael Servetus'un (1511-155~) Unitariusçuların inançlarını yaymak için t<Ui"an'ltlnıkgôstermesinin Müteferrika'yı da etkilemişolabileceği kuşku!IJz, düşü. ~~~ '. . KaMncilikle'tJnitariusçuluğun birbirlerinin tam ~rşltl d9ktriı:tlerolquğunu belirtmek için Michael Servetus'un.diridiri yakıldığını da eklemek~kmektedir_ . Ahmed Rasim'inkonuiiLerine kaldığımız yerden sürdürelim: , . . i yazmış olduğu makaleden yaptığımız alıntıları "1128'deAvuSÜ!rya ile zuhureden'muharebedeBelırat'ta içtimaedenMacar- lara tercümanlık vazifesiyle.memur olarıfs"(1129) Orta Macar Kralı meşhur Rakoçi de Dersaadet'e gelmekle ıbrahimmumiileyhyanında kalmJŞtır:HattaRakoçi'nin Tekfur dağında ikameti esnasındadahi ıbrahim Efendi'yi beraberindebulmuş ise de ek. ser vaktini ıstanbul'da Sadrazamıbrahim PaŞa neztiinde geçinniştir. Macar efazlM müverrihinden(erdemlitı.tihçilerinden)olatı Doktor Karaçuiı Efendi bunları yudıktarı sonradiyor ki:' ' , Sadrazam OS1tanlılararasmda ıılOm Yefün~n Jle Avnıpa medeniyetinin taammüm ettirilmesi {genelf!'Ştirilmesi)arzu!IJı-ı~raz .veizhar etmekte idi. Fransa Sefaretindenavdeteden Yinn ise~'Mehmed Çelebi ile ~Iu Said Efendi' ki bilahare sadıazam olanSaid Paşa'Clır- ıbrahim Paşa'yıteşvikederlerdi. - 'ıbrahim. Efendi bu m~sudaniilolmakiçin SadQz~a bir matbaa teşkilw tesisi hakkında müracaat etti. Sadrazamtısvipedip icrasını emreyJedive bu ~!lJS için ıbrahim Efendi'ye kifayet miktırı para ,dahi verdiğinden ıbrahim Efendi Viyana'dan hikkiklar ile humfat'dökmecUeri ve ma1b..acdarıgetirip işe başlamak istedi; Ama gayrıme'mulmiinanaatauğradı(beklenmeyenengellerie kaI'Şllaştı),..6 ' Alıntıdan da anlaşılacağı üzere Ahmed Rasim kaynak olarak Karaçun Efendi'den yararlanmıştır. (6). Ahmed Rasim, a.g.y. ss. 812-814 , YAŞAR 157 A. TONTA Qgy:ı;ınh sınırıaniçindey.aş,,"aY~ ,baştathktan Doğal ol~rak ibrahim Müteferrika sonra çeşitli kademelerde birçok görev yapmıştlı;.Başarıh hi~metleri; dolayıs1y,!a(jır ki kendisine "müteferrika"ltk görevi verilmiştir.' ' Bilinen o ki, ibrahimMüteferrikaOsm;ın1thizmetine girmezdençok öocelerii3asma scuıatıni1atımaktadır. Berkes,Müteferrika'nın hangi nedenlerlematbaayı tanmış. olabileceğini şöyle anlatmaktadır. "Erdel ve Macaristan,Orta Avrupa'da matbaac;ılığ1l1çjlbukyerleştiği yer Qbnuştur. Oımanlt tarihçisi Peçevi'nin doğlim yeri olan Peç'şehrinin /;>aşIıca,kilisesi 1570 de Unjtariusakidesinikabuletmişti. Bir,yüzyılsonra o Zil11anll)en tanınmış ,harf dökümcüsii ve basınıcl$lol;ın, Misztotf~ulu'Mihail Kiss, Kofojvarşehrinde, 1689 dao ~amanınen önemli U nitarius basımevini kurmuştur. Ora~! ibrani, Enneni V~ Gürcü harfleri. döken bu ~. bastığı kutsal kitapmetni dolay,ısıyla KaMn,istlerio hücumunauğranııştl; ıbraHimMüteferrikadaha Türk olmazdan önce basmazenaatını değilse bile basımeviniyakındanbilen birKişi idi. Yirmisekiz Çeleb1 Metımet Efendi .' ye basımevi açılması fikriniogetirmiştir.'" . .' Kaldı ki, Selim 'NiizhetGerçek'a.,gôre Müte.ferrika,dah.a1719-17:20 yıllarında, ıŞ şimşir üze~nekazllmışo,lan19.x48,5Grn' boyuüındclbir.harita ba!lt1 ~'altına da şu notu düşmüştür: "Btnim devleduefendimeğerfennanınlZolursa dahi büyüRI~. yapılır. Sene 1132.,,8 Ki bu tarih 1719'a karşıltk gemektedir. Oysa Said Efendi'nin . Paris'tendöomesi 1725 ylllarınararilam~dır'~.Müteferrikabu 'sır~d,a~ı.:Ieniz ,9 MütefefJi~'nın S~d . haritasın'ı basmakla meŞlJlldjjr,;OS01aı:1,ErS9Y daeşerinde, Efendi'den önce matb~aişleri ilej 19ilendiği.nedair bQtduğu ~ betgedensöze~ekte.. . dW. . Bu'dunımda,matbaa açı.ması fikrinin Said- 'Efendi'yeMüteferrika tarafından açllmfŞolabilec~i 'düşünÜleibiID'. EğerŞaid Efendi'nin matbaanın kurulrn~inda bir rolÜ olduysa - ki ,oImuş1Ur.". bu rol: 1) Matbaanın açılmaSt iÇingerekli se.",ayenin Said Efendi ıarafmCJahbulu'nması;2) Said Efendi'niıf(deVıet kademesindeiyi bir yerde görevli olmasından dolayı) bürokratik engellerin aşılmasında 'yardımcı. olma; 'itorpil" göreviniüsdenmesişek,lindegerçekleşmiş olabilir. ' ('7). Berkes, a.g.Y. s." 57 , . ' (8). Selim Niizhet ~çek, Türk MatbaaedılıI; Müteferrikil Matbfi481,:İataiıbül1939. s. 34. ~Eaerin vesikalar bölümünde Marmara Dımizi Haritasmm 'klişesifaksimile olarakvardır.) " (9). Osman &soy, 1firldye~yeMatboanmGirişive Ilk BaSıfanEserler, Ankara,1959 s. 31 ' , AHrv1 ET , ,~,'\si ~,1'iN.i EJRAr-'lir,1 ,,1 üTEFER;:ıj KA VE Mp,TBAACJLI K Ahmed Rasim Karaçun Efendi'yi kaynak göstererek Müteferrika'nın Sadrazama matbaa kurulmasıkonu9:indaba-ş vurduğunu; Sadr#amın bu başvuruyu ,onaylayarak kun.ılmasını'ernrettiğini ve dahası; bu i,ş için Müt~ferrika'ya yeterli miktarda para bile verdiğini kaydetmektedir. Ancak, neSadrazamınmatbaanın kuıulmasını emret~, tiği ve ne de bu husu,s için Müteferrika'ya yeterli miktarda para verdiği belgelencrıri!. memektedir. i Hakkikların ve hurutat dökmedlerinn nerede~getjrildiği konu'sunda, yapılan , araştırmalarda çok değişik görüşler ortaya atılmıştır. Ahmed Rasim'in kaynak göster. diği KaraçunEfendi. hikkikların ve hurufat dökmedlerinin Viyana'dan getirtildiği. ni; Selim Nüzhet Gerçek Lork'a~dayanarak "istanbL1l" da isaga edild1ğini"(kalıba d,ö. olduğunu, küldüğünü), 10 NecipAsım harfleri dökeninArabo~lu adındabirErmeni i 1 Carleson ise harflerin ehlıyetli Alman LIstalarınc~ döküldüğünü iddia etmektedir,. ler. ,1Z Osnı an Ersoy da elindeki belgeleredayanarali harflerin isıanbu i'da döküldüğü ' ve yapılmaSı' mümkün otanbUtiiı materyalin ifstanbul!'da yapıldığı sonucuna varmak ta- clır. 13 i, ' Hikkik ve hurufat dökmedlerıtıin Viyana d..n getirtitdiğihu'su~na olumlu yönden yaklaşılacak olul'5i1şunlar söylenebilir:Avus-ı..ryave italyao devirdematbaa. cılığın en fazla getiştiği ülkeler durumunda-ydt.EğetViyana'da Arap harfleriyle ba. slmCllık daha ileride idıyse, belki de bu nedenle hakkaklar ve hurufat döl-,,,'edleri Viyana'dan getirtilmiş olabilir. Ancak Duolasılık çokzayıf görijnmekte~ir. Öte. yandan S.N.Gerçek,Müteferrika'nınkitapbasma izni almak için verdiği difekçenin çok giizetıolartfesih.ya:zılatı ileilk basılan,eserlerin yazıların," birbirine benzemesindendolayı harflerinıstanbul'da dökiirnüş olabUeceğini söylemektedir.1,4 Şu da ,bir ,gerçek ki.is~nbul halkı matbaayı çbk önce~itarihlerden beri tanınıaktıdır. Çünkü, o devirde ıstanbul'da ~lnlıklartırafJr1dan kurulmuş vefaaliyette olan matbaalar bulunmaktadır. 1,5 ErsoY'jigöreilk Türk matbaaSl ku.rulurker:ıazınlık, lardanyardımgörü1rn~tür. Ersoy bu t~ini şöyle desteklemektedir: (10) Gerçek, (l.g.y. s. 60 (ll) Ersoy,(l.g.y.s. 35 , . (12) Edvard Carleson, ıbrahim Müteferrika Basıme!)i ue Bastığı ilk ESerler, Yayma .' Hazırlayan: M.Akbı.ılut, Ankara 1979. (13) Ersoy, a.g.y.s.35 (14) Gerçek, a ,g.y. s. 60. (15) İstanbul'cia kı.ırulim Musevi, &nem ye Rummatbaalım konusunda S,N, Gerçek ineserine bakılabilir. (s.26)' Ayrıca, matbaanın yayılmasıyla ilgili olarak bkz. Ersoy, a.g.)!. Ek i. yAŞAR A.TQNTA "... ibrahim Müteferrika tarafından' verilen istidanın ortalarındaki bir pafagrafta matbaa işlerinden anlayan edavat ve alat vc mühimmat san'atında mahir yonaadlı 16 bir Yahudinin yardımındanbahse<l'iImektedir." Ahmed Rasinı daha sonra makalesine şöyle devam etmektedir: "Bu menılekette tarik~ ilmiyye ricali zinüfuz olmakla (bilim adamları etkin olmakla) bu ihtir.a-ı cedidc(yenr buluşa) birtürlü cevaz (izin)vcrmediler. Matbaanın müslünıanlar arasındaisti'mal olunmamasını'(kullanılinam;ısın'ı) Sadrazama alettekrar (tekrar tekrar~arı ettiler. Ve lüzumundanfazla kitapların meyclan-ı tedavüle vaz' edilmesi ile (dolaşıma konulması ile) asayiş~ umumi (geneldü1;cn) vemuamelat-ı diniyyeye (dinselişlemlere) ihtidi-ı mezkurun (adı gççcn buluşun) tehlikeli olacağını ıikı reylediler." . 7' . . Sait Maden'e göre Ahmed Rasimyukarıdaki bilgileri De Saussure'danalarak ver. mektedir. Sait Maden, De Saussure'un bir süre Tekirdağ'da Rakoçi'nin yıımnda. bulunmuşbir Macar soylu9J olduğunu, bu arada M~tcferrika ilc de .tanışmış otabi. lec~ini, fakat verdiği bilgilerinbelli tarihlerdenve belgelerdenyoksun olduğunu kay: detmektedir. 1.8 Bu nedenle de De Saussure'a pek güvenmernekgerekmektedir. Dikkati çeken diğer bir nokta da, DeSaussure'uri,irıatb;ıanın bulurıı.şununüzerindenüç yüzyıla yakın bir süre geçmesinekarşın, matbaadan.hiila "ihtira-ı cedid" diye söz . . '. etmesidir. '.' Yukarıdak paragTilfta,matbaanın işler halegeçmesiyle birlikte gereğinden fazla kitapların piyasaya çıkmasıylagenel düzenin ve dinsel işlemlerin b~zulacağından sözedilmektedir. Ancak gereğinden fazla ki.tıplarınneşekilde zararı dokunacağı konusunda.ne yazık ki ayrıntılı bilgi verilmemiştir. Dolay.ısıylabasılacakkitapların . nasılıararlı olacağı anlaşılmamaktadır. Ahmed Rasinı,matbaaya karşı çıkllmasıyJa ilıjli olarak~akate$inde şu satırlara . da yer vermektedir. "ıstanbul'da ozamanlar yüzlerce adarnl,n maişeti (geçimi). yalnız kitapların istinsahı(çoğaltılması) ve yazısı olmayanlarınmektuplarınıyazmojehusuSlna nıüıiha~ıridi. aunlar da matbaanın küşadrndan(açılmasından)mutazarrır (zararlı)olacaklarından vemedar-ı maişetlerini (geçinı,yollarını) kaybedeceklerinden haif oldukJarın. dan (korktUklarından)hoşnutcilmaddar.Bunlar Sadrazaının matbaayı iltizam ettiği. (ı 6) E~oy ~O.g.)'.s. 35 (17) Ahmed Rasim, (Ü3)l\Iad.~n,a.g.m,s.87 . a.g.y. s. 814 . i -~ 160 AHMET ~-~ RASI'M'iN.iBRAHiMMOTEFERRiKA ~---:-~~- --, _.- VE MATBAACtLIK l1i (gerekli' bulduğunu) görüncegüya din4rrübinitl (islamdininin) muhafazası bahanesi ileavamı ma1baa aleyhineteşviketmeğe:başladılar, Fiilen biraz patırtı ve çok dedikodu oldıı. Artık bunlarla taraftarlarını, tes1dnetmek için"müsag-ı şerıiyi mutazammm fetva-yı şerife olmadıkça (dinsel izni içeren fetva olmadıkça) hiçbir kitabın tab'olunamayacağını vaad ettiler. Ve bLJ. veçhile matbaa 1139 senesinde ıbrahim . Efendi'nin kendi evinde işe başladı. "19 /. .' . Geçimlerini kitaplann çoğaltılmasından. ve mektupyazmaktan sağlayan birçok kimseni!) matbaanın faaliyete geçmesi nden hoşnut olmayacaklan açıktır.Nitekim kitap esnafı da elikolubaği. du rmamıştır. Bu arada bir de gösteri "yapmışlardır. Gösterinin yapıldığı.tar.ihhakkında kesin bir bilgi olmamakla birlikte, inceleyebildiğimiz kaynakların tümünde böyle bir olayın m~ydana geldiği dOğrulanmak tadır. Selim Nüzhet Ger~ek'e &öre hattatlar ve esnaflar "ka~)t ve kalemlerialat ve edavat-ı kitabeti bir tabutı koyarak bir cenazealayı" düzenlemişlerdir. Ancak bu gÖsteriye katılanlar "birazr>atırtı ve birçok dedikQ(luya sebebiyet 'verdikleri haldesonraları süktııet" . bulmuşlardır. 20 . . . Jale Baysal da Müteferrika'dan I. Me.ş~ tiyete Kadar Osmanlı '.flirlderinin Bastıkları. Kitaplar: adlıeserindePatrona HaJit Isyan, slraslJida Sadrazam Damat ıbrahim Paşa'nın öldürülmesinekarşın basımevinin kınJıp dökülmedensağlamkaimış olduğuna dik.kati çekerek; sözkonusu düşmanlığın geniş halk kitlelerini etkilemediğini wrgulamaktadır. Bu düşmanlığın halk kitlelerini etkilememesine neden olarak da, toplumun basılı kitapla fazla ilgilenmediğini; bir yüzyıl boyunca basılan kitaplann sayısının 180'~ gewı ediğin i ve Müteferrika öldüğünde terekesindebastığı kitapları dan birçoğunun satılmadan kal(lığIDI göstennektedir. ..~ . , Toplum bilimciler kültüriiı m~diunsurlarıı:ıın toplumsal yaşamı belirleyici ol. duğunu sawnmaktadırlar. Nesnelkoşullar gerektirmedikçe dışarıdan alınan kültürel Yenilikler toplumsal yaşamda benimsenememektedir.Başkabir deyişle,- nezamali ki ,toplum matbaayagerekslmne wymuş ve bu yeniliği benimsemeyehazırhale,gelmiş, on.daıı.sonra.matbaa kunılabilmiştir. . . ., . Kuşkusuz matbaanınkurulmasındaki gecikmede birtakım sosyo.ekonomik nedenle~ roloynamıştır. Birbaşka neden olarak da Herkes, Osmanlı devlet sis,temine özgü lonc.asınırlamaliirıni Qmekgöstermektedir.2 2 Oysa Türklerle ya~yana yaşayan, amazer1aatvetic.aretleu~aşan Rum, Ermeni ve Yahudi ~!nhklar matt>aarıınbulunu- (19) Ahmed Rasim, a.g.y. 8.815 (20) Gerçek, o.g.y. 8.59 (21) Jale Baysal, Müteferrika 'da,n 1. Meş~ Kitaplar, İstanbul 1968. 88.13-14. (22) Berkes, a.g.y. s. 6.0 . . _ tiyet Kadar Osmanlı '.flirklerinin Bastıkları . ,- -"-~---~--f" -. , YAŞAR 161 A. TONTA malliaakunnuşlardır. Bunda, Osmanlı yönetiminde. yaşayan şunda,\hemen,sonra azınlıklara gösterilen hoşgörünün büyük bir payrolsa gerek. Yoksabazı araş~nnacıla~ rın saviJnduğu gibi, Osmanh devletyöı:1etimidinsel nedenlerle matbaanın girişini geıştır , kanısındayız. Sözün özü, müslüman top,ciktirmek gibi bir dekadansa uğram <it1 lum o devirlerde kitaba değil, {akat basıh kitaba \ gereksinim duymamaktadır. , "', ' Şeyhülislamın fetVası "av~ı matbaa aleyhine te~vik" eden kimseleriyatıştırmak ve adı geçen gösteriy! Onlemekiçinçıkartılmıştır, kanısındayız. Fakat fetvada dinsel kitapların basılarnayacağına ilişkin herhangi birhükiin bJ,ıluıvnadığl hU!1Uu, 23 ' ' fetvan ın V~n Kulu sözlüiij1d~kj~r~tinden Yararlanılarak ortaya .çıkarılmlŞ tır'. ~~~d Rasim'd,en yaptı~ımız alıntıda, esnaf tarafından yapılan gösteriyeulema--dan bazı kimselerin de~trldığından SÖZ edilmek~ir. Hiçbirt>illm adamınınbllimselaraştırmalara geniş bOyutlar kazandıracak böyle bir ~rişimin(TürkjYe'de Türkçe yayın yapacak bir matbaa kurulması girişiminin) karşısında olabileceğini düşij1mek mümkün &.örünmemektedir, Ma1b~, b,aşta da beli,rttiğimiz gibi, düşüncelerin kitlesel~ leşmesinde önemli rol oynamaktadır. Gerçek bilim a~ının da yaptığı dUşij1sel üretimi ne kadar çok kimseye yayabilirse kendisin~ o kadar başarılı sayması gerekir. Ahmed Rasim 'in verdiğ i bilgileri ak tarmaya devam,edc;lim : "Ulum ve füıunun ve her nev'i efkar.. medeniyyenin sür'at.. intişarına (her türlü uygar düşüncelerinhızla yayılmaSına) hizmet etın'işolan ihtlraat.. miiıimmeden (önemli ,buluşlardan)biri de matbaacılık dediğimiz ~bt-ı tıbOGt(basmsanatı)dır. Bu sanat'atı ilk icad eden G.ıttenberg namında Harlem şehri .-halisindenbir zattıi'. Tarih-4icadi 844 sene.j hicriWe ve 1440sene.j iniladiYYedir.Fakatbizdeki tarih.j icadı pek sonradır. , , , "OSınanlılardahuruf-u arabiWeyi (arap harflerini)san'at-ı tıbaata tatbikVe. ilk tesis edenler bilahare mesned-4sadarete irtika (en yüce makama yükselen) ve 1170 tarihinde dar-i bekaolmuş(ölmüş} Said Mehmed'Paşa ile DergaJ1-ıAli kapıcıbaşılarından Müteferrika ıbrahim Ağa'dır. Kibar-ıhacegan-ı divan-ıhümayundan (dvan-ı hUmayunefendilerinin büyüklerinden)Yirmisekiz Çelebi Mehmed Efendi sefaret1e Paris'e izarn olunduğu (gönderildiği)zaman mandumu{oğlu) Said Efendi'yi de kethüdalık hizmetiylem.aiyetinde götürmüş idi, Said Mehmed E~ndi'nin bu seyahatte (23) Hidayet Yavuz Nub~lu, "Müteferrika Matbaasınm Kurultnası Için Verilen Fetva tjzerine~ "Türk Kiitiiphaneciler Demeği Basım lie Ya)'lncılığıml%lR 250. Yıiı Bilimşel 'TopIQntfSllQ-l1 Anılık 1979, ,Ankanı 'Bildiriler,ADkara.1980 ss. 119-126. ' " '-" 162 'AH~,1ET RASiM'iN iBRAHiM --- ~-~ MüTEFERRiKA VEMATBAACALIK en evvel naz;ır-ı dikkatini celbeden şeyParis'in matba~iları oldu. istanbUl'a avdctinde(döntişühde} rhüşa!ıedatını (gö&rirJerini) ve bu babdakiefkarmı (bu konOda:ki dÜşüncelerini),M(jteftrrikiıibrahimAğa'ya;ınlattl."24 , , \ Buradabirazdurahmvc birJki noktaya deginclfm:Va,ptığırrıızalıntıcla matbaanın dışarıdaki "tarih .,jkadı" iie bizdeki. "tı:'rih.,j kadl"miıfaf"klftYlduğu Söylenmektedir. DÜnyanın herhangi bir ülkesinde yapılan yeni bir buluş bilimin evrenselliği yasasına tcrs dÜşmeden düriyanmher,tarafına yayılır., Bizde yapılan da,matbaacıh. ğı n rfrcvcÜtteknikleriÖğreniler~k ü'kerniidekendi idt<tplarıimzıri'basımı için hizmete sokulmasıdir. Demek istCdiğ imii ,'ülkı!rriiidç inatb~ kunılmadan önce diğer ülkelerde matbaa kurulmuştürve faaiiyettediı',:YOk;a'rnatba'aikinci kez icadedilmemiştir. Ayrıca, 15. yÜzyıl ronlarınt1aO!irn'anh iinparatorlÜgu coğrafyasında çalişmakta olan birçok matbaanın olduğu da bilinmektedir. , 'Öteya'ri~n ,Said Efend,i'ninPiU"ismatbaalarınl merakla ,görmesi ve istanbul'a dÖnlişiipde 'gö'rd,iildei'inima1baakonuıııylaı;iddibir şeki/ge ilgilenenibrahim Müteferrika'ya anlaımaSldikkat çekicidir~ Bu da~ibrahim Müteferrika'nın matbaayı daha önceden.tıı)ıdiğını gösterin'ekted,k.' Nite~l>u 'SjlvımlZldoğrulayan belgeler de bu1unmaktadır: Ersoy'un e.serinde'bu husu'-s/ailgili gen'iş bilgi verilmiştir. 2 s Kaldı ki, bu husu sla Ilişki! i olarak ,şusonı da' akla g~lmekt~ ir: Ac~a Said Efendi Paris dönüş'ü gözlernierini ne~ren,birb~şkasına değil' ,de Mütcfernka'ya anlatmıştır? Demek ki, Mü'. c - . <"'c,"- . _'. .' . _ ~. .'. ." ,'.' , _ . '_', _ . .. -' '" _ teferrika'nın bu sanatı daha öncedentanıdığı devict yönetiminin üst kademelerinde görevalan bazı kimselercede bHinrrıekted,ir. , ~ Ahmed Rasim matbaanın kunıluş çalışmalarını da şöyle anlatmaktadır: "ibrahim Ağa Vesiletii't-Tıbaanamıyı.r'bir risaleyazaralcbunda :Saniat...tıbaatın fevaidini (basım'sanatının yararlarını) say<ft<İöktü:Bu(Vesiletü't-Tıbaa)sadrazam ot zamanolan .ibrahimPaşa'ya :takdim.olundu. Müşaruıiileyh(adıgeçefl) Paşa bunun ebemmiyetini takdir itehemen-Q devrin uk<1l~veflizelasından (akdlive erdemlilerinden) bir meclis~ meşveret(danışmakuıulu) teşkileyledi. Bunun efkar-i ammeee (kamuoyunca) mazhar-ıhüsn-ükabul olması ve kavaid.,jşFr'i mübine (islamdini ku. ra!1~rına ).t;atl? ik, ,ve tevfik ,ile tesisi için şeyhülişlamdan ,isti~ya (fetVıl istenmesine) meclisçe karar, verildi. _Şeyhwislam Abpu/lah, Efendi de fetva verdi, Bir de takriz (takdim:yetak~jjry~ısı) y~dı'. U~emaiJaJ:) j)ek ç~ ~~vatdatakrizler,yazdıl~r.' . " ',. ... - ile beraber '''\Tesileiii/t-Tıb,QClmeclis.,j meşveretin ,mazb~tası ve fetva- ve, takrizler SultınAhm~'Salis'e, (Üı. Ahm,efe).ari'olunarakbkhatt-ıhümayun He Said Meh. meçIEfendi ve şetiki'(orıağ I)Miitefe.rrj}Q Jbr<ıhimAğa 'yamcı~aa tesisi, ve kitap tab ',I için ruhSat ita kılındı (verildi)."26, ' (24) Ahmetl Rasiın,a.g,y. FeritBey'in ss, .815-'-816 N€vSal.iOsmallJadh (25 )Ersöy', a.g,y., 8,,31 (28) Ahmed Rasinı, a,g.y, 8; 817 Ahmed Rasim yukarıdakibilgileri e8eril1d~naktarmaktadır. ' Oıman , YAŞAR A. TONTA Yukarıya aldığımız ik i paragrafta Ahmed Rasim ,ibrahim Miiteferrika' nın basım sanatının yararlarını anlatan bir risale yazarak zamanınsadrazam i Damat ibrahim Paşa'yasunduğunu; Sadrazamın Vesilet4't"Tıbdaadını ta~lyanQurisalenin önemine binaen bir danışma kurulu kurarak şeyhiilislanıdan fetva istenmesine karar verildiği. . ni ve şeyhülislamın da fetva verdjğiniyazmaktadır.' Vesiletü't-Tıbaa ad1ıil risalenin b ıgüne kadar bir yazma nüshası bulunamamışt~r; Ayrıca, günümüzde nadir eserler kita piyasasında baş tarafındaadı geçen risale basılı olan V~n Kulu sözlükleri. olmayan ra oranla dahadeğerli sayılmaktadır. Kanımızca Vesiletü't-Tıbaabasıla ilgili tüm hazırlıklar tamamlandıktan sonra Damat ibrahim Paşa'ya su~lmuştıı .Matbaa konusunda kamuoyu 01uştıırmakanıacıyla yazılmıştır. Ortada maıbaa Ileilgili hiçbir hazırlık yokken yazılıp Sadrazama sunulmuş değildir. Toplandığıiddia edilen danış akurulunun herhangi bir konu Uzerindckar.ar verme yetkisi buh.ınmamaktadır.Daışma kurulu boyle bir karar çıkarama)'"acağı gibi şeyhülislamı fetvavermeye de orlayamaz. Şeyhülislamdan devlct ışleriylc ilgili olarak saltpadişah fetva isteyebil r. Kaldı ki, danışma kUl\1lunun b" yle bir yetkisininolabiıeceğini kabullensek bile söz konusu kararla ilgili mazhata rtada bulunmamaktadır. Verilen bilgilerdeise, danışma kuruhınun şeyhülislamclan aldığı fetvayı kendi karar mazbatasıylabirlikte Sultan ııı. Ahmct'e sunduğu; padiş ın da verd1ği hatt':' hÜ01ayun ile Said Mehmed Efendi. ile ortağı Mütefeıtika'nınbas"mevi kurarak kitap basmalarını' onayladığından söz edilmektedir. Bu bilgiler Osmanlıyönetim erkinin yapısınauy~n gözükmemektedir. Şöyle ki; danışma kuıulu taratnmn fetVa padişaha sunulmaktadır. Padişah da bu fetvaya boyuneğerek fermanver ektedir. Yani, kararı, fetvayı veren şeytiülisıam onaylamakta. padişahise uygulam tadır.Böyle bir durumun -Osmanlı yönetimirÜn hiyerarşik örgütlenişi içerişinde yeri lmayacağı kanısındayız'. Ahmed Rasim makalesinin ilgili bilgiler vermektedir nda., Müteferrika matbaasında basılan eserlerle . ~ "Bunun üzerine mÜ01aileyhimaa (adı geçen iki kişiye) edavat-ı tab'iyyeyi (basım araçlarını) ihzar ve ikmal ile ilk Osmanlı matba~sınıSultanSeiim civarında ibra. him Ağa'nın hanesindeve 1140 tari ~ hicriyyesinde tesis.cttiler (ki tarih~icadından ,üç asır sonda demektir).-aumatbaa a 1140'ta ilk basılan kitap meşihlr-i ulemadan (ünlü bilginlerden) meşhur Vani Me ed bin Mustafa Efendi'nin Van Kulu nam lu- " gatıdırki Sılıah-, Cevhen ismindeki 19at kitabının Türkçeye tercemcsidir. Bin tane basılmıştır (1141 senc) ikinci tabi edilen Katip Çeleb 'nin: Tunfetü'l bahriye tarihidir. . ' . ,-- Kibar fı Esfari'l-Bihar "'"\m 164 AHMET RASiM'iN ıBRAHiM MüTEFERRiKA Oçüncü:Terceme~ Tarih~ Seyyah VE MATBAACILIK . Dördüncü: Tarih~ Hind~ Gaıbi EI.Müsenıma bi Hadis~ Nev. Beşinci: Tarih~ Timur,Gürgan. Altıncı : Tarih ~.M Isru'I.Kad im ve Mısru 'I-Cedid . Yedinci: Gülşen~ Hülefa. . Sekizinci: Usulü'I.Hikem fi Nizamü'I-'Omem(ibrahifn. Dokuzunaı: Füyuzat-ı Mıknatısiyye (keza). \ Müteferrika'nın). Ownaı : Cihann~.a (Katip Çelebi~nin). . Onbirinci:Tarih~Naima,cild-ieweL Onikinci: Tarih-i Naima, cild-i sani. Tarih-i Raşid ,(cild~ievvel ve sani)." 27 Ahmed Rasim'in konumuzia ii~1i olarak Osmanlı Tarihi adlı eserinde yer alan makalesi yukarıdaki satırlarla son bul~~tadır. Ancak, yazısının sonundaki birkaç çelişkiye de değinmek gerekmektedir. -Müteferrikamatbaasında basılan kitaplanh kaynakçası Ersoy'uneseiinde veril. 'mektedir. 28 Ayrıca,adı geçen matbaada basılan ,ve özellikle resimliolan kitapların. ayrıntılı özellikleri hakkında-geniş bilgi, konuyagrafik sanatının doğuşu açısından yaklaşan Sait Maden'in Qevre dergisindeyayıml~nandizi yazısında bulunmaktadır.29 Ahmed R~im, matbaanın faaliyete geçiş tarihiyle ilgili üç değişik tarih vermektedir.ilkind e, "... Ve bu veçhile matbaa 1139 senesinde ibrahim Efendi'nin kendi evinde işe baş~/\demektedir.3o Verdiği butariht~nbirxaç sayfasonra; "...ilk Osmanlı matba.; Sultan, Se6m civannda ibrahim Ağa'nın hanesinde ve 1140 tarih-i hicriy'yesinde tesis ettiler" 3 i cümlesibulunmaktadır. Birkaç satır sonrada ilk basılan eser olan VanKulu sözlüğününbasım tarihini verirken: "Bintane basılmıştır (1141 se06)"3 2 .demektedir. 1139, 1140 ve :1141. 1141 tarihii!k basılan eser olan Van Kulu'nun yayın ti\rihiolarakdoğrudur. 1139 Ve 1140taiihlerindekiçelişki di. ğer kaynaklardada karşımıza çıkmaktadır. Yukarıda,Vesileffl't-Tıbaaının matbaa " konusunda kamuoyu faratmak,dilekçe ire fetvanın da herhangi birtepkiyi.önlemek amacıyla ele alındığını ifade etmiştik. Aynca, Vesilem't.Tıbaa'nınmevtut nüshalarına dade~inmiştik. Bu bilgilerimatbaanın kuruluşunu doğru saptamak amacıyla kullana- (27) Ahmed Rasim,a.g.y.ss. 818~19 (28) Ersôy,a.g.y.ss. 3845 (29) Sait Maden, "Başlangıcindan .BugüneTürk' Grafik Sanatı (2); Müteferrika: Dönemi, "çeure Cilt I, No. 2, ss. 83-87;ve:Ciltl,No. 3, ss. 87~90, 1980. . . . (30) Ahmed. Rasim, a.g.y. s. 815 (31) a.y. 818 (32)0.y. . . .' YAŞAR 165 A. TONTA biliriz. Vesiletii 't-Tıbaa 'rlRlbasılı kopyesinin elde bulunmaması ; ayrıca, dilekçede örnek. . nüshaların takdim edildiğinden söz edilmesi b,izi, fetvaya dayanan .emr~ hümayun alınmasından önce matbaanın kurulmuş ve faaliyetegeçmiş olabileceği noktas.ınagô, türmektedir. Matbaanın hartlerdolayısıy.1a Avrupa'dan ithaldeğil, tamamen yerli bir tesis olarak' kurulduğu, ve bunun için gerekli olan zama,n da göz'önündealınırsa, ma1baanın vertlen, tarihlerdenç~k daha önceki bir tarihte kuıulduğu söylenebilir .Hat- ta bu tarihi, bir olasılık da olsa, saifÇelebi'nin Paris'ten dönüşüolarak da düşünmek mümkündür. . . , Ahmed Rasim, Müteferrika matbaasında basılan eserlerin kaynakçasını verirl<en, dikkat edileceği üzere Gtammaire Turquie'ten hi~ söz etmemektedir. Bilindiği gibi Müteferrika, Nôima Tarihi'ne değin bastığıkitapların kolofonunu Naima Tarihi' \ nin sonundavermiştir.Bu kolofonda Grammaire Turquie gösterilmemiştirP Bunun üzerine, Ahmed Rasm'inbasllaf1 kitapların listesini Nôima Tarihi'nde bulunan kolofondan y.a~rlimarak verdiğini bu nedenle de, Grammaira Thrquie'ü listesine alm4d1ğını düşünmüştük; Fakat hemen 'sonra, S.N.Gcrçek'ineserinin "vesikalar" bölümüoü.karJş,tırırkenbuldu,ğumuz.kolofolJun faksimilini inceieyerek,Ahmed Ra- sim'in baSılan esenerinlistesiniverirkenbukolofondan yarananmitdığını saptamıŞ i bulunmaktayız,. " Çünkü kolofondaQrammaire Thrquie verilmediği halde T"kvimü't-Tevôrih adlı eser Cihannümô,'dan sonra,"Takvimü't-Tcvarih .Ii Kitip Çelebi cild 500" şeklinde verilmektedir. Oysaki, Ahmed Rasim'in verdiği listede Takvimü't-Tevarih'in de adı geçmemektedir. OolaYISly1a, listenin hangi kaynaklara dayanılarak hazırlandığı saptanamamıştır. .. Ayrıca verilen.listede, basılan ka,çıncı eser olduğu belirtilmeksizin Tarihi Raşid' it birinci ve ikinci ciltlerinin vadığından söz edilmektedir. Basılandiğer eserler konusunda ise araştırma yapma gereği duyuımam ıştır. ',,- Ahmed Rasipı'in rhakal~sihakkında' genelolarak şunlar söylenebilir: Incelenen kaynaklar eleştiri süzgecinden geçirilmeksiıin olduğ~ gibi kab",i edilmjştir. Bu nedenle dekaynaklarda mevcutbaiıçe6şkilerin farkına varılamamıştır; Basımevinin kurulUşu ~nu_5Unda eldeki belgelere dayal'Illarak doğrulu kesin olarak bilinen vefakat AhmedRasim tarafından hatalı olarak aktarılan bilgilere de , rastlanmaktadır.Ömeğin, Ahmed Rasim, De Saussure'di1n aktararak, istanbul halkının çokeskiden beri ıanıd1ğımatbaayı "ibtira-ıcedid" diye netilendirmeldedir. ıstanbul'da yaşayan Musevi, Ermeni ve Rum azınlıkların 15. yüzyılın sonundan itibaren istanbul;da kitap bastıklarına 'daha önce değinilmişti. Ayrıca, Avrupa'da basılmış Şark, eserlerinin Istanbul kit;ı.pçdarınd;ı. sattıdığı ve bunların, itibar görmediği de . bilinmektedir. ';133) Gerçek, a.g.y .s. 84 , 166 AHMET RASir,:'iN iBRAHiM MÜTEFERRil<A VE MATBAACtLlK Keza,.. Ahmed Rasim' in maKalesindebeli rttiğigib i, matbaan ın ku ıt/lmasına Said EfendiJ1in düşüncesiylehaşlanmadığı hu G;Isuna yukarıda ayr' tl" bir biçimde yer ve- rilmişti. . l Tarihsel kaynakların iyi değerlendirilememesincJenve yapılan araştırmalarda bilimsel kuşkuculukla hareket edilmemesindendolayı yan,lgilariıdüşülmüştür.Oysaki, verilen bilgiler o giiJıkübelgelerdensağlanmıştır. KuşkusllZ doğru bilgiler de bu . belgelerde bulunmaktadır.. . SONÜÇ Vlusal kültür yaşamımızclaç()k önemlibir yer tutanmatbaanın kurulUşu, .ülke. mizdeki düşünselgelişmeye ~üyük bir hıt kazandırmış ve toplum eskiye oranla daha fazla ve nispeten daha ucuza kitap okuyabilme olanağ.u\a ka.wşmuştUr.~ önemli bu. luşun uygulanmasında :ıynı zamandabir bilim adamı olan ibrahim Mütefernka'nın ro. iü kuşkusuz çok fazladır. Her konuda derin birbilgiyesahip olan Mütefe~rika'oın bel~ leğinde daha öğrencilikyıll:ırındagöi'çlüğübas,rnevidetin bulunduguridada buSanatı "lt ra'.' etmiştir. .. . izler ~lrakırilŞve olanak ibr;ıhim Mteferrika devlet kademelerinde üstlendiği görevleri ve matbaatılığı birarada yürütmüştür,.kanısındayız.Buna karşın azımsanamayacak.sayıdavebirbirin. den değerli kitaplar basmıştır. öyleki, :bastığı eserlerdenbir kısmı bugünbile birinci el kaynakolmaözelliğinikorumaktadır. . Devingertharflimatbaanın ülkeırtiz(lefaaliy~te geçmesiyle birlikte basılan kitiplarda hızı. bir artıŞ olduğu sôylenebilir. bk ylUama çok y.avaşolan bu hız, yazıda mekanizasyonun yararlan toplum tarafından gÖrüldükçeartmaya başlamıştır. Hele hele kutsalkitaplarabir zarar gelmediğini ve saygısızlık edilmediğini gören kimseler basılı kitaba daha fazla ilgigöstermişlerdir. Gii1ünüzderurkiye'de 2500'e yakın sade. tegazetertin ba-sllmasl,matbaan4okültürel~lişmemizcieoynactığı rolünen açık kanı. . . tı olarak gösterilebilir. kanısındayız. KAYNÇ ADIVAR,A. ADNAN OsmanlıTürklerin'de Ilim,geli ştirilmiş 4. bs. istanbul: Remzi Kitabevi, 1982.s. 166-170. AHMED RASiM OsmanlıTarihi, Cılt 1I,<istanbul; Şems Matbaasl,1326_ 1329.55.811-819. BA YSA L, JA LE BERKES, NiYAZi BERKES, NiYAZi Müteferrika 'dtin Birinci Meşru tiyete 'Kada~ Türiderinin Bastıkları Kitaplar, istanbul, 1968. Osmanlı "ilk TütkMatbaa KUıucu~nun Dini w Fikri Kimliği," BeIleten Cilt26.No.104, ss. 720-721, 1962. Türkiye 'de Çaf{claşlaşnıa, istanbul,1978. . YASARA' 167 TONTA' CARLESON, EDV ARD ERSOY, OSMAN GERÇEK, SELiM . lbrahiın kliiteferrika Basımevi ve Bastığı/lk Eser/er, Yayına hazırlayan: Mustafa Akbulut. Ankara: Türk Kütüphaneeilel' Derneği, 1979. Tiliniye'ye Matbaanın Girişi ve 1II~Bası/an Eser/er, Anka. ra,1959 NOZHET TilrkiUatbaacllıjJı 1939. i; Müteferrika Matbaası, Istanbul, . ' "ibrahim Müteferrika," lş.ldm Aniihlopedisi, cm 5, ss KUN, HALASi 896-0 MADEN, SAiT UBaşlangıcındanBugi~e Türk Grafik Sanatı (2); Müteferrika Dönemi, "Çevre Ciltt,No. 2, SS83-87, Mart-Nisan . 1980: MADEN, SAiT "Başlangıcından BugüneTürk Grafik Sanatı (3); Mütefer., rika Dönemi 2,u Çevre Cilt i, No. 2, ss. 87 -90, M,W+laZ 1980. SIVAVUşGIL,SABRI TVRKKVTVPHANECILER ... ESAT "Ahmed Rasim," Islam Ansiklopedisi Cilt 1, s.201. DERNEG/ Basımve YayıncılIRımIZin 250. Yılı Bilimsel ToptanhsJ 10-11' Aralık 1979, Ankara Bildiriler, Ankara, 1980, ss. 119-126.. 

W3009565379.txt

https://journal.uwgm.ac.id/index.php/KESMAS P-ISSN: 2477-1880; E-ISSN: 2502-6623 December 2019, Vol. 5 No. 2 Hubungan Antara Pengetahuan, Motivasi Pasien dan Dukungan Keluarga Terhadap Kepatuhan Minum Obat Anti Tuberkulosis (OAT) Pada Penderita Penyakit TB Paru BTA (+) di Puskesmas Pasundan Kota Samarinda Nindi Elvira Fitriani 1, Tumpak Sinaga 2, Abdul Syahran 3 nindielviraf7@gmail.com, Universitas Widya Gama Mahakam, Indonesia1 siganatumpak@yahoo.com2, abd_syah@yahoo.co.id3, Dinas Kesehatan Provinsi Kalimantan Timur, Indonesia 2 3 Abstrak Latar Belakang: Data Puskesmas Pasundan Kota Samarinda, angka keberhasilan pengobatan tahun 2017 sebesar 47,60%, yang masih dibawah target Nasional sebesar 85%. Tujuan : Tujuan penelitian ini mengetahui hubungan antara pengetahuan, motivasi pasien dan dukungan keluarga terhadap kepatuhan minum obat anti tuberkulosis (OAT) pada penderita penyakit TB Paru BTA (+) di Puskesmas Pasundan Kota Samarinda tahun 2019. Metode Penelitian: Jenis penelitian kuantitatif dengan desain cross-sectional. Populasi adalah seluruh penderita penyakit TB Paru dengan BTA Positif yang berkunjung ke di Puskesmas Pasundan Kota Samarinda dari bulan Januari sampai Oktober tahun 2018 dengan jumlah 31 penderita. Teknik pengambilan sampel total sampling. Analisis data menggunakan uji chi-square. Hasil : Hasil penelitian tidak ada hubungan pengetahuan terhadap kepatuhan minum obat anti tuberkulosis (p value : 0,056 > α : 0,05), tidak ada hubungan motivasi pasien terhadap kepatuhan minum obat anti tuberkulosis (OAT) (p value : 0,057 > α : 0,05) dan ada hubungan dukungan keluarga terhadap kepatuhan minum obat anti tuberkulosis (OAT) (p value : 0,002 < α : 0,05). Kesimpulan: Tidak ada hubungan pengetahuan, motivasi pasien dan Ada hubungan dukungan keluarga terhadap kepatuhan minum obat anti tuberkulosis (OAT) pada penderita penyakit TB Paru BTA (+) di Puskesmas Pasundan Kota Samarinda tahun 2019. Kata kunci: Pengetahuan, Motivasi, Dukungan Keluarga, Kepatuhan Minum Obat Anti Tuberkulosis. Abstract Background: Data from Pasundan Health Center in Samarinda City, the success rate of treatment in 2017 is 47.60%, which is still below the national target of 85%. Objectives: The purpose of this study was to determine the relationship between knowledge, patient motivation and family support for adherence to taking anti-tuberculosis drugs (OAT) in patients with pulmonary TB disease (+) in Pasundan Health Center, Samarinda City in 2019. Research Metodes: This type of quantitative research with cross-sectional design. The population was all patients with pulmonary TB with positive smear who visited Pasundan Health Center in Samarinda City from January to October 2018 with a total of 31 patients. Total sampling technique sampling. Data analysis using the chi-square test. Results: The results of the study there is no relationship of knowledge of adherence to taking anti-tuberculosis drugs (pvalue: 0.056> α: 0.05), there is no relationship between patient motivation and adherence to taking antituberculosis drugs (OAT) (p-value: 0.057> α: 0.05 ) and there is a relationship of family support for adherence to taking anti-tuberculosis drugs (OAT) (p-value: 0.002 <α: 0.05). Conclusion: There is no relationship of knowledge, patient motivation and there is a relationship of family support for adherence to taking anti-tuberculosis drugs (OAT) in patients with pulmonary TB disease (+) in Pasundan Health Center, Samarinda City in 2019. P-ISSN: 2477-1880 E-ISSN: 2502-6623 Kesmas Uwigama : Jurnal Kesehatan Masyarakat Kesmas Uwigama : Jurnal Kesehatan Masyarakat P-ISSN 2460-0350, E-ISSN 2477-5819 December 2019, Vol. 5 No. 2 Kata kunci: Knowledge, Motivation, Family Support, Compliance with Anti-Tuberculosis Medication. DOI : http://dx.doi.org/10.24903/kujkm.v5i1.838 Received : October 2019 Accepted : November 2019 Published : December 2019 Copyright Notice This work is licensed under Creative Commons Attribution 4.0 International License. P-ISSN: 2477-1880 E-ISSN: 2502-6623 PENDAHULUAN Data Kementerian Kesehatan tahun Paru pada tahun 2013 sebesar 96,12%, 2011 menunjukkan bahwa insiden semua kembali mengalami penurunan pada tahun tipe TB adalah 450.000 kasus atau 189 per 2015 menjadi 82,57% dan meningkat 100.000 penduduk, angka prevalesi semua kembali pada tahun 2016 menjadi 83,86%. tipe TB 690.000 atau 289 per 100.000 Angka keberhasilan pengobatan TB Paru penduduk dan angka kematian TB sebesar (Succes Rate) per Kabupaten/Kota. Kota 64.000 atau 27 per 100.000 penduduk atau Samarinda berada di urutan ke 2 terendah 175 orang per hari. Jumlah kasus TB di setelah Kabupaten Paser dengan jumlah Indonesia menurut Laporan WHO tahun keberhasilan pengobatan (Succes Rate) 2015, diperkirakan ada 1 juta kasus TB sebesar 71,55%. (Data Profil Kesehatan baru pertahun (399 per 100.000 penduduk) Provinsi Kalimantan Timur Tahun 2016). menurun pada tahun 2014 menjadi 90,64%, dengan 100.000 kematian pertahun (41 per Menurut Data Dinas Kesehatan 100.000 penduduk) angka notifikasi kasus Kota Samarinda tahun 2016, penemuan (Case Notification1 Rate/CNR) dari semua kasus TB BTA (+) di Kota Samarinda kasus, sebanyak dilaporkan sebanyak 129 per 457 kasus, menurut jenis 100.000 penduduk. Jumlah seluruh kasus kelamin, jumlah kasus pada laki-laki lebih 324.539 kasus, diantaranya 314.965 adalah tinggi daripada perempuan yaitu 270 kasus kasus baru. Adapun jumlah kasus TB paru pada berdasarkan microskofik 2016 perempuan. Menurut kelompok umur, sebanyak 759/100.000 penduduk kasus TB Paru pada kelompok umur 25-34 tahun (Kemenkes RI, 2016). Menurut Data Dinas Kesehatan Provinsi Kalimantan Timur tahun 2016, laki-laki dan 187 kasus pada tahun. Sedangkan kasus TB Paru pada anak-anak 0-14 tahun sekitar 3% dari total penemuan kasus. Sedangkan angka presentase keberhasilan pengobatan TB P-ISSN: 2477-1880 E-ISSN: 2502-6623 Kesmas Uwigama : Jurnal Kesehatan Masyarakat 125 Kesmas Uwigama : Jurnal Kesehatan Masyarakat P-ISSN 2460-0350, E-ISSN 2477-5819 December 2019, Vol. 5 No. 2 keberhasilan pengobatan (Succes Rate) lanjutan 4-6 bulan berikutnya. Pengobatan pada tahun 2017 Kota Samarinda sebesar yang teratur pada pasien TB paru dapat 42,20%, dengan penemuan BTA (+) sembuh secara total, apabila pasien itu berjumlah sendiri mau patuh dengan aturan-aturan 144 kasus (Data Dinas Kesehatan Kota Samarinda Tahun 2017). tentang pengobatan TB Paru. Sangatlah Menurut Data Puskesmas Pasundan penting bagi penderita untuk tidak putus Samarinda, keberhasilan berobat dan jika penderita menghentikan pengobatan (Succes Rate) pada tahun 2017 pengobatan, kuman TB Paru akan mulai sebesar 47,60%. Nilai tersebut masih berkembang sangat jauh dari target Nasional yaitu penderita mengulangi pengobatan intensif sebesar 85%. penemuan kasus TB BTA (+) selama 2 bulan pertama. Tanpa pengobatan, pada tahun 2015 sebanyak 24 kasus, dan setelah lima tahun 50% dari penderita TB meningkat pada tahun 2016 sebanyak 34 akan meninggal, 25% akan sembuh sendiri kasus, dan terus meningkat pada tahun dengan daya tahan tubuh tinggi, dan 25% 2017 sebanyak 57 kasus, dan pada tahun sebagai kasus kronik yang tetap menular. 2018 dari bulan Januari sampai dengan Sebaliknya, jika penderita melaksanakan Bulan Oktober berjumlah 31 kasus (Data pengobatan dengan baik atau pengobatan Puskesmas Pasundan Tahun 2018). dengan pengawasan minum obat secara Kota angka Mengingat tingginya kasus TB paru langsung biak lagi yang sehingga berarti mampu dan risiko penularan terhadap orang lain mempertahankan diri terhadap penyakit, yang cukup tinggi maka pemerintah mencegah masuknya kuman dari luar dan mengeluarkan kebijakan dapat menekan angka kematian yang penanggulangan TB dalam paru melalui disebakan oleh TB Paru (Septia, 2017). pengadaan obat anti tuberkulosis (OAT). Penelitian terdahulu yang dilakukan Kebijakan ini sejalan dengan rekomendasi Ahsan dkk (2012) menyatakan bahwa salah WHO dimana penggunaan obat anti satu faktor yang dapat meningkatkan tuberkulosis strategi kepatuhan pengobatan pada penderita (Directly Observed Treatment Shortcourse) dengan penyakit kronik ialah adanya DOTS mengurangi dukungan keluarga yang baik. Dukungan penyebaran penyakit TB paru. TB paru keluarga sangat diperlukan terutama pada adalah penyakit yang dapat diobati dan penderita disembuhkan. Pengobatan TB paru dapat penyakit kronik dan mengharuskan ia diberikan dalam 2 tahap, yaitu tahap mengkonsumsi obat dengan jangka waktu intensif 2 bulan pengobatan dan tahap yang lama, karena keluarga merupakan lini (OAT) bertujuan dalam untuk P-ISSN: 2477-1880 E-ISSN: 2502-6623 Kesmas Uwigama : Jurnal Kesehatan Masyarakat TB yang juga merupakan 126 Kesmas Uwigama : Jurnal Kesehatan Masyarakat P-ISSN 2460-0350, E-ISSN 2477-5819 December 2019, Vol. 5 No. 2 pertama bagi mendapatkan meningkat penderita masalah apabila kesehatan kesehatan itu atau sendiri. Merupakan salah satu fungsi keluarga untuk mendukung anggota keluarga yang sakit dengan berbagai cara, seperti memberi dukungan dalam mengkonsumsi obat. Berdasarkan uraian tersebut, maka HASIL PENELITIAN Hubungan pengetahuan terhadap kepatuhan minum obat anti tuberkulosis (OAT) pada penderita penyakit TB Paru BTA (+) di Puskesmas Pasundan Kota Samarinda tahun 2019. Tabel 1. Hubungan pengetahuan terhadap kepatuhan minum obat anti tuberkulosis (OAT) pada penderita penyakit TB Paru BTA (+) peneliti tertarik untuk melakukan penelitian tentang mengetahui pengetahuan, hubungan motivasi pasien antara Kepatuhan Minum Obat Anti Tuberkulosis No Pengetahuan dan minum obat anti tuberkulosis (OAT) pada penderita penyakit TB Paru BTA (+) di Tinggi 2 Rendah Puskesmas Pasundan Kota Samarinda % Patuh % Tidak Patuh % 7 22,6 5 16,1 12 38,7 4 12,9 15 48,4 19 61,3 11 35,5 20 64,5 31 100 dukungan keluarga terhadap kepatuhan 1 Total Jumlah p value 0,056 tahun 2019. METODE PENELITIAN Metode penelitian yang digunakan adalah dengan menggunakan metode Hubungan motivasi terhadap kepatuhan minum obat anti tuberkulosis (OAT) pada penderita penyakit TB Paru BTA (+) di Puskesmas Pasundan Kota Samarinda tahun 2019. Adapun besarnya sampel pada penelitian Tabel 2. Hubungan motivasi terhadap kepatuhan minum obat anti tuberkulosis (OAT) pada penderita penyakit TB Paru BTA (+) ini adalah sebesar 31 responden. Jumlah No Motivasi 1 Tinggi 9 29 8 2 Rendah 2 6,5 11 35,5 kuantitatif dengan desain cross-sectional. tersebut sama dengan jumlah populasi, yaitu seluruh pasien penderita penyakit TB Jumlah Paru dengan BTA Positif yang berkunjung Kepatuhan Minum Obat Anti Tuberkulosis Tidak Patuh % % Patuh Total % 25,8 17 54,8 12 38,7 14 45,2 20 64,5 31 100 p value 0,057 ke di Puskesmas Pasundan Kota Samarinda dari bulan Januari sampai Oktober tahun 2018 dengan jumlah 31 penderita. Analisis ini digunakan untuk mengetahui hubungan atau korelasi antara variabel bebas dan variabel terikat dengan uji Chi Square (yang disajikan dalam bentuk kontigensi 2x2. P-ISSN: 2477-1880 E-ISSN: 2502-6623 Kesmas Uwigama : Jurnal Kesehatan Masyarakat Hubungan dukungan keluarga terhadap kepatuhan minum obat anti tuberkulosis (OAT) pada penderita penyakit TB Paru BTA (+) di Puskesmas Pasundan Kota Samarinda tahun 2019 Tabel 3. Hubungan dukungan keluarga terhadap kepatuhan minum obat anti tuberkulosis (OAT) pada penderita penyakit TB Paru BTA (+) tabel No Dukungan Keluarga Kepatuhan Minum Obat Anti Tuberkulosis Tidak Patuh % % Patuh Total 127 % p value Kesmas Uwigama : Jurnal Kesehatan Masyarakat P-ISSN 2460-0350, E-ISSN 2477-5819 December 2019, Vol. 5 No. 2 1 Tinggi 10 32,3 5 16,1 15 48,4 2 Rendah 1 3,2 15 48,4 16 51,6 11 35,5 20 64,5 31 100 Jumlah responden pengetahuan tentang TB Paru 0,002 rendah, namun patuh minum obat anti tuberkulosis PEMBAHASAN Hubungan pengetahuan terhadap kepatuhan minum obat anti tuberkulosis (OAT) pada penderita penyakit TB Paru BTA (+) di Puskesmas Pasundan Kota Samarinda tahun 2019 Berdasarkan hasil penelitian terlihat tinggi, proporsi tertinggi pada responden yang patuh minum obat anti tuberkulosis berjumlah 7 responden (22,6%), hal ini dikarenakan responden tahu dan memahami tentang TB Paru sehingga berupaya agar sembuh dari penyakit tersebut dengan patuh minum obat anti tuberkulosis. Terdapat responden pengetahuan tentang TB Paru tinggi, namun tidak patuh minum obat anti tuberkulosis berjumlah 5 responden (16,1%). Hal ini dikarenakan sering lupa waktu minum obat, malas dan bosan dari 19 responden pengetahuan tentang TB Paru rendah, proporsi tertinggi pada responden yang tidak patuh minum obat anti tuberkulosis berjumlah 15 responden (48,4%), hal ini dikarenakan responden kurang tahu dan kurang memahami responden jadwal minum obat yang dianjurkan dokter, mematuhi saran dokter dan tidak pernah kesal ataupun bosan dengan pengobatan tersebut. Hasil uji statistik dengan menggunakan uji fisher diperoleh hasil p value : 0,056 > α : 0,05 sehingga Ho diterima yaitu tidak ada hubungan pengetahuan terhadap kepatuhan minum obat anti tuberkulosis (OAT) pada penderita penyakit TB Paru BTA (+) di Puskesmas Pasundan Kota Samarinda tahun 2019. Hasil penelitian ini sesuai dengan penelitian terdahulu yang dilakukan Wulandari (2015) menunjukkan bahwa tidak ada hubungan pengetahuan dengan kepatuhan pasien Tuberkulosis Paru tahap lanjutan untuk minum obat di RS Rumah dengan pengobatan tersebut. Adapun 4 (12,9%). Hal ini dikarenakan selalu ingat dari 12 responden pengetahuan tentang TB Paru berjumlah tentang TB Paru sehingga kurang berupaya agar sembuh dari penyakit tersebut dengan tidak patuh minum obat anti tuberkulosis. Terdapat Sehat Terpadu. Begitupula penelitian Dewi (2011) menunjukkan bahwa tidak ada hubungan antara pengetahuan dan sikap penderita dengan kepatuhan penderita minum Obat Anti Tuberkulosis Paru di Puskesmas Lidah Kulon Surabaya. Berdasarkan hasil penelitian juga diketahui adanya kecenderungan bahwa pada penderita TB yang memiliki tingkat pengetahuan tinggi cenderung patuh dalam pengobatan TB. Hal ini terlihat bahwa pada P-ISSN: 2477-1880 E-ISSN: 2502-6623 Kesmas Uwigama : Jurnal Kesehatan Masyarakat 128 Kesmas Uwigama : Jurnal Kesehatan Masyarakat P-ISSN 2460-0350, E-ISSN 2477-5819 December 2019, Vol. 5 No. 2 penderita TB yang memiliki tingkat cenderung baik. Penderita memiliki pengetahuan tinggi sebanyak 22,6% patuh pengetahuan yang kurang akan tetapi dalam menjalani pengobatan TB dan penderita memahami sedikit informasi penderita TB yang memiliki tingkat yang mereka dapat dengan baik. Sehingga pengetahuan rendah sebanyak 48,4% yang saat penderita memiliki pemahaman yang tidak patuh dalam menjalani pengobatan baik maka diharapkan aplikasi juga baik TB. (Noorhizmah, 2016). Pengetahuan mempengaruhi penderita pemahaman dapat penderita Tidak adanya hubungan antara tentang penyakit TB Paru, semakin rendah pengetahuan dengan kepatuhan minum tingkat pengetahuan tentang kesehatan Obat Anti Tuberkulosis. Hal ini bisa terjadi maka akan kurang cara pemahaman dikarenakan oleh pengawasan yang kurang terhadap suatu penyakit, sehingga dengan atau penderita sudah bosan, sehingga harus pengetahuan yang tinggi maka akan ditingkatkan lebih lanjut. Dimana ada menunjang terhadap kepatuhan minum kecenderungan bahwa penderita Obat Anti Tuberkulosis (OAT) (Wulandari, memiliki pengetahuan tahu, memahami, 2015). dan aplikasi kurang memiliki tingkat Salah satu penunjang keberhasilan pengobatan TB Paru adalah dengan mengetahui seberapa jauh pengetahuan penderita tentang TB Paru. Pengetahuan penderita mengenai istilah TB Paru, gejala dan penularannya di Puskesmas Pasundan tergolong rendah. Padahal kemampuan dan pengetahuan masyarakat atau penderita akan gejala bagaimana penyakit cara TB Paru penularannya dan sangat mutlak dipahami. Karena dalam tahap ini, dapat dilakukan pencegahan terjangkitnya penyakit TB Paru atau setidaknya penyakit penderita dapat diketahui lebih dini sehingga pengobatan yang akan dilakukan tidak terlalu lama dan memiliki resiko kegagalan lebih kecil. Tingkat memahami yang kepatuhan yang tidak patuh. Hubungan motivasi terhadap kepatuhan minum obat anti tuberkulosis (OAT) pada penderita penyakit TB Paru BTA (+) di Puskesmas Pasundan Kota Samarinda tahun 2019 Berdasarkan hasil penelitian mengenai hubungan motivasi terhadap kepatuhan minum obat anti tuberkulosis (OAT) pada penderita penyakit TB Paru BTA (+) di Puskesmas Pasundan Kota Samarinda tahun 2019 terlihat dari 17 responden motivasi kesembuhan TB Paru tinggi, proporsi tertinggi pada responden yang patuh minum obat anti tuberkulosis berjumlah 9 responden (29%), hal ini dikarenakan responden memiliki tekat yang kuat dalam diri sendiri untuk sembuh sehingga responden patuh minum obat anti penderita berdasarkan hasil penelitian P-ISSN: 2477-1880 E-ISSN: 2502-6623 Kesmas Uwigama : Jurnal Kesehatan Masyarakat 129 Kesmas Uwigama : Jurnal Kesehatan Masyarakat P-ISSN 2460-0350, E-ISSN 2477-5819 December 2019, Vol. 5 No. 2 tuberkulosis. Terdapat responden yang penelitian berbanding terbalik, yaitu pasien motivasi kesembuhan TB Paru tinggi yang motivasinya tinggi banyak yang tidak namun tidak patuh minum obat anti patuh, sebaliknya pasien yang motivasinya tuberkulosis rendah banyak yang patuh dalam menjalani berjumlah 8 responden (25,8%). Hal ini dikarenakan responden pengobatan. mudah lupa waktu minum obat. Octaswari (2015) tidak ada hubungan Adapun dari 14 responden motivasi kesembuhan TB Paru rendah, proporsi Begitupula penelitian antara motivasi diri dengan kepatuhan minum obat penderita TB paru. tertinggi pada responden yang tidak patuh Adapun faktor-faktor yang minum obat anti tuberkulosis berjumlah 12 mempengaruhi ketidakpatuhan antara lain responden (38,7%), hal ini dikarenakan pemahaman tentang instruksi dan kualitas responden kurang memiliki tekat yang kuat interaksi. Mungkin motivasi yang dimiliki dalam diri sendiri untuk sembuh sehingga pasien untuk berobat tinggi, tetapi jika responden tidak patuh minum obat anti dalam menyerap instruksi dari tenaga tuberkulosis. Terdapat responden yang kesehatan tentang program pengobatan motivasi kesembuhan TB Paru rendah kurang, akan sangat sia-sia motivasi yang namun patuh minum obat anti tuberkulosis dimiliki. berjumlah 2 responden (6,5%). Hal ini motivasinya tinggi dan ditunjang oleh dikarenakan responden selalu ingat waktu kualitas interaksi yang baik antara tenaga minum obat. kesehatan dan pasien, maka pemahaman Hasil uji statistik Tetapi sebaliknya, jika dengan tentang instruksi yang diberikan akan menggunakan uji fisher diperoleh hasil p sangat tinggi. Kualitas interaksi antara value : 0,057 > α : 0,05 sehingga Ho professional diterima yaitu tidak ada hubungan motivasi merupakan bagian yang penting dalam terhadap kepatuhan minum obat anti menentukan derajat kepatuhan (Latifatul tuberkulosis Muna, 2014). (OAT) pada penderita penyakit TB Paru BTA (+) di Puskesmas Pasundan Kota Samarinda tahun 2019. Hasil penelitian penelitian terdahulu kesehatan dan pasien Dalam penelitian ini dijelaskan bahwa suatu kondisi akan menimbulkan ini didukung suatu keinginan, dorongan perilaku tertentu yang dilakukan yang mengarah pada kondisi tersebut. Latifatul Muna (2014) menunjukkan bahwa Perilaku tidak ada pengaruh motivasi dengan mengarahkan mengarah pada perilaku. kepatuhan berobat pada pasien TB Paru di Dalam penelitian ini didapatkan hasil Poli Paru BP4 Pamekasan. Dari hasil bahwa P-ISSN: 2477-1880 E-ISSN: 2502-6623 Kesmas Uwigama : Jurnal Kesehatan Masyarakat dan respon motivasi klien intrinsik TB akan dalam 130 Kesmas Uwigama : Jurnal Kesehatan Masyarakat P-ISSN 2460-0350, E-ISSN 2477-5819 December 2019, Vol. 5 No. 2 melaksanakan program pengobatan Pasundan Kota Samarinda tahun 2019 dipengaruhi oleh dua faktor yaitu faktor terlihat dari 15 responden dukungan internal dan faktor eksternal. Faktor keluarga tinggi, proporsi tertinggi pada internal yaitu meliputi keinginan dalam diri responden yang patuh minum obat anti sendiri, pengetahuan individu, tingkat tuberkulosis pendidikan, pengelolaan diri dan juga usia. (32,3%), hal ini dikarenakan keluarga yang Sedangkan untuk faktor eksternal tinggal berjumlah serumah selalu 10 responden mengingatkan meliputi faktor ekonomi, agama faktor jadwal minum obat dan memberikan pendukung keluarga dan perawat. Motivasi semangat kepada responden agar sembuh. untuk penderita TB dipengaruhi oleh dua Terdapat responden responden dukungan hal tersebut yaitu dari dalam diri penderita keluarga tinggi namun tidak patuh minum itu sendiri dengan adanya dorongan, obat keinginan untuk berobat, keinginan untuk responden (16,1%). Hal ini dikarenakan melakukan hal yang baik guna mencapai respondne mulai malas dan bosan, sehingga kesembuhan dan dukungan dari keluarga, anjuran keluarga kurang ditaati. masyarakat dan petugas kesehatan dalam anti tuberkulosis Adapun dari 16 responden dukungan seorang penderita TB dikatakan baik tertinggi pada responden yang tidak patuh apabila dapat minum obat anti tuberkulosis berjumlah 15 mengendalikan dirinya untuk mencapai responden (48,4%), hal ini dikarenakan kebaikan dirinya yaitu keembuhan dengan keluarga yang tinggal serumah tidak cara mengingatkan jadwal minum obat dan tidak rutin meningkatkan tersebut minum obat. Untuk motivasi penderita TB rendah, 5 menangani penyakit tersebut. Motivasi penderita keluarga berjumlah proporsi memberikan semangat kepada responden diperlukan adanya penyuluhan tentang agar penyakit dan bahayanya penyakit tersebut. dukungan keluarga rendah namun patuh Hubungan dukungan keluarga terhadap kepatuhan minum obat anti tuberkulosis (OAT) pada penderita penyakit TB Paru BTA (+) di Puskesmas Pasundan Kota Samarinda tahun 2019 Berdasarkan hasil penelitian sembuh. Terdapat responden minum obat anti tuberkulosis berjumlah 1 responden (3,2%). Hal ini dikarenakan responden memiliki semangat yang tinggi untuk cepat sembuh. Hasil uji statistik dengan mengenai hubungan dukungan keluarga menggunakan uji fisher diperoleh hasil p terhadap kepatuhan minum obat anti value : 0,002 < α : 0,05 sehingga Ho ditolak tuberkulosis penderita yaitu ada hubungan dukungan keluarga penyakit TB Paru BTA (+) di Puskesmas terhadap kepatuhan minum obat anti 131 (OAT) pada P-ISSN: 2477-1880 E-ISSN: 2502-6623 Kesmas Uwigama : Jurnal Kesehatan Masyarakat Kesmas Uwigama : Jurnal Kesehatan Masyarakat P-ISSN 2460-0350, E-ISSN 2477-5819 December 2019, Vol. 5 No. 2 tuberkulosis (OAT) penderita dimana keluarga inti maupun keluarga penyakit TB Paru BTA (+) di Puskesmas besar berfungsi sebagai sistem pendukung Pasundan Kota Samarinda tahun 2019. bagi anggota keluarganya. Fungsi dasar Dukungan pada keluarga sangat keluarga yaitu fungsi perawatan kesehatan. diperlukan terutama pada penderita TB Fungsi yang juga merupakan penyakit kronik dan kemampuan mengharuskan ia mengkonsumsi obat anggota keluarga yang mengalami masalah dengan jangka waktu yang lama, karena kesehatan. Keluarga perlu memberikan keluarga merupakan lini pertama bagi dukungan yang positif untuk melibatkan penderita apabila mendapatkan masalah keluarga sebagai pendukung pengobatan kesehatan atau meningkat kesehatan itu sehingga sendiri. Merupakan salah satu fungsi pemantauan pengobatan antara petugasdan keluarga anggota keluarga yang sakit. untuk mendukung anggota perawatan kesehatan keluarga adanya untuk kerjasama adalah merawat dalam keluarga yang sakit dengan berbagai cara, seperti memberi dukungan dalam mengkonsumsi obat (Septia, 2017). Dukungan keluarga yang positif diharapkan baik mengantar langsung untuk periksa di puskesmas maupun di rumah sakit, dokter atau petugas kesehatan lainnya dan berpartisipasi penuh pada pengobatan penderita. Dukungan keluarga memilki hubungan dengan kepatuhan minum obat pasien TB dimana PMO sebaiknya adalah anggota keluarga sendiri yaitu anak atau pasanganya dengan alasan lebih bisa dipercaya. Selain itu adanya keeratan hubungan emosional sangat mempengaruhi PMO selain sebagai pengawas minum obat juga memberikan dukungan emosional kepada penderita TB (Septia, 2017). Dukungan keluarga merupakan salah satu faktor yang mempengaruhi KESIMPULAN 1. Tidak ada hubungan pengetahuan terhadap kepatuhan minum obat anti tuberkulosis (OAT) pada penderita penyakit TB Paru BTA (+) di Puskesmas Pasundan Kota Samarinda tahun 2019. 2. Tidak ada hubungan motivasi pasien terhadap kepatuhan minum obat anti tuberkulosis (OAT) pada penderita penyakit TB Paru BTA (+) di Puskesmas Pasundan Kota Samarinda tahun 2019. 3. Ada hubungan dukungan keluarga terhadap kepatuhan minum obat anti tuberkulosis (OAT) pada penderita penyakit TB Paru BTA (+) di Puskesmas Pasundan Kota Samarinda tahun 2019. kepatuhan untuk pengobatan TB Paru, P-ISSN: 2477-1880 E-ISSN: 2502-6623 Kesmas Uwigama : Jurnal Kesehatan Masyarakat 132 Kesmas Uwigama : Jurnal Kesehatan Masyarakat P-ISSN 2460-0350, E-ISSN 2477-5819 December 2019, Vol. 5 No. 2 REFERENSI Aditama, TY.2012. Tuberkulosis: Diagnosis, Terapi Dan Masalahnya . Edisi IV, Cetakan I. Jakarta: Penerbit Ikatan Dokter Indonesia. Ahsan, A., dan Putu Ari Sadhu Permana Hany. 2012. Hubungan Dukungan Keluarga dengan Kepatuhan Minum Obat pada Pasien Hipertensi di Poli Jantung RSSA Malang. Tesis Alsagaff, H. dan Abdul M. 2016. DasarDasar Ilmu Penyakit Paru.Cetakan IV. Surabaya: Airlangga University Press. Arifin, Zainal. 2011. Penelitian Pendidikan. Bandung: Remaja Rosda. Arikunto, S. 2010. Manajemen Penelitian. Edisi keempat. EGC: Jakarta. Asra, Septia. 2017. Hubungan dukungan keluarga dengan kepatuhan minum obat pada penderita TB Paru di Rumah Sakit Umum Daerah Arifin Achmad. JOM PSIK Vol 1 No. 2 Adherence to medication in stroke survivors: a Qualitative comparison of low and high adherence”. Conger, J. 2011. Adolencence and Youth. New York: Harper and Row Publisher Inc. Data Dinas Kesehatan Kota Samarinda Tahun 2017. penemuan kasus TB BTA (+) di Kota Samarinda. Samarinda. Data Puskesmas Pasundan Tahun 2018. Data Puskesmas Pasundan Kota Samarinda : angka keberhasilan pengobatan (Succes Rate). Samarinda. Dedewijaya, 2009. Apa Itu Motivasi?. http://www.dengan-tulisanmewarnai-dunia-dan-mengubahdunia.htm Depkes RI, 2011, Pedoman Nasional Penanggulangan Tuberkulosis, Jakarta. Dewi. 2011. Hubungan pengetahuan dan sikap penderita dengan kepatuhan minum Obat Anti Tuberkulosis Paru P-ISSN: 2477-1880 E-ISSN: 2502-6623 Kesmas Uwigama : Jurnal Kesehatan Masyarakat di Puskesmas Lidah Kulon Surabaya. Skripsi Universitas Airlangga. Glick, I.D, Anya H. Stekoll, dan Spencer Hays. 2011. The Role of the Family and Improvement in Treatment Maintenance, Adherence, and Outcome for Schizophrenia. Journal of Clinical Psychopharmacology Volume 31, Number 1, February 2011. Gough, A. dan Garri Kaufman 2011 Pulmonary Tuberculosis: clinical features and patient management. Nursing Standard. July 27: vol 25, no 47. Hasibuan, Malayu S.P. 2010. Manajemen Sumber Daya Manusia. Edisi Revisi. Jakarta: Bumu Aksara Kementrian Kesehatan RI, 2009, Keputusan Menteri Kesehatan Republik Indonesia Nomor 364/MENKES/SK/V/2009 tentang Pedoman Penanggulangan Tuberkulosis (TB). Kementerian Kesehatan RI, 2013, Riset Kesehatan Dasar 2013,Jakarta : Badan Penelitian dan Pengembangan Kesehatan. Latifatul, Muna. 2014. Motivasi dan Dukungan Sosial Keluarga Mempengaruhi Kepatuhan Berobat Pada Pasien TB Paru di Poli Paru BP4 Pamekasan. Jurnal Ilmiah Kesehatan, Vol 7, No 2. Mubarak. 2011. Ilmu Kesehatan Masyarakat : Teori dan Aplikasi. Jakarta : Salemba Medika. Noorhizmah. 2016. Faktor-faktor Yang Berhubungan Dengan Kepatuhan Penderita Tuberkulosis Paru Dengan Melakukan Pemeriksaan Ulang Dahak Pada Akhir Pengobatan Tahap Intensif Di Puskesmas Wonosobo, Skripsi, Universitas Diponegoro, Semarang. Notoatmodjo, Soekidjo. 2010. Metode Penelitian Kesehatan. Rineka Cipta. Jakarta. 133 Kesmas Uwigama : Jurnal Kesehatan Masyarakat P-ISSN 2460-0350, E-ISSN 2477-5819 December 2019, Vol. 5 No. 2 Notoatmodjo, S. 2012. Promosi Kesehatan dan Perilaku Kesehatan. Jakarta: PT Rineka Cipta. Nursalam. 2013. Metodologi Penelitian Ilmu Keperawatan: Pendekatan Praktis. Jakarta : Salemba Medika. Octaswari. 2015. Hubungan Antara Dukungan Sosial dan Motivasi Diri dengan Kepatuhan Minum Obat Pasien Tuberkulosis Paru di RSUD Panembahan Senopati, Puskesmas Sewon I dan II Bantul. http://repository.unjaya.ac.id/2682/ Perhimpunan Dokter Paru Indonesia, 2014. Tuberkulosis Pedoman Diagnosis dan Penatalaksanaan di Indonesia, Jakarta : Direktorat Jendral Pengendalian Penyakit dan Penyehatan Lingkungan. Price, S.A. dan Wilson, L.M. 2012. Patofisiologi: Konsep Klinis Proses-proses Penyakit. Jakarta: Penerbit Buku Kedokteran EGC. Rachmawati. 2012. Metodologi penelitian kuantitatif dalam riset keperawatan. Rajawali Pers. Jakarta. Rahmayuni. 2016. Hubungan Motivasi Diri Dengan Kepatuhan Minum Obat Anti Tuberculosis (OAT) Pada Pasien TB Paru Di Puskesmas Andalas Padang Tahun 2016. Diploma thesis, Universitas Andalas. Robbins, Stephen P, 2010. Perilaku Organisasi. PT Indeks. Syam dan Fauziah. 2009. Pengaruh Komitmen Organisasi dan Kepuasan Kerja Terhadap Kinerja Manajerial. Jakarta: Jurnal Riset Ankutansi Indonesia. Widoyono, 2011, Penyakit Tropis Epidemiologi, Penularan, Pencegahan Pemberantasannya, Erlangga, Jakarta. Wijaya, Agung A, 2012, Merokok dan Tuberkulosis, Vol. 8, Maret 2012 World Health Organization (WHO). 2010. Indonesia TB Country Profile.(Online),http://whqlibdoc.w ho.int/publications/2010/97892415 47833_eng.pdf. Wulandari, Dewi Hapsari. 2015. Analisis Faktor-Faktor yang Berhubungan dengan Kepatuhan Pasien Tuberkulosis Paru Tahap Lanjutan Untuk Minum Obat di RS Rumah Sehat Terpadu Tahun 2015. Jurnal Administrasi Rumah Sakit Volume 2 Nomor 1. Septia A. 2017. Hubungan Dukungan Keluarga dengan Kepatuhan Minum Obat pada Penderita TB Paru. Jurnal Online Mahasiwa PSIK. Scheurer, D., Niteesh Choudhry, Kellie A. Swanton, Olga Matlin, dan Will Shrank. 2012. The American Journal Of Managed Care Vol. 18, No. 12 Sugiyono. 2014. Statistika Untuk Penelitian. Alfabeta. Bandung. P-ISSN: 2477-1880 E-ISSN: 2502-6623 Kesmas Uwigama : Jurnal Kesehatan Masyarakat 134 

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GENOME SEQUENCES crossm New Reference Genome Sequences for 17 Bacterial Strains of the Honey Bee Gut Microbiota Kirsten M. Ellegaard,a Philipp Engela a Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland We sequenced the genomes of 17 strains isolated from the gut of honey bees, including strains representing the genera Lactobacillus, Bifidobacterium, Gilliamella, Snodgrassella, Frischella, and Commensalibacter. These genome sequences represent an important step forward in the development of a comprehensive reference database to aid future analysis of this emerging gut microbiota model. ABSTRACT T he honey bee gut is colonized by a remarkably simple community dominated by only 8 to 10 bee-specific phylotypes (1). However, genome-level analyses have shown that several of the phylotypes comprise highly divergent strains (2–4). As such, the honey bee is a promising future model for studying strain-level evolution and function in gut-associated bacterial communities (5). Here, we present 17 new genome sequences of strains isolated from the gut of honey bees, which were generated to facilitate the development of a reference genome database for this community. All strains were isolated from honey bees collected from our apiary in Lausanne, Switzerland, by culturing gut homogenates on agar plates (6) under microaerophilic or anaerobic conditions (7). Four strains of the genus Lactobacillus (Table 1) were selected for sequencing with PacBio 20K (Pacific Biosciences) single-molecule real-time (SMRT) technology. The strains were grown overnight in MRS broth supplemented with fructose and cysteine (8) at 35°C under anaerobic conditions, and total genomic DNA was extracted using a cetyltrimethylammonium bromide-based extraction protocol (7). De novo genome assembly was done using the Hierarchical Genome Assembly Process (HGAP) version 2.3. Another 13 strains representing the genera Bifidobacterium, Gilliamella, Snodgrassella, Frischella, and Commensalibacter (Table 1) were selected for sequencing with Illumina technology. The strains were cultured as described previously (7), and total genomic DNA was extracted with the GenElute bacterial genomic DNA kit according to the manufacturer’s instructions (Table 1). Sequencing libraries were prepared with the TruSeq DNA kit and sequenced on the MiSeq platform (Illumina) using the paired-end 2 ⫻ 250-bp protocol. All 13 genomes were sequenced to a minimum depth of 50⫻ (Table 1). The resulting FASTQ files were trimmed with Trimmomatic (9) to remove eventual adapter sequences and low-quality reads using the following parameters: LEADING, 20; TRAILING, 20; SLIDINGWINDOW, 4:15; and MINLEN, 50. The reads were assembled with SPAdes version 3.7.1 (10) using the “-careful” flag and multiple k-mer sizes (21, 33, 55, 77, 99, 127). Small contigs (less than 500 bp) and contigs with low k-mer coverage (less than 5⫻) were removed from the assemblies, resulting in 6 to 40 contigs per assembly, with a median N50 of 529,190 bp. For strains with related complete genome sequences or scaffolds available, the contigs were reordered with Mauve (11). Assembly qualities were checked by remapping reads to assemblies with the Burrows-Wheeler Aligner (12) and by GC-skew visualization with DNAplotter (13). For strain ESL0184, the main chromosome was cut into three contigs due to assembly Volume 7 Issue 3 e00834-18 Received 14 June 2018 Accepted 22 June 2018 Published 26 July 2018 Citation Ellegaard KM, Engel P. 2018. New reference genome sequences for 17 bacterial strains of the honey bee gut microbiota. Microbiol Resour Announc 7:e00834-18. https://doi.org/10.1128/MRA.00834-18. Editor J. Cameron Thrash, Louisiana State University Copyright © 2018 Ellegaard and Engel. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Philipp Engel, philipp.engel@unil.ch. mra.asm.org 1 Ellegaard and Engel TABLE 1 Genome assembly statistics and strain information Genus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Bifidobacterium Bifidobacterium Bifidobacterium Bifidobacterium Bifidobacterium Gillamella Gillamella Gillamella Gillamella Gillamella Snodgrassella Frischella Commensalibacter Species L. apis L. helsingborgensis L. melliventris L. kulllabergensis B. asteroides B. asteroides B. asteroides B. asteroides B. indicum/ B. coryneforme G. apicola G. apis G. apis NAd NA S. alvi F. perrara Commensalibacter sp. Phylotypea Firm5 Firm5 Firm5 Firm5 Bifido Bifido Bifido Bifido Bifido Sublineage Firm5-1 Firm5-2 Firm5-3 Firm5-4 Bifido-1 Bifido-1 Bifido-1 Bifido-1 Bifido-2 Strain ESL0185 ESL0183 ESL0184 ESL0186 ESL0170 ESL0198 ESL0199 ESL0200 ESL0197 Extracted DNA (␮g)b 17.9 19.3 19.8 16.1 1.1 1.3 5.3 4.7 1.1 No. of contigs 1 2 4 1 7 12 7 16 6 N50 (bp) 1,683,102 1,856,015 1,505,590 2,018,944 1,162,986 618,428 558,059 500,320 1,389,647 Assembly size (bp) 1,683,102 1,867,232 2,036,181 2,018,944 2,175,262 2,235,610 2,167,340 1,933,421 1,715,238 Coverage (ⴛ) 420 300 320 290 200 280 50 300 300 GC content (%) 37 37 36 36 60 60 59 60 61 No. of genesc 1,578 1,780 2,015 1,915 1,771 1,820 1,741 1,621 1,408 Gilliamella Gilliamella Gilliamella Gilliamella Gilliamella Snodgrasella Frischella Commensalibacter Gilli-1 Gilli-2 Gilli-2 Gilli-3 Gilli-3 NA NA NA ESL0178 ESL0169 ESL0172 ESL0177 ESL0182 ESL0196 ESL0167 ESL0284 0.3 3.9 1.8 2.0 1.2 3.7 3.1 1.3 18 13 17 19 31 15 40 13 364,598 481,163 374,672 953,736 255,373 1,281,809 277,847 471,180 2,885,657 2,430,778 2,685,772 3,086,198 3,537,173 2,446,304 2,558,525 1,948,862 200 270 200 50 160 130 200 50 34 35 34 35 35 41 34 38 2,602 2,227 2,468 2,868 3,257 2,224 2,313 1,767 aBased on 16S rRNA amplicon sequencing. amount of extracted DNA. cGene count based on the JGI Microbial Genome Annotation Pipeline. dNA, not applicable. bTotal uncertainty generated by a duplicated prophage sequence. Strains ESL0183, ESL0185, and ESL0186 were submitted as complete genomes, with strain ESL0183 having a small plasmid contig of 11.3 kb. Core phylogenies were generated for the Lactobacillus, Bifidobacterium, and Gilliamella strains, including previously published isolates derived from honey bees, using OrthoFinder (14) for ortholog prediction and RAxML (15) for phylogenetic inference. Based on the phylogenies, the Lactobacillus and Bifidobacterium strains represent members of previously reported sublineages, whereas two strains of the genus Gilliamella (ESL0177 and ESL0182) represent a new sublineage, with strain ESL182 having the largest genome size reported for this genus to date (3.5 Mbp) (Table 1). Data availability. The complete genome sequences for the strains reported here have been deposited in GenBank under the accession numbers CP029476, CP029544/ CP029545, and CP029477, and the whole-genome shotgun projects have been deposited under the accession numbers QGLH00000000, QGLJ00000000, QGLK00000000, QGLL00000000, QGLI00000000, QGLG00000000, QGLQ00000000, QGLN00000000, QGLO00000000, QGLP00000000, QGLR00000000, QGLS00000000, QGLM00000000, and QGLT00000000. Additionally, the genomes were annotated using the JGI Microbial Genome Annotation Pipeline, where they have been deposited under the genome identification numbers 2684622912, 2684622914, 2684622911, 2684622916, 2684622918, 2684622919, 2684622920, 2684622917, 2684622913, 2684622925, 2684622922, 2684622923, 2684622924, 2684622926, 2684622927, 2684622921, and 2756170209. ACKNOWLEDGMENTS This work was funded by Human Frontier Science Program (HFSP) Young Investigator grant RGY0077/2016, the European Research Council Starting Grant (ERC-StG) “MicroBeeOme,” Swiss National Science Foundation grant 31003A_160345, and the Fondation Herbette at the University of Lausanne. REFERENCES 1. Kwong WK, Moran NA. 2016. Gut microbial communities of social bees. Nat Rev Microbiol 14:374 –384. https://doi.org/10.1038/nrmicro.2016.43. 2. Ellegaard KM, Tamarit D, Javelind E, Olofsson TC, Andersson SGE, Vasquez A. 2015. Extensive intra-phylotype diversity in lactobacilli and bifidobacteria from the honeybee gut. BMC Genomics 16:284. https:// doi.org/10.1186/s12864-015-1476-6. Volume 7 Issue 3 e00834-18 3. Engel P, Stepanauskas R, Moran NA. 2014. Hidden diversity in honey bee gut symbionts detected by single-cell genomics. PLoS Genet 10: e1004596. https://doi.org/10.1371/journal.pgen.1004596. 4. Engel P, Martinson VG, Moran NA. 2012. Functional diversity within the simple gut microbiota of the honey bee. Proc Natl Acad Sci U S A 109:11002–11007. https://doi.org/10.1073/pnas.1202970109. mra.asm.org 2 Microbiology Resource Announcement 5. Ellegaard KM, Engel P. 2016. Beyond 16S rRNA community profiling: intra-species diversity in the gut microbiota. Front Microbiol 7:1475. 6. Engel P, James RR, Koga R, Kwong WK, McFrederick QS, Moran NA. 2013. Standard methods for research on Apis mellifera gut symbionts. J Apicult Res 52:1–24. https://doi.org/10.3896/IBRA.1.52.4.07. 7. Kesnerova L, Mars RAT, Ellegaard KM, Troilo M, Sauer U, Engel P. 2017. Disentangling metabolic functions of bacteria in the honey bee gut. PLoS Biol 15:e2003467. https://doi.org/10.1371/journal.pbio.2003467. 8. Olofsson TC, Alsterfjord M, Nilson B, Butler E, Vasquez A. 2014. Lactobacillus apinorum sp. nov., Lactobacillus mellifer sp. nov., Lactobacillus mellis sp. nov., Lactobacillus melliventris sp. nov., Lactobacillus kimbladii sp. nov., Lactobacillus helsingborgensis sp. nov. and Lactobacillus kullabergensis sp. nov., isolated from the honey stomach of the honeybee Apis mellifera. Int J Syst Evol Microbiol 64:3109 –3119. https://doi.org/10 .1099/ijs.0.059600-0. 9. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114 –2120. https://doi.org/10 .1093/bioinformatics/btu170. 10. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Volume 7 Issue 3 e00834-18 11. 12. 13. 14. 15. Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455– 477. https://doi.org/10.1089/cmb.2012.0021. Darling AE, Mau B, Perna NT. 2010. progressiveMauve: multiple genome alignment with gene gain, loss and rearrangement. PLoS One 5:e11147. https://doi.org/10.1371/journal.pone.0011147. Li H, Durbin R. 2009. Fast and accurate short read alignment with BurrowsWheeler transform. Bioinformatics 25:1754–1760. https://doi.org/10.1093/ bioinformatics/btp324. Carver T, Thomson N, Bleasby A, Berriman M, Parkhill J. 2009. DNAPlotter: circular and linear interactive genome visualization. Bioinformatics 25: 119–120. https://doi.org/10.1093/bioinformatics/btn578. Emms DM, Kelly S. 2015. OrthoFinder: solving fundamental biases in whole genome comparisons dramatically improves orthogroup inference accuracy. Genome Biol 16:157. https://doi.org/10.1186/s13059-015-0721-2. Stamatakis A. 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688 –2690. https://doi.org/10.1093/bioinformatics/btl446. mra.asm.org 3 

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Eur. Phys. J. C (2014) 74:3153 DOI 10.1140/epjc/s10052-014-3153-3 Regular Article - Theoretical Physics Formalism of gauge-invariant curvatures and constructing the cubic vertices for massive spin- 23 field in AdS4 space I. L. Buchbinder1,2,a , T. V. Snegirev1,b , Yu. M. Zinoviev3,c 1 Department of Theoretical Physics, Tomsk State Pedagogical University, Tomsk 634061, Russia National Research Tomsk State University, Tomsk, Russia 3 Institute for High Energy Physics, Protvino, Moscow Region 142280, Russia 2 Received: 9 June 2014 / Accepted: 24 October 2014 / Published online: 28 November 2014 © The Author(s) 2014. This article is published with open access at Springerlink.com Abstract We study the interaction of a massive spin-3/2 field with electromagnetic and gravitational fields in the four dimensional AdS space and construct the corresponding cubic vertices. The construction is based on a generalization of Fradkin–Vasiliev formalism, developed for massless higher spin fields, to massive fermionic higher spin fields. The main ingredients of this formalism are the gaugeinvariant curvatures. We build such curvatures for the massive theory under consideration and show how the cubic vertices are written in their terms. construct a gauge invariant object that we will call curvature and denote as R. Moreover, with the help of these curvatures a free Lagrangian can be rewritten in explicitly gauge-invariant form as  R ∧ R. L0 ∼ Using these ingredients one can construct two types of nontrivial1 cubic vertices: – Abelian vertices that have the form L ∼ R ∧ R ∧ , 1 Introduction – non-Abelian ones that look like Till now most of the investigations of consistent cubic interaction vertices for higher spin fields were devoted to bosonic massless fields (see e.g. [1–9]). The results on massive higher spin interactions are not so numerous, although there exists a classification of massive and massless cubic vertices in flat space by Metsaev [10–12] as well as some concrete examples [13–21]. At the same time there exist just a small number of papers devoted to fermionic higher spin interactions [11,22– 24]. At least one of the reasons for this is that technically such investigations appear to be much more involved. In this paper, using massive spin 23 as a simple but physically interesting and non-trivial example of massive fermionic higher spin fields, we apply the so-called Fradkin–Vasiliev formalism [25,26] (see also [21,27–32]) to the construction of electromagnetic and gravitational cubic vertices. Let us briefly recall the basic properties of this formalism. A higher spin particle is described by a set of fields that we collectively denote  here and for each field one can a e-mail: L ∼ R ∧  ∧ . For the massless bosonic fields Vasiliev [33] has shown that any such non-Abelian vertex can be obtained as a result of a deformation procedure that can be described as follows. – Construct quadratic in fields deformations for the curvatures and linear corrections to the gauge transformations R ∼  ∧ , δ1  ∼ ξ, so that the deformed curvatures R̂ = R + R transform covariantly, δ R̂ = δ1 R + δ0 R ∼ Rξ. snegirev@tspu.edu.ru c e-mail: yurii.zinoviev@ihep.ru (2) At this step most of the arbitrary parameters are fixed, however, there still remains some ambiguity. The reason is that covariance of the transformations for deformed joseph@tspu.edu.ru b e-mail: (1) 1 We call a vertex trivial if it can be constructed using gauge-invariant curvatures only, i.e. L ∼ R ∧ R ∧ R. 123 3153 Page 2 of 9 Eur. Phys. J. C (2014) 74:3153 curvatures guarantees that the equations of motion will be gauge-invariant but it does not guarantee that they will be Lagrangian. – Non-Abelian cubic vertex arises then one put these deformed curvatures into the free Lagrangian and using explicit form of the gauge transformations requires it to be gauge-invariant L∼  R̂R̂ ⇔ δL ∼  RRξ = 0. Note, finally, that Vasiliev has shown [33] that any non-trivial cubic vertex for three fields with spins s1 , s2 and s3 having up to s1 +s2 +s3 −2 derivatives can be constructed as some combination of Abelian and non-Abelian vertices. However, it has been pointed out that the Vasiliev construction [33] was initially formulated only for massless higher spin field theories. In this paper we generalize the Vasiliev approach to massive higher spin theories on the example of cubic electromagnetic and gravitational coupling for a massive spin- 23 field in the AdS4 space. Let us stress that in this paper we systematically restrict ourselves to cubic terms in the Lagrangians and terms linear in the fields in the gauge transformation laws, so all the Lagrangians we obtain are gauge-invariant in the linear (first non-trivial) approximation only. As is well known for higher spin theories in four or greater dimensions to proceed beyond this linear approximation one has to introduce an infinite number of fields with ever increasing spins and the corresponding gauge algebra becomes infinite dimensional. Thus in what follows all the higher order terms (quartic terms in the Lagrangians and quadratic terms in the gauge transformation laws) will be discarded. The paper is organized as follows. In Sect. 2 we give all necessary kinematics formulas. Here we introduce two gauge-invariant objects (for the physical field ψμ and Stueckelberg one φ) and rewrite the free Lagrangian using these objects. Sect. 3 devoted to the electromagnetic interaction for such massive spin- 23 field (for previous results see e.g. [34–36]2 ). In Sect. 3.1, as an independent check for our calculations as well as for instructive comparison, we consider this task using straightforward constructive approach. Then in Sect. 3.2 we consider the same task in the Fradkin–Vasiliev formalism. At last in Sect. 4 we construct gravitational cubic vertex in the Fradkin–Vasiliev formalism. normalized so that3   1  a 2 a a [Dμ , Dν ]ξ = λ e[μ ξν] + Γμν ξ , λ2 = − . (3) 2 3 In (3) eμ a is a background (non-dynamical) tetrad linking world and local indices and has the standard definition, eμ a eν b g ab = gμν , where gμν and g ab are, respectively, curved world AdS metrics and flat local one. Wherever it is convenient local indices are converted into the world ones by eμ a and its inverse eμ a , in particular the matrix γμ with world index is understood as γμ = eμ a γ a . Also we introduce the following notation for antisymmetric combinations of eμ a : μν  μνα  [μ ν] [μ ν α] ab = e a e b , abc = e a e b e c . In the expression (3) Γ ab = 21 γ [a γ b] is an antisymmetric combination of two gamma matrices and is a particular case of the more general definition which we will use, 1 [a1 γ . . . γ an ] n! where n = 2, 3, 4 for four-dimensional space. Let us present the main properties of such Γ -matrices: Γ a1 a2 ...an = Γ ab1 ...bn = − g a[b1 Γ b2 ...bn ] + γ a Γ b1 ...bn , Γ ab1 ...bn = g a[b1 Γ b2 ...bn ] + (−1)n Γ b1 ...bn γ a , γ a Γ b1 ...bn = 2g a[b1 Γ b2 ...bn ] + (−1)n Γ b1 ...bn γ a , (4) γa Γ ab1 ...bn = (d − n)Γ b1 ...bn , Γ ab1 ...bn γa = (−1)n (d − n)Γ b1 ...bn . The identities (4) are used in the paper to verify the gauge invariance of the Lagrangians. All fermionic fields are anticommuting and Majorana. We also use the Majorana representation of the γ -matrices with hermitian conjugation defined as follows: (γ a )† = γ 0 γ a γ 0 (Γ a1 a2 ...an )† = γ 0 Γ an ...a2 a1 γ 0 = −γ 0 Γ a1 a2 ...an γ 0 . In this case γ 0 γ a and γ 0 Γ ab are symmetric in the spinor indices, while γ 0 , γ 0 Γ abc , and γ 0 Γ abcd are antisymmetric. 2 Kinematic of massive spin-3/2 field Notations and conventions We work in four-dimensional Anti-de Sitter space with cosmological constant . The AdS covariant derivatives Dμ is For a gauge-invariant description of a massive spin-3/2 field besides the master vector-spinor field ψμ one has also introduced Stueckelberg spinor field φ. The free Lagrangian is 2 3 We use Greek letters μ, ν, . . . for world indices and Latin letters a, b, . . . for local ones. Summation over any repeated indices is implied. For indices in (square) round brackets we use the convention of complete (anti)-symmetrization without normalization factor. Spinor indices of (tensor)-spinor fields are omitted. Some aspects of higher spin equations of motion in an external electromagnetic field have been studied in [37]; however, these equations are non-Lagrangian. 123 Eur. Phys. J. C (2014) 74:3153 Page 3 of 9 3153 known and has the form Using the Bianchi identities for the curvatures (7) we obtain   μνα  μναβ ¯ abcd ¯ μν Γ abc α να + 8i M abc  −3m abcd  μν Γ μν  ¯ μ Γ ab ν = 0. +8m ab  i μνα  i abc Dν ψα + eμ a φ̄γ a Dμ φ abc ψ̄μ Γ 2 2 3M μν  μ a ψ̄μ Γ ab ψν − M φ̄φ. +3ime a ψ̄μ γ φ − 2 ab L0 = − (5) This Lagrangian is invariant under the following gauge transformations: δ0 ψμ = Dμ ξ + δ0 φ = 3mξ 3 Electromagnetic interaction iM γμ ξ 2 3.1 Constructive approach where M 2 = m 2 +λ2 = m 2 −/3 and m is a mass parameter. Note that such a description works in de Sitter space as well, provided m 2 > /3, m 2 = /3 being the boundary of the unitarity region. Using the explicit form of gauge transformations one can construct two gauge-invariant objects (curvatures): m iM γ[μ ψν] , μν = D[μ ψν] + Γμν φ + 6 2 iM γμ φ, μ = Dμ φ − 3mψμ + 2 (6) which satisfy the Bianchi identities D[μ να] D[μ ν] m iM = Γ[μν α] − γ[μ να] , 6 2 iM γ[μ ν] . = −3mμν − 2 i μνα  ¯ μ γ a δφ. ¯ μν Γ abc δψα − ieμ a   2 abc We prefer to work with Majorana fermions; thus for the description of electromagnetic interactions we will use a pair of them, ψμ i , φ i , where i = 1, 2 is the SO(2) index. Let us switch to the minimal electromagnetic interaction by the standard rule, Dμ ψν i ⇒ Dμ ψν i + e0 εi j Aμ ψν j , and similarly for φ i . The Lagrangian (5) with the above replacement for the covariant derivative is not invariant under the free gauge transformations, and the variation of the Lagrangian has the form ie0 i j μνα  i abc ε abc ψ̄μ Γ Fνα ξ j 2 = −3ie0 εi j eμ a ψ̄μ i Γ abc F bc ξ j . δ0 L0 = − (7) Note that the curvatures (6) are closely related with the Lagrangian equations of motion. It can be seen from the total variation of Lagrangian (5) that can be written as follows: δL0 = − As will be seen in the next section sometimes this ambiguity may be important in order to reproduce the most general cubic vertex, so we will not fix it here. (8) (11) To compensate for this non-invariance let us introduce nonminimal interactions: μν  L1 = εi j ab ψ̄μ i (b1 F ab + b2 Γ abcd F cd )ψν j +ieμ a ψ̄μ i (b3 F ab γ b + b4 Γ abc F bc )φ j + b5 φ̄ i (Γ F)φ j , (12) Moreover, using these curvatures the free Lagrangian (5) can be rewritten in explicitly gauge-invariant form. The most general ansatz looks like   μνα  μναβ ¯ abcd ¯ μν Γ abc α L0 = c1 abcd  να + ic2 abc  μν Γ μν  ¯ μ Γ ab ν . +c3 ab  (9) where (Γ F) = Γ ab Fab , which produce the following variations: The requirement to reproduce the original Lagrangian (5) partially fixes the parameters −iεi j eμ a Dμ φ̄ i (b3 F ab γ b − b4 Γ abc F bc )η j   M(b3 + 2b4 ) + + 6mb5 εi j φ̄ i (Γ F)η j 2 3c3 = −8c1 , 6c2 m = 1 − 16c1 M. 2 (10) The remaining freedom in the parameters is related with the identity   μναβ abcd ¯ β ] = 0. abcd Dμ [να Γ δ0 L1 = 4b1 εi j ψ̄μ i η j (D F)μ − ib3 εi j φ̄ i γμ η j (D F)μ μν  +2εi j ab Dμ ψν i (b1 F ab + b2 Γ abcd F cd )η j +iεi j eμ a ψ̄μ i ((2Mb1 + 3mb3 )F ab γ b +(2Mb2 + 3mb4 )Γ abc F bc )η j where we have (D F)μ = Dν F νμ as well as the corresponding corrections to the gauge transformations: δ1 ψμ i = iα1 εi j (Γ F)γμ ξ i , δφ i = α2 εi j (Γ F)ξ j , δ1 Aμ = β1 εi j (ψ̄μ i ξ j ) + iβ2 εi j (φ̄ i γμ ξ j ), (13) 123 3153 Page 4 of 9 Eur. Phys. J. C (2014) 74:3153 which in turn give us δ1 L0 = β1 εi j ψ̄μ i η j (D F)μ + iβ2 εi j φ̄ i γμ η j (D F)μ μν  −6α1 εi j ab Dμ ψ̄ν i (2F ab + Γ abcd F cd )η j −3iεi j eμ a ψ̄μ i [(4Mα1 − 2mα2 )F ab γ b −(2Mα1 + mα2 )Γ abc F bc )]η j −iα2 εi j eμ a Dμ φ̄ i (2F ab γ b + Γ abc F bc )η j −2Mα2 εi j φ̄ i (Γ F)η j . From these explicit expressions one can see that gauge invariance can be restored and that gives the solution for arbitrary coefficients: b1 = 6α1 , b2 = 3α1 , b3 = −2α2 , b4 = α2 Mα2 , β1 = −24α1 , β2 = −2α2 , b5 = 3m provided the following important relation holds: 4Mα1 + 2mα2 = e0 . approximation only. Indeed, the introduction of cubic terms in the Lagrangian and linear terms in the gauge transformation laws generate quadratic variations of the form δ1 L1 . To compensate for these variations one has to introduce quartic terms and/or additional fields and so on. In supergravities (i.e. theories containing spins not higher than 2 and 3/2) such a program can indeed be realized leading to models with spontaneous supersymmetry breaking (see e.g. [38,39] and references therein). But for the theories with spins greater than 2 such a program will require the introduction of an infinite number of fields with ever increasing spins. The basic aim of our paper is to construct first a nonvanishing term in the interacting Lagrangian. It means that we consider only those terms in the Lagrangian and gauge transformations which are required for the realization of this aim. The higher order terms in the gauge transformations do not affect the form of the above cubic interaction vertex, and therefore we do not study these higher order terms here. (14) 3.2 Formulation in terms of curvatures The Lagrangian (12) together with the correction, stipulated by the minimal switch to the interaction in the free Lagrangian, is the final cubic electromagnetic interaction vertex for the spin-3/2 field. As we see, this vertex contains two arbitrary parameters. A few comments are in order. – Calculating the commutator of two gauge transformations we obtain [δ(ξ1 ), δ(ξ2 )]Aμ = −8εi j (6α1 2 + α2 2 )(ξ2 i γ ν ξ1 j )Fμν , and it means that for non-zero electric charge e0 any such model must be a component of some (spontaneously broken) supergravity. – From the supergravity point of view the remaining freedom in the parameters α1 and α2 is clear: in general our vector field is a superposition of the graviphoton (i.e. a vector field from the supermultiplet ( 23 , 1)) and the goldstino’s superpartner (i.e. a vector field from the supermultiplet (1, 21 ) (see e.g. [38,39] and references therein). – From the expression for the parameter b5 one can see that we have an ambiguity between flat and massless limits. Indeed, in the flat case b5 = α32 and does not depend on the mass any more, while for the non-zero cosmological term this parameter is singular in the massless limit. – The most simple case—α2 = 0, i.e. the vector field is just the graviphoton. In this case the electric charge e0 = 23 Mα1 becomes zero at the boundary of the unitarity region. Now we reformulate the results of Sect. 3.1 on the base of Fradkin–Vasiliev formalism. Following the general procedure we begin with a deformation of the curvatures so that they transform covariantly; see (1), (2). In the case under consideration R = {μν i , μ i , Fμν }. For the fermionic curvatures the deformations correspond simply to the minimal substitution: μν i = e0 εi j A[μ ψν] j , μ i = e0 εi j Aμ φ j , (15) while the transformations for the deformed curvatures look like ˆ μi = 0 ˆ μν i = e0 εi j Fμν ξ j , δ  δ (16) ˆ μν i = μν i + μν i ,  ˆ μ i = μ i + μ i , and where  i i μν , μ are given by (6). The most general deformation for the electromagnetic fields strength quadratic in the fermionic fields can be written as follows: Fμν = εi j [a1 ψ̄[μ i ψν] j + ia2 ψ̄[μ i γν] φ j + a3 φ̄ i Γμν φ j ]. (17) The corresponding corrections to the gauge transformation have the form δ1 Aμ = εi j [2a1 ψ̄μ i ξ j − ia2 φ̄ i γμ ξ j ]. (18) The deformed curvatures will transform covariantly As we have already noted in the Introduction the Lagrangians obtained in this paper are gauge-invariant in the linear 123 i ¯ μν ¯ [μ i γν] ξ j ], δ1 F̂μν = εi j [2a1  ξ j − ia2  (19) Eur. Phys. J. C (2014) 74:3153 Page 5 of 9 3153 provided the following relation holds: 3ma3 = − ma1 − Ma2 . 6 (20) Here F̂μν = Fμν + Fμν . Now let us consider the Lagrangian   1 μναβ ˆ¯ i abcd ˆ i αβ L = − F̂ μν F̂μν + c1 abcd  μν Γ 4 μνα  ˆ i abc i ˆα ¯ μν Γ  +ic2 abc  μν  ˆ i ab i ˆ ¯ Γ   +c 3 ab μ ν (21) where all initial curvatures (including the electromagnetic field strength) are replaced by the deformed ones, and let us require that this Lagrangian be invariant. Non-trivial variations arise only with respect to transformations with the spinor parameter ξ i . Using the explicit form of the covariant transformations (16), (19) we obtain 1 μν  ¯ μν i (a1 F ab − 48e0 c1 Γ abcd F cd )ξ j δL = − εi j ab  2 ¯ μ i (a2 γ b F ab + 6e0 c2 Γ abc F bc )ξ j . (22) +iεi j eμ a  To compensate these terms one needs to introduce nonminimal corrections (note that they have exactly the same form as in the previous subsection): δ1 ψμ i = iα1 εi j (Γ F)γμ ξ j , δ1 φ i = α2 εi j (Γ F)ξ j , (23) Let us extract the cubic vertex. Using the relations (10), (25) we get μν  L1 = 3α1 εi j ab ψ̄μ i (2F ab + Γ abcd F cd )ψν j −iα2 εi j eμ a ψ̄μ i (2γ b F ab − Γ abc F bc )φ j Mα2 i j i ε φ̄ (Γ F)φ j + 3m ie0 μνα  + εi j abc Aμ ψ̄ν i Γ abc ψα j 2 ie0 i j μ + ε e a Aμ φ̄ i γ a φ j . (26) 2 Here the last two lines correspond to the minimal interactions, while the other terms are non-minimal corrections. The Lagrangian L1 (26) up to the minimal interaction is the same as obtained in Sect. 3.1 for the electromagnetic cubic vertex. 4 Gravitational interaction 4.1 Kinematics for gravity Let us briefly review basic features of gravity in the AdS4 space at free level. In the frame formulation the gravitational field is described by dynamical frame h μ a and Lorentz connection ωμ ab being antisymmetric in local indices. The free Lagrangian in AdS space has the form L0 = 1 μν  ac bc 1 μνα  ab ωμ ων − ωμ Dν h α c 2 ab 2 abc μν  +λ2 ab h μ a h ν b (27) and is invariant under the gauge transformations δ0 ωμ ab = Dμ η̂ab − λ2 eμ [a ξ̂ b] , which in turn produce the following variations: δ1 L0 = μν  ¯ μν i (6F ab + 3F de Γ abde )ξ j −α1 εi j ab  ¯ μ i (2γ b F ab + Γ abc F bc )ξ j . −iα2 εi j eμ a  δ0 h μ a = Dμ ξ̂ a + η̂μ a . (24) Tμν a = D[μ h ν] a − ω[μ,ν] a . a1 = −12α1 , a2 = 2α2 , (25) Thus we see that the choice of the parameters α1 and α2 is related with the choice of parameters c1 and c2 in the free Lagrangian. Moreover, if we use the relation 6mc2 = 1 − 16Mc1 , 2 The gauge-invariant objects (linearized curvature and torsion) have the form Rμν ab = D[μ ων] ab − λ2 e[μ [a h ν] b] , Comparing with (22) we conclude α1 = 8e0 c1 , α2 = 6e0 c2 . (28) (29) They satisfy the Bianchi identities D[μ Rνα] ab = λ2 e[μ [a Tνα] b] , D[μ Tνα] a = −R[μν,α] a . (30) Note that on the mass shell for the auxiliary field ωμ ab by virtue of (30) we have Tμν a = 0 ⇒ R[μν,α] a = 0, D[μ Rνα] ab = 0. (31) we again obtain Finally, the free Lagrangian can be rewritten as follows: 4Mα1 + 2mα2 = e0 . L0 = c0  μναβ abcd  Rμν ab Rαβ cd , c0 = 1 . 32λ2 (32) 123 3153 Page 6 of 9 Eur. Phys. J. C (2014) 74:3153 4.2 Gravitational coupling for massive spin-3/2 Following the general scheme (1), (2) we begin with the deformation of the curvatures that in the case under consideration are R = {μν , μ , Rμν ab , Tμν a }. As in the case of electromagnetic interactions deformations for spin-3/2 curvatures correspond to the minimal substitution rules, i.e. to the replacement of the covariant derivative D → D + ω and background tetrad eμ a → eμ a + h μ a : μν  = a1 ω[μ ab Γab ψν] + 2Mi h [μ a γa ψν]  2m a a − h [μ Γν] φ , 3 μ = a1 (ωμ ab Γab φ + 2Mi h μ a γa φ). (33)  2m a Γμ φ ξ̂a δ1 ψμ = −a1 Γ ab ψμ η̂ab + 2i Mγ a ψμ ξ̂a − 3  −ωμ ab Γab ξ − 2i Mh μ a γa ξ , (34) while the transformations for the deformed curvatures will have the form  2m ˆ μν = −a1 Γ ab μν η̂ab +2i Mγ a μν ξ̂a + Γ[μ a ν] ξ̂a δ 3  −Rμν ab Γab ξ − 2i M Tμν a γa ξ , ˆ μ = −a1 (Γ ab μ ηab + 2i Mγ a μ ξ̂a ). δ (35) ˆ μ = μ + μ and μν i , i ˆ μν = μν + μν ,  Here  are given by (6). Now let us consider the most general deformations for gravitational curvature and torsion quadratic in the spin-3/2 fields: Rμν ab = b1 ψ̄[μ Γ ab ψν] +ib2 e[μ [a ψ̄ν] γ b] φ +ib3 ψ̄[μ Γν] ab φ +b4 e[μ a eν] b φ̄φ + b5 φ̄Γμν ab φ, Tμν a = ib6 ψ̄[μ γ a ψν] + b7 e[μ a ψ̄ν] φ +b8 ψ̄[μ Γν] a φ + ib9 φ̄Γμν a φ. (36) Note that there are three possible field redefinitions ωμ ab ⇒ ωμ ab + iκ1 φ̄Γμ ab φ, h μ a ⇒ h μ a + κ2 ψ̄μ γ a φ + κ3 eμ a φ̄φ that shift parameters b3 , b6 and b7 . 123 δ1 ωμ ab = 2b1 ψ̄μ Γ ab ξ − ib2 eμ [a φ̄γ b] ξ − ib3 φ̄Γμ ab ξ, δ1 h μ a = 2ib6 ψ̄μ γ a ξ + b7 eμ a φ̄ξ + b8 φ̄Γμ a ξ. (38) Then the curvature and torsion will transform as follows: ¯ μν Γ ab ξ +ib2 e[μ [a  ¯ ν] γ b] ξ −ib3  ¯ [μ Γν] ab ξ, δ R̂μν ab = 2b1  ¯ μν γ a ξ −b7 e[μ a  ¯ ν] ξ + b8  ¯ [μ Γν] a ξ, (39) δ T̂μν a = 2ib6  provided the following restrictions on the arbitrary parameters hold: The corrections to the gauge transformations will look like δ1 φ = −a1 (Γ ab φ η̂ab + 2i Mγ a φ ξ̂a ), In order that deformed curvatures transform covariantly we have to introduce the following corrections to the gauge transformations: (37) 3mb2 = b6 (M 2 − m 2 ) − b1 M 3mb8 = −b6 M + b1 , mb1 mb6 + 2b7 (M 2 − m 2 ) 6mb9 = − − 2b3 , 6mb4 = 3 3 mb1 − 2b3 M. (40) 6mb5 = − 3 Here R̂μν ab = Rμν ab + Rμν ab , T̂μν a = Tμν a + Tμν a and Rμν ab , Tμν a are given by (29). The general solution to these relations has four free parameters, for example, b1,3,6,7 . But as we have already noted three of them are related with possible field redefinitions, so we have one non-trivial parameter b1 only. Let us consider the following Lagrangian:   μνα  ˆ μναβ ˆ¯ abcd ˆ ˆα ¯ μν Γ abc  L = c1 abcd  να + ic2 abc  μν Γ   μν  ˆ ˆ ν + c0 μναβ R̂μν ab R̂αβ cd ¯ μ Γ ab  +c3 ab  abcd   μναβ ¯ +ic4 abcd μν Γ abc α h β d μνα  ¯ μ Γ ab ν h α c +c5 abc  (41) where the first four terms are just the sum of free Lagrangians for the massive spin- 23 case and the massless spin-2 case with the initial curvatures replaced by the deformed ones, while the last two terms are possible Abelian vertices. Note that in dimensions d > 4 we would have to introduce one more Abelian vertex:   μναβγ ¯ μν Γ abcd αβ h γ e . abcde Now we require that this Lagrangian be gauge-invariant. To fix all coefficients it is enough to consider variations that do not vanish on-shell. For ξ -transformations we have   μναβ ¯ ab cd δL = (−24c1 a1 + 4b1 c0 ) abcd  αβ Rμν Γ ξ μνα  +i(6c2 a1 − 8c0 b3 ) abc Rμν ad ξ̄ Γ bcd α μνα  +i(6c2 a1 − 8c0 b2 ) abc Rμν ab ξ̄ γ c α ; Eur. Phys. J. C (2014) 74:3153 Page 7 of 9 3153 the last two terms vanish on-shell and for the second term it can be seen from the identity:   μνα  μναβ 0 = abcd Rμν,α a ξ̄ Γ bcd β = 3 abc Rμν ad ξ̄ Γ bcd α . The first term does not vanish on-shell so we have to put b1 c0 = 6c1 a1 . (42) Calculating the variations for the η̂ab -transformations we obtain   μναβ ¯ abce αβ η̂de δL = −8c1 a1 abcd  μν Γ μνα  ¯ μν Γ abd α η̂cd −i(12c2 a1 − 3c4 ) abc  μν  ¯ μ Γ ac ν η̂bc . +(8c3 a1 − 2c5 )  ab − 2. For this vertex we obtain   μναβ L = (−48c1 a1 + 8c0 b1 ) abcd Dμ ων ab ψ̄α Γ cd ψβ = 0. Vertex 3 2 − 3 2 This expression vanishes due to the obtained expressions and the restrictions for the arbitrary coefficients. Vertex 21 − 21 − 2. In this case we have μν  L = (−c3 a1 + 8c0 b5 ) ab Dμ ων cd φ̄Γ abcd φ μν  +(8c3 a1 − 2c5 ) ab ωμ ac Dν φ̄Γ bc φ μν  +(2c3 a1 + 16c0 b4 ) ab Dμ ων ab φ̄φ. The second term drops out due to the relations among the coefficients, the first term vanishes on-shell for the gravitational field due to the identity μνα  0 = abc Rμν,α d φ̄Γ abcd φ μνα  = 2 abc (Dμ ων,α d + λ2 eμ d h ν,α )φ̄Γ abcd φ μν  = 6 ab Dμ ων cd φ̄Γ abcd φ, The first term vanishes in d = 4 due to the identity   μναβγ ¯ abcd αβ η̂γ e 0 = abcde  μν Γ   μναβ ¯ abce = 4 abcd  αβ η̂de , μν Γ while the third term can be removed by the field redefinition while the last two terms give the restrictions h μ a → h μ a + κ3 eμ a φ̄φ. c4 = 4c2 a1 , c5 = 4c3 a1 . (43) Finally we consider variations under the ξ̂ a transformations    3 μναβ ¯ abc δ L = −i 16c1 a1 M + mc4 abcd  αβ ξ̂ d μν Γ 2 μνα  ¯ μν Γ ab α ξ̂ c +(32c1 a1 m + 3mc5 ) abc  μνα  ¯ μν Γ abcd α ξ̂ d +(−4Ma1 c2 + Mc4 ) abc  μν  ¯ μ Γ abc ν ξ̂ c −i((4a1 c2 − c4 )m + (4c3 a1 − c5 )M) ab  μν  ¯ μ γ a ν ξ̂ b . +i(2(8a1 c2 + c4 )m − 4(2c3 a1 + c5 )M) ab  Taking into account (10), (40), (42), and (43) we obtain simply   i μναβ  ¯ μν Γ abc αβ ξ̂ d +2ia1 μν  ¯ μ γ a ν ξ̂ b . δL = − abcd  ab 2 To compensate these variations one can use the following corrections: − 2. For the last possibility we get μνα  L = i(−24c2 a1 + 6c4 ) abc ωα da Dμ ψ̄ν Γ bcd φ μνα  +i(16c0 b2 − 12c2 a1 ) abc Dν ωα ab ψ̄μ γ c φ μνα  −i(16c0 b3 − 12c2 a1 ) abc Dν ωα dc ψ̄μ Γ abd φ. Vertex 3 2 − 1 2 The first term drops out due to the relations among the coefficients, the last term vanishes on-shell for the gravitational field, and the second term can be removed by the field redefinition h μ a → h μ a + κ2 ψ̄μ γ a φ. As a result, the expression (41) is the final form for Lagrangian of coupled spin-2 and spin-3/2 fields in AdS4 space including the cubic interaction vertex. The parameters c1 , c2 , c3 are fixed in the free gravitational field Lagrangian (27), the parameter c0 is given by (32). The parameters c4 , c5 , and the parameters of the gauge transformations are expressed through the single parameter a1 , which is the only free parameter of the theory. δ1 ψμ ∼ μν ξ̂ ν , δ1 φ ∼ μ ξ̂ μ . Thus the requirement that the Lagrangian be gaugeinvariant fixes the coefficients c4,5 for the Abelian vertices and also relates the coefficients b1 and c1 (which is just a manifestation of the universality of the gravitational interaction). Note that by the Metsaev classification [11] in general dimensions d > 4 we would have two non-minimal vertices with two derivatives as well as the minimal one. But in d = 4 dimensions these two derivative vertices are absent (on-shell and up to possible field redefinitions) as we will now show. 5 Conclusion In this paper we have constructed the cubic vertices for a massive spin-3/2 field coupled to electromagnetic and gravitational fields in AdS4 space. The corresponding vertices are gauge-invariant due to the presence of Stueckelberg auxiliary fields and contain a number of free parameters. The results are given by the expressions (12) or (26) for the electromagnetic interaction and (41) for the gravitational interaction. 123 3153 Page 8 of 9 The construction of the vertices is based on a generalization of the Fradkin–Vasiliev formalism [25,26] where the main building blocks of the Lagrangians are the gaugeinvariant curvatures. Although this formalism was known only for massless higher spin theories, we have shown, for the example of a spin-3/2 field, that the gauge invariant curvatures can in principle be constructed for massive higher spin fields as well. Certainly the case of massive fermionic fields appears to be the most technically involved one. Nevertheless it seems worth to apply this formalism to massive fermionic fields with spins higher than 3/2. As we pointed out, the constructed vertices contain some number of arbitrary parameters (two for the electromagnetic coupling and one for gravitational coupling) which cannot be fixed only by gauge invariance. In principle one can hope that the additional constraints for those parameters can appear from the requirements of causality. The possibility of such constraints was already demonstrated in the papers [17,20]. Acknowledgments I.L.B and T.V.S are grateful to the grant for LRSS, project No. 88.2014.2, RFBR grant, project No. 12-02-00121-a and RFBR-Ukraine grant, project No. 13-02-90430-a for partial support. Their research was also supported by grant of Russian Ministry of Education and Science, project TSPU-122. T.V.S acknowledges partial support from RFBR grant, project No. 14-02-31254. Work of Yu.M.Z was supported in part by RFBR grant Nj. 14-02-01172. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. Funded by SCOAP3 / License Version CC BY 4.0. References 1. M.A. Vasiliev, Cubic interactions of Bosonic higher spin gauge fields in Ad S5 . Nucl. Phys. B 616, 106 (2001). arXiv:hep-th/0106200. (Erratum-ibid. B 652, 407 (2003)) 2. K.B. 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(IJACSA) International Journal of Advanced Computer Science and Applications, Vol. 6, No. 10, 2015 Design of ANFIS Estimator of Permanent Magnet Brushless DC Motor Position for PV Pumping System TERKI Amel MOUSSI Ammar TERKI Nadjiba Genie Electric department Biskra University Biskra, Algeria Genie Electric department Biskra University Biskra, Algeria Genie electric department Biskra University Biskra, Algeria Abstract—This paper presents a new scheme for PMBLDC (permanent magnet brushless direct current) rotor position estimation based an ANFIS (adaptive network fuzzy inference system) estimator. The operation of such motor requires accurate rotor position knowledge. However, most of rotor position sensors produce undesirable effects such as mechanical losses and have other disadvantages. In order to overcome the disadvantages, sensorless scheme seems to offer great advantages. This work present an ANFIS estimator design. Combining the adaptive capability of the neural network to gather with the reasoning ability of the fuzzy logic in ANFIS modeling results in a fast responding and flexible model. This procedure lends itself perfectly adapted for complex system such as PV pumping systems. Keywords—Photovoltaic system; Brushless DC motor; ANFIS estimator; Speed controller I. INTRODUCTION In early1980s, DC motors are widely used for PV pumping applications either with or without intermediate converters [1]. Starting, steady state, and transient performances of solar power fed DC motors for linear and centrifugal pump load torques were analyzed and showed a perfect matching [2]. However these motors a bulky, and require frequent maintenance. Nowadays solar power fed permanent magnet brushless DC (PMBLDC) motors were being used instead [2]. PMBLDC machine is more popular due its simple structure and low cost [1, 3]. These machines have the advantages of light weight, small size, simple mechanical construction, easy maintenance, good reliability, and high efficiency [1, 3, 4, 5]. In general, the motors are equipped with mechanical position detecting devices to provide proper commutation for power devices in the bridge inverter [6]. These mechanical devices, such as Hall Effect sensor, optical or inductive sensor produce undesirable effects such as mechanical losses and also have other disadvantages like change in mechanical design and requirement of maintenance. In order to overcome these drawback, there is a need to develop a sensorless scheme for estimating rotor position. In recent years, many sensorless drive methods have been proposed. Of these, the most popular one in the back emf based Filter (EKF) is used [7]. This method provides excellent speed response but requires heavy online matrix computing. An offline FEM assisted position and speed observer has also been studied in the literature [8]. Zero crossing of line to line PM flux linkage is used for estimating the speed and position [9]. Flux Linkage Observer (FLO) based on the integration of back EMF, with a simple start-up method is proposed [10]. In this paper the design of an ANFIS estimator of permanent magnet brushless DC motor in PV pumping system is presented. The Adaptive Neuro Fuzzy Inference System (ANFIS), is a neural network that is functionally the same as a Takagi–Sugeno type inference model. The ANFIS is a hybrid intelligent system that takes advantages of both ANN and fuzzy logic theory in a single system, by using the ANN technique to update the Takagi–Sugeno type inference model parameters. In order to explain the concept of ANFIS structure, five distinct layers are used. The first layer in the ANFIS structure is the fuzzification layer; the second layer performs the rule base layer; the third layer performs the normalisation of membership functions (MFs); the fourth and fifth layers are the defuzzification and summation layer respectively [11]. This rest of the paper is organized as follows. In section 2, the configuration of PV pumping systems is presented, The Adaptive network fuzzy inference system estimator is described in section 3. Section 4 shows the experimental performance of Adaptive network fuzzy inference system estimator. Finally, Section 5 concludes our contribution and merits of this work. II. GENERAL SYSTEM LAYOUT The full system mainly consists of the solar cell array generator, DC/DC converter with MPPT (maximum power point tracker) command, PMBLDC motor with its bridge inverter coupled to a centrifugal pump load. The motor is controlled through a hysteresis current loop and an outer speed with PI type controller as shown in fig1. 136 | P a g e www.ijacsa.thesai.org (IJACSA) International Journal of Advanced Computer Science and Applications, Vol. 6, No. 10, 2015 Photovoltaic Generator DC/DC PMBLDC Inverter Pump ia=-(ib+ic) MPPT i a* ⍵ref + e Speed reference Current generator Tref controller - ib* fem hysteresis Current controller 3 ia ib ic ic * ⍵ ᶱ d/dt ANFIS Estimator Fig. 1. Overall system configuration Vbn  Rib  pb  eb A. PV generator model The characteristic of the Photovoltaic generator can be presented by the following nonlinear equation [4]: I  I sc  I o [exp( (V  Rs I ) (V  Rs I ) )  1]  Vth Rsh (1) Isc PV array short circuit current, PV array reverse saturation current, The flux expressions are given by the following expressions: Rs PV array series resistance, Vth PV array thermal voltage. The thermal voltage Vth and t reverse saturation current Io are successively identified by [4]: I o  I sc  (Vop  Rs I op )    I oP  exp    Vrh   (2) ( ) (10) ( ) (11) ( ) (12) Where Ls: the self-inductance and M: the mutual inductance. And (13) (3) B. Permanent magnet Brush-Less DC motor model The simplified schematic of PMBLDC motor who has a trapezoidal electromotive force, the use of Park transform is not the best approach in modelling the machine. Instead the natural approach in used where the emf is generated with respect to rotor position [4]. The operating sequences of the machine can be subdivided into six cycles with respect to rotor position as shown in table II. Therefore by substituting Eq.13 in Eqs 10, 11 and 12: ( ) (14) ( ) (15) ( ) (16) From the electrical equations 4, 5 and 6, the following system is obtained [ ] [ ][ ] [ ][ ] [ ] (17) With Leq = LS –M From this system, the decoupled phase equations are obtained and the explicit current equations are given by: The electric of the motor can be described by [4] Van  Ri a  pa  ea (7) (8) Vcn  Vc0  Vn0 (9) Where R: per phase stator resistance. ia,b,c and a,b,c are respectively phase currents of phases a, b and c and total flux linkage of a,b and c. p: Laplace operator. Rsh PV array equivalent shunt resistance, (Vop  R s I op  Voc ) I op log(1  ) I sc Van  Va0  Vn0 Vbn  Vb0  Vn0 IPV Array output current, Vth  (6) With Where: Io (5) Vcn  Ric  pc  ec (4) 137 | P a g e www.ijacsa.thesai.org (IJACSA) International Journal of Advanced Computer Science and Applications, Vol. 6, No. 10, 2015 ⁄ ⁄ [ ] [ [[ ⁄ ] [ ][ ] [ ]] (18) ] The mechanical part is expressed by the following equation: (19) With: Te: electromagnetic torque. Tr: Load torque : speed J : moment of inertia B : viscose friction coefficient Neglecting the frictional coefficient and taking where P is the pole pairs number, (19) can written as: ⍵ ( )⁄ ⍵ ( ) ⍵ ( ) ⍵( ) ( ) ( ) ( ) The value of the torque reference is given by [4]: ( ) ( ) ( ) ( ) (25) ) the speed error of previous interval is, Where ( ( ) is the speed error of working interval. and are speed controller gains. D. Current control Several techniques can be used to control the phase current of the PMBLDC motor. In this paper a hysteresis current controller is used. It has the major advantage of not requiring machine parameters to be known. However the commutation frequency is not constant [4]. It depends on many factors such as the applied voltage, the back emf, hysteresis band ...etc. (20) ) ⁄⍵ (21) And the angular position is expressed by ⍵ (23) (24) Maximum value of commutation frequency is obtained at starting and is given by [4]: The developed torque can be expressed by ( compared with the reference speed ⍵ ( ) and the resulting error is estimated at the nth sampling instant as: (22) (26) The commutations are obtained by comparing actual currents ia.b.c to a rectangular reference i*a.b.c and by keeping them in hysteresis band . The commutation sequences of switches are summarised in the table I. [5]. TABLE I. THE CMMUTATION SEQUENCES OF SWITCHES * Si ia  (ia  I ) T1 on T4 off Va= U/2 *  I ) Si ia  (ia Si i  (i*  I ) b b Si i  (i*  I ) b b * Si ic  (ic  I ) Si ic  (ic*  I ) T1 off T4 on Va= -U/2 T2 on T5 off Vb= U/2 T2 off T5 on Vb= -U/2 T3 on T6 off Vc= U/2 T3 off T6 on Vc= -U/2 E. Pump model A centrifugal type is used. It can be described as an aerodynamic load which is characterised by the following load equation [4]: Fig. 2. Back e.m.f and current waves forms for phase a, b and c C. Speed control PI speed controller is widely used in industry due to its ease in design and simple structure. The rotor speed ⍵( ) is (27) Where A is the pump constant 138 | P a g e www.ijacsa.thesai.org (IJACSA) International Journal of Advanced Computer Science and Applications, Vol. 6, No. 10, 2015 TABLE II. III. OPERATING SEQUENCES WITH RESPECT TO ROTOR POSITION ANFIS ESTIMATOR The adaptive network fuzzy inference system play a significant role in the field of artificial intelligence. It combines the advantages of a fuzzy controller as well as quick response and adaptability nature of artificial neural network [11]. Hence using this scheme one can avoid the use of a position sensor which produces mechanical losses in the motor. Different configurations of adaptive network fuzzy inference system have been tested and the best one that gives the minimum error value is presented in fig6. The Algorithm of the model structure was constructed as shown in Fig.3. The membership functions were obtained from the data set of the back emfs which were first normalized for loading Data. Then generate Fis, built on sugeno structure, which is obtained by creating of input membership function: number (tree inputs) & type (triangular) and output type(linear). The choice of optimization method (hybrid) is obtained by Train Fis. If the results are satisfactory (error is minimum) after test Fis, denormalized procedure is done and go to application thereafter. The ANFIS structure consists of 5 layers of neurons, each of which having a very specific behavior. Original Data Normalization Load Data (testing, training, checking) Generate Fis Input: 1. Number of Mfs 2. type of Mf Output: Mf type Train Fis Optimization Methode Epoch number: error Test fis From these, second, third and fifth layer have constant behavior, while layers 1 and 4 have varying parameters. Layer 1: consists of five adaptive neurons in which the fuzzification is performed, that is: the grade of membership to the defined membership functions of the input is evaluated. f i1  µ Ai (x) No yes Denormalization (28) Where x is the input to ith neurons and Ai is membership function correspond to variable x. Application Fig. 3. Algorithm of the ANFIS model The membership function µAi (x) is triangular. Layer2: consists of 125neurons, each node output represents the firing weight of a rule gives than: Wk = µ Ai (x)  µ Bj (y) Minimum Error (29) Layer3: consists of 125 neurons, every node calculates the ratio of every rule firing weight to the sum of all rule firing weights. k : represent the number of rule, i : represent the number of x partition and j : number of y partition. Wk  Wk  Wi (30) 139 | P a g e www.ijacsa.thesai.org (IJACSA) International Journal of Advanced Computer Science and Applications, Vol. 6, No. 10, 2015 Layer 4: consists of 125 neurons. Nodes in layer 4 are adaptive nodes in which the consequent evaluation inference is calculated, its output is defined as: f k4  Wk  f k  Wk   pk x  qk y  rk  (31) Wk is the output of layer3 and pi , qi , ri  is called consequent parameters. 5  Wk  f k4 (32) k IV. 150 100 Eab(v) 50 0 -50 -100 Layer5: Finally, is single node in layer 5, where sums all the outputs from 4th layer to compute the overall output of the network: f 200 SIMULATION RESULTS The entire ANFIS network architecture is represented in the Fig.6. The inputs are the three line to line back emfs of the motor namely Eab, Ebc and Eca Fig4. And the desired output is the estimated rotor position angle. For producing the above relationship with least possible error, a five layer feed forward adaptive network fuzzy inference system is used. The input layer consists of five neurons that have their inputs as the three line to line back emfs, the three hidden layers consists of 125 each and the output layer consists of a single neuron whose output is the estimated position of the rotor. The relationship between the back emf and the position of the rotor is shown in the Fig.5. Fig.7 shows estimated rotor position by adaptative network fuzzy inference system and the error, along with real rotor position it’s a case without regulation. Fig.8 shows the estimated rotor position by adaptive network fuzzy inference system and the error, along with real rotor position the case is with PI speed control. In the comparison of the cases with control (PI regulator) and at without control, The controlled system attains the steady state in (0.04s) greater than the case without regulation the system attains the steady state in(0.023s), so the PI regulator slows the dynamic responses. The performance of thThe performance of the trained adaptive network fuzzy inference system is found to have very minimal errors witch is about 1.303e-7 as seen in the Fig.7 (without control). -150 -200 0 1 2 3 4 Angle teta (rd) 5 6 7 Fig. 5. The back emf Eab For Fig.8 (with PI controller), the error is 7.8386e -9. The error of output of ANFIS and real rotor position sensor are compared through Fig.7 and Fig.8 respectively. It is clear that the error in Fig.8 is too small (7.8386e -9) than these in Fig.7 (1.303e-7). The proposed scheme works efficiently where the usage of neural network topology together with fuzzy logic in The adaptive network, ANFIS, not only includes the characteristics of both methods, but also eliminates some disadvantages of their lonely-used case. Input1, MF Int1 mf1 Eab Int2 mf2 Aggregated Output1 Ebc teta The back fems (V) 150 100 50 Eac 0 -50 Int3 mf5 -100 Rule 125 -150 0 0.02 0.04 0.06 0.08 Time (sec) 0.1 0.12 0.14 Output MF 125 Fig. 6. Structure of ANFIS model proposed Fig. 4. The inputs curves 140 | P a g e www.ijacsa.thesai.org (IJACSA) International Journal of Advanced Computer Science and Applications, Vol. 6, No. 10, 2015 la position réelle erreur 7 0.06 6 0.04 5 erreur(rd) teta(rd) 0.02 4 3 2 0 -0.02 1 -0.04 0 0 0.01 0.02 0.03 0.04 temp(sec) 0.05 0.06 0.07 0.08 -0.06 0 0.02 0.04 0.06 la position estimée 7 0.08 0.1 0.12 temp(sec) 0.14 0.16 0.18 Fig. 8. Simulation Results with PI controller 6 5 V. teta(rd) 4 3 2 1 0 -1 0 0.01 0.02 0.03 0.04 temp(sec) 0.05 0.06 0.07 0.08 erreur 0.05 0.04 0.03 0.02 erreur(rd) 0.01 CONCLUSION The developed ANFIS models were successful in estimating rotor position of PMBLDC motor used in PV pumping system. In this paper the design of adaptive network fuzzy inference system based sensorless scheme was developed for permanent magnet brushless DC motor. The proposed scheme works efficiently for both with control (PI regulator) and at without control. It was found that the error is very minimal and this is smaller in system with regulation, than these without regulation. Also adaptive network fuzzy inference system based estimator is very flexible. 0 -0.01 -0.02 -0.03 -0.04 -0.05 0 0.01 0.02 0.03 0.04 temp(sec) 0.05 0.06 0.07 0.08 Fig. 7. Simulation Results without control la position réelle This scheme not only eliminates the position sensor, thereby cutting the cost but also transforms the drive into a highly efficient drive by eliminating losses caused by position sensors. The estimation error is found to be very minimal proving that the developed system is a very efficient and a reliable one. [1] 7 6 [2] teta(rd) 5 4 [3] 3 2 1 [4] 0 0 0.02 0.04 0.06 0.08 0.1 0.12 temp(sec) 0.14 0.16 0.18 0.2 la position estimée [5] 7 6 teta(rd) 5 [6] 4 3 [7] 2 1 0 0 0.02 0.04 0.06 0.08 0.1 0.12 temp(sec) 0.14 0.16 0.18 0.2 REFERENCES A.Moussi, A. Saadi, A. achour, Greg Asher, Photovoltaic pumping systems technologies trends, Larhyss journal ,2003,pp.127-150. PackiamPeriasamy, N.K.Jain,I.P.Singh, A review on development of photovoltaic water pumping system, Renew.and Sustainable.Energ. Reviews, Vol. 43 ,2015, pp. 918–925. MetinDemirtas,AslanDenizKaraoglan, Optimization of PI parameters for DSP-based permanent magnet brushless motor drive using response surface methodology, Energy Conversion and Management, Vol. 56, 2012, pp. 104–111. A. Terki, A. Moussi, A. Betka, N. Terki, An improved efficiency of fuzzy logic control of PMBLDCfor PV pumping system, Appl.Math.Model , Vol. 2, 2012, pp. 934-944. A.Moussi, A.Terki, Greg Asher, Hysteresis current controller of permanent Magnet Brushless DC motor PV pumping system, ASME, 2005, pp. 523-528 [International Solar Energy conference]. T. Hiyama, Neural network based estimation of maximum power generation from PV modules using environmental information, IEEE Trans. EC, Vol.12, 199, pp. 241–247. A. Ungurean, V. Coroban-Schramel and I. Boldea, Sensorless control of a BLDC PM motor based on I-f starting and Back-EMF zero-crossing detection, OPTIM ’10, 2012, pp. 377-382 [Optimization of Electrical and Electronic Equipment Conference,OPTIM ’10.IEEE 12th International]. 141 | P a g e www.ijacsa.thesai.org 0.2 (IJACSA) International Journal of Advanced Computer Science and Applications, Vol. 6, No. 10, 2015 [8] [9] AlinStirban, Ion Boldea, and Gheorghe-DanielAndreescu, MotionSensorless Control of BLDC-PM Motor With Offline FEM-InformationAssisted Position and Speed Observer, IEEE Trans. Ind. Appl, Vol. 48, 2012, pp. 1950-1958. LiviuIoanIepure, Ion Boldea and FredeBlaabjerg, Hybrid I-f starting and observer-based sensorless control of single phase BLDC-PM Motor drives, IEEE Trans. Ind. Electron, Vol.59, 2012,pp. 3436 – 3444. [10] SreepriyaR&RagamRajagopal, Sensorless Control of Three Phase BLDC Motor Drive with Improved Flux observer, ICCC, 2013, pp.292297 [IEEE International Conference on Control Communication and Computing]. [11] Ali M. Abdulshahed, AndrewP. Longstaff, Simon Fletcher, Alan Myers, Thermal error modelling of machine tools based on ANFIS withfuzzy cmeans clustering using a thermal imaging camera, Appl. Math. Model, Vol. 39, 2015, pp. 1837–1852. APPENDIX The PV generator, motor and pump used in this study have the following parameters: PV generator Modules AEG-40. (Temperature T=25°Cand solar insolation E=1000W/m².) Open circuit voltage 22.40 V Short circuit current 2.410 A Series resistance 0.450 Current temperature coefficient 0.06%/ C Voltage temperature coefficient 0.40%/ C Centrifugal pump Rated speed 3000 rev/min Rated power 521 W Flowrate 2.597 l/s Head 14.11 m Efficiency 69% Brushless DC motor Rated power 690 W Rated speed 3000 rev/min Rated voltage 200-220V Rated current 4.8 A Per phase resistance 1 Per phase inductance 5 mH Poles number 6 E.m.f constant 0.47 142 | P a g e www.ijacsa.thesai.org 

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ISSN:2349- 0012 Vol..2, Issue 5 ИССЛЕДОВАНИЯ ПРОБЛЕМ ОХРАНЫ ТРУДА РАБОТНИКОВ СЕЛЬСКОХОЗЯЙСТВЕННЫХ ОТРАСЛЕЙ Чориев Ислом Нормуминович ассистент Каршинский инженерно-экономический институт кафедра «Экология и охрана труда» Республики Узбекистан Аннотация: В данной статье рассмотрены уровень подготовленности по охране труда руководителей, фермеров и других работников в сфере сельского хозяйства. А также проведено исследования несчастные случаи и травмы, возникающие при выращивании сельскохозяйственной продукции и в перерабатывающей промышленности, определяются причины травм и меры по их устранению. Ключевые слова и фразы: Сельское хозяйство, охрана труда, несчастные случаи, сельскохозяйственной продукции, травмы, повреждения, опасные факторы, вредные факторы, перерабатывающей промышленности, химические элементы, биологические вещества, RESEARCH OF PROBLEMS OF LABOR PROTECTION OF WORKERS IN AGRICULTURAL INDUSTRIES Choriyev Islom Normuminovich assistant Karshi Engineering and Economic Institute 539 MAY ISSN:2349- 0012 Vol..2, Issue 5 department "Ecology and labor protection" Republic of Uzbekistan Annotation: This article discusses the level of preparedness for labor protection of managers, farmers and other workers in the field of agriculture, a special assessment of jobs, as well as accidents and injuries that occur when growing agricultural products and in the processing industry, determines the causes of injuries and measures to their elimination. Key words and phrases: Agriculture, labor protection, accidents, injuries, damages, agricultural products, processing industry, dangerous factors, harmful factors, chemicals, biological elements, machinery and equipment. Актуальность темы: Сельское хозяйство является одной из важнейших отраслей, определяющих уровень жизни населения страны, его благосостояние и обеспечение продовольственную безопасность. Сельскохозяйственная продукция перерабатывается во многих отраслях народного хозяйства (производство соков, табака, кофе, шоколада, сушка фруктов и др.). В то же время многие промышленные объекты (машиностроение, нефтегазовое, производство удобрений и др.) работают на сельское хозяйство. Получается, что прямо или косвенно в сельском хозяйстве заняты сотни тысяч инженеров и рабочих. Особое место в обеспечении занятости населения занимает сельскохозяйственная отрасль. В сельскохозяйственном секторе занято 3,7 млн населения республики, 27,2% из всех занятых (рис-1). 540 MAY ISSN:2349- 0012 Vol..2, Issue 5 Рисунок -1. Доля занятых в сельском Рисунок-2. хозяйстве, % (2021 г.) труда в Производительность сельском хозяйстве в долларах США (2021 г.) Из этой диаграммы видно, что, несмотря на то, что уровень занятости населения значительно выше, чем в развитых странах, экономические показатели находятся на очень низком уровне. Это и есть модернизация сельского хозяйства, охрана труда работников, занятых в отраслей , создание для них достаточных условий на рабочем месте, оплата и поощрение их труда, защита работников от отравления удобрениями и ядохимикатами. вопросов используется обеспечения для средства сельскохозяйственных индивидуальной культур, защиты а требуют рассмотрения. Кроме того, в отраслях также задействованы тысячи сложной тяжелой техники, опасного технологического оборудования и информационнокоммуникационных технологий. Поэтому сельское хозяйство является основной отраслью экономики страны со сложной системой. Техногенные аварии в такой ситуации неизбежны. Также не оставляют своего влияния первичные 541 и вторичные факторы опасных природных явлений MAY ISSN:2349- 0012 Vol..2, Issue 5 (геологические, гидрометеорологические, зпизотические, эпифитные). В таких условиях среди работников могут фиксироваться различного рода несчастный случай, травмы, отравления и смерти. Кроме того, известны случаи отравления удобрениями, используемыми в сельском хозяйстве, и химикатами, используемыми для борьбы с вредителями. Получается, что все вышеизложенное показало, насколько актуален вопрос анализа охраны труда и техники безопасности для агропромышленного комплекса. Целью данной статьи является анализ законодательства об охране труда в сфере сельского хозяйства на предмет его актуальности, подготовка выводов и предложений на основе анализа. Исходя из цели статьи, рассматриваются следующие задачи: - анализ проблем применения законодательства об охране труда в сфере сельского хозяйства; -анализ несчастных случаев, происшествий, травм, повреждений и профессиональных заболеваний, произошедших в сфере сельского совершенствованию методики хозяйства; -разработка предложений по мероприятий в области охраны труда в сфере сельского хозяйства и другие задачи определены. Основная часть: Сельскохозяйственный сектор обширен и является одним из основных звеньев экономической сети страны, а также локомотивом, который тянет за собой промышленность страны. Безусловно, важно отметить специфические черты, определяющие экономические и организационно-правовые отношения по управлению столь сложной и широкий экономической системой. 1) наряду с экономическими законами, который управляет сельским хозяйствым отраслей, существуют и независимые от человека естественные 542 MAY ISSN:2349- 0012 Vol..2, Issue 5 (биологические) законы (погодные условия, эпидемиологическая ситуация, загрязнение окружающей среды и др.) 2) это также существенно в связи с ограниченной возможностью неукоснительного соблюдения трудовых отношений и трудового законодательства в сельскохозяйственных условиях; 3) земледелие представляет собой среду, в которой основное производство территориально ограничено, не может быть ничем заменено и имеет свойство повышать производительность только при правильном его использовании; 4) использует в сельском хозяйстве такие средства, как радиационнохимические соединения, опасные для живых организмов; 5) производство сельскохозяйственной продукции рассредоточено на больших территориях с различными природно-климатическими условиями; 6) производственные процессы в сельском хозяйстве и конечные результаты труда могут не совпадать; 7) созданный продукт часто является промежуточным продуктом и перерабатывается в сельском хозяйстве; 8) занятость в сельском хозяйстве носит сезонный характер; 9) отсутствие постоянных рабочих мест (рабочих мест) у большинства занятых в сельском хозяйстве. 10) большинство работников, работающих в сфере сельского хозяйства, имеют среднее образование и не обладают достаточными знаниями о мире, что влияет на соблюдение трудового законодательства. Реализация вышеуказанных мер требует правового регулирования трудовых отношений по таким признакам в сельском хозяйстве, разработки и применения правил охраны труда, защиты прав работников в этой сфере. Известно, что сельское хозяйство является отраслью, которая поставляет и перерабатывает продукты питания населению. Создание 543 MAY ISSN:2349- 0012 Vol..2, Issue 5 кластеров, терминалов, транспортно-логистических систем для обеспечения населения продовольственными товарами, статистические данные последних 10 лет показывают, что среднегодовой показатель составляет 9...10%. Также с каждым годом увеличивается объем экспорта продукции. В связи с этим в нашем государстве приняты важные указы и решения, государственные программы, последовательно обеспечивается их выполнение. В частности, согласно постановлению Президента Республики Узбекистан от 14 марта 2019 года «О мерах по развитию сельскохозяйственной кооперации в области плодоовощного производства» в Ферганской, Андижанской и Наманганской области в 2020 году и 100 в 2021 году по стране за счет средств международных финансовых институтов. Сегодня в 55 районах нашей республики эффективно работают кластеры, специализирующиеся на производстве плодоовощной продукции. К этим кооперативам и кластерам присоединились фермеры- сельхозпроизводители, реализующие свою продукцию на внутреннем и внешнем рынках, эффективно использующие земельные и водные ресурсы, модернизирующие техническое оснащение. Наряду с повышением урожайности сельскохозяйственных культур находится решение проблем охраны труда работников. Это обеспечивает снижение динамики несчастных случаев в сфере сельского хозяйства. По данным Государственной службы статистики, количество пострадавших в сельскохозяйственных организациях за последние пять лет уменьшилось на 48,0% (рис- 3). Но этого показателя недостаточно. 544 MAY Vol..2, Issue 5 ISSN:2349- 0012 Рисунок -1. Динамика сокращения численности сельскохозяйственных рабочих и травматизма в результате несчастных случаев Именно поэтому управление охраной труда в аграрном секторе имеет особое значение. Однако если действия в этом направлении осуществляются только в одностороннем порядке, не может быть достигнута достаточная безопасность работников при исполнении служебных обязанностей: в этом случае государственные органы, органы местного самоуправления и негосударственные органы, работники и работодатели должны соблюдать действующие законы , стандарты, правила и требования. Высокий уровень травматизма и возникновения профессиональных заболеваний среди сельскохозяйственных рабочих связан с уровнем воздействия опасных и вредных факторов. Их можно разделить на следующие группы: - факторы, связанные с работой машин и механизмов. - неблагоприятные природно-климатические факторы. -необходимость контакта с удобрениями, пестицидами, опасными химическими и биологическими соединениями, используемыми в сельском хозяйстве; 545 MAY ISSN:2349- 0012 Vol..2, Issue 5 -риск заражения опасными заболеваниями при контакте с нездоровыми животными; -высокий риск для здоровья или жизни в результате укусов (укусов) диких животных (насекомых, грызунов, рептилий) в полевых условиях; -высокий потенциальный риск, связанный с необходимостью выполнения определенной работы в часто движущихся механизмах; -соблюдение законодательства об охране труда и технической безопасности, следовательно, низкий контроль в области внутреннего порядка и дисциплины; - специфика сельскохозяйственных работ, в том числе высокая степень тяжести и напряженности работ, связанная с их сезонностью; - другие факторы. В целях минимизации негативного влияния на профилактику такого травматизма и профессиональных заболеваний в сельском хозяйстве особое внимание следует уделять требованиям, установленным законодательством. Конечно, в сельском хозяйстве, как и в других сферах, общие вопросы организации безопасного труда регулируются нормами Трудового кодекса. Однако соблюдается и приказ Минтруда от 27 октября 2020 г. № 746 «Об утверждении правил охраны труда в сельском хозяйстве», учитывающий специфику труда для сельского хозяйства. Охрана труда в сельском хозяйстве включает следующие направления: - организация рабочего процесса. Соблюдение этих требований должно находиться под контролем работодателя; - требования к работникам; - дополнительные требования к отдельным видам работ. Требования к организации рабочего процесса устанавливает работодатель. В целях минимизации вреда, который может быть нанесен работникам под воздействием негативных факторов, работодатель обязан 546 MAY ISSN:2349- 0012 Vol..2, Issue 5 приложить все усилия для повышения уровня автоматизации и механизации рабочих процессов, при наличии опасных химических соединений и других угроз, использовать методы работы, исключающие непосредственный контакт с такими угрозами, или к работе допускается только специально обученный персонал. Работники, вступающие в контакт с машинами, химическими соединениями и другими опасностями, представляющими опасность для их здоровья в связи с выполнением служебных обязанностей, должны быть обеспечены средствами индивидуальной и коллективной защиты за счет средств работодателя. Оборудование, предоставляемое работодателем для выполнения определенного работодатель задания, должен должно быть обеспечивать в исправном работникам состоянии, разумное а сочетание режимов труда и отдыха. По собственной инициативе работодатель может вводить дополнительные нормы в области охраны труда, не противоречащие требованиям действующего законодательства. Работники обязаны соблюдать требования охраны труда в агропромышленном комплексе. В частности, они несут ответственность за: - своевременное прохождение учебных занятий по охране труда в соответствии с графиком, утвержденным работодателем; - выполнение должностных инструкций, в том числе по организации труда и использованию оборудования; - правильное использование средств индивидуальной и коллективной защиты соотрудниками; - сообщать непосредственному вышей организацию о нештатных ситуациях, возникающих в процессе работы. По данным ряда зарубежных исследователей, работники сельского хозяйства подвергаются большему риску, чем большинство работников 547 MAY ISSN:2349- 0012 Vol..2, Issue 5 других отраслей. (тяжелый физический труд, повышенный уровень шума и вибрации, неблагоприятные микроклиматические условия, различные химические и биологические вещества). Кроме того, несчастные случаи с участием аварии тракторов и прицепов являются причиной большинства несчастных случаев с участием сельскохозяйственных животных и полевых работ.[4] Но, в силу ряда объективных и субъективных причин, часто случающиеся практически не обрашается внимание на присходящехся на несчастных случаев, а статическая отчетность не находится под строгим контролем. например других отраслях строго провирается и проводит анализ, Среди отраслей сельского хозяйства наиболее опасной отраслью является животноводство и растениеводство, в которых в 2021 году зафиксировано в 1,7 раза больше смертельных травм. Факторами, вызывающими гибель и тяжкие телесные повреждения работников, являются: - отсутствие препятствий в подвижных частях оборудования; - отсутствие средств защиты или они не в рабочем состоянии; - неисправность машин, механизмов, инструментов и приспособлений; - создание рабочих мест, не отвечающих требованиям охраны труда; - низкий уровень обучения и инструктажа работников по безопасным методам работы, организации и контроля за соблюдением требований охраны труда и трудовой дисциплины; При возделывании сельскохозяйственных культур чаще всего получают травмы водители сельскохозяйственных тракторов, 74,0 % от общего числа пострадавших и 16,5 % подсобных рабочих (рис-4). 548 MAY ISSN:2349- 0012 Vol..2, Issue 5 Рисунок- 3. Самые вредные профессии в растениеводстве Снижение уровня травматизма и профессиональных заболеваний в сельском хозяйстве является одной из приоритетных задач в области охраны жизни и здоровья работников сельского хозяйства. Человечество существует, потому что его основная деятельность состоит из труда. Однако сохранение жизни, здоровья и трудоспособности работников в процессе труда не происходит само по себе, это требует формирования умения работать с соблюдением правил охраны труда и техники безопасности. По результатам многочисленных исследований причин производственного травматизма и острых профессиональных заболеваний все виды можно разделить на три основные группы. Первая группа причин: связанные с неправильными действиями непосредственного исполнителя в определенном трудовом процессе. Вторая группа причин: связанные с неправильной организацией работы; 549 MAY ISSN:2349- 0012 Vol..2, Issue 5 Третья группа причин: связана с техническими, аппаратными, приборами, неисправностями оборудования или внезапным отказом или нестабильным течением технологических процессов. Хронические профессиональные заболевания - возникают в результате отсутствия доступа к безопасному технологическому оборудованию или того, что это экономически нецелесообразно, а работники не используют средства индивидуальной защиты и другие меры безопасности. Стоит отметить, что не бывает техногенных аварий, причиной которых является человек, в итоге попавший в ту или иную аварию. Потому что он «что-то не сделал» или «сделал не так». либо является результатом неправильных действий отдельных сотрудников. Кроме того, не следует забывать, что безопасный труд включает в себя физическую и психологическую подготовку работников к работе «по правилам» и выполнению своих функций в соответствии с требованиями охраны труда, при достаточной профессиональной грамотности и осознанной мотивации. Поскольку полностью исключить риски с помощью технических и организационных мер абсолютно невозможно, безопасность работника зачастую определяется только его поведением. При устранении и предупреждении причин травматизма (психологических и психофизиологических) это связано с подбором работников по их профессиональным качествам, а также обучением и переподготовкой работников, инструктажем и формированием их безопасного поведения. [10] Мировой опыт показывает, что особое значение имеют требования техники безопасности и охраны труда, обучение оказанию первой помощи пострадавшим, а самое главное – своевременные профилактические мероприятия. В сельском хозяйстве анализ случаев травмирования показывает, что 550 MAY ISSN:2349- 0012 Vol..2, Issue 5 более 70% пострадавших не обучены вопросам охраны труда, не проходят инструктаж по охране труда. Это следствие того, что многие производители не объединены в крупные кластеры, логистические центры и корпорации и холдинги. Потому что при регистрации НС и ПЗ не проводится проверка и контроль по охране труда, а контролирующий орган не требует документов. В результате исследований охрана труда в сельскохозяйственных субъектах оставлена как есть, и в целом отмечается недостаток учебной литературы и научно-исследовательских работ в этой области. Отсюда следует, что в дальнейшем необходимо реформирование всей системы сельского хозяйства на государственном уровне. Практика исследований показывает, что многие несчастные случаи, когда внешне легкие травмы ограничиваются оказанием первой помощи в начальный период, в дальнейшем травмы становятся серьезными и переходят в несчастный случай, закончившийся летальным исходом. Сегодня в следственных процессах рассматриваются только несчастные случаи, повлекшие за собой временную или постоянную утрату трудоспособности или смерть. Из общего количества несчастных случаев 2,5% могут быть расследованы контролирующими органами в соответствии с законодательством, а остальные 75%, на практике, ни комиссия, ни акт Н-1 не выдаются, и к виновнику не принимаются меры. ответственные сотрудники. По этой причине детальный анализ несчастных случаев в подобных ситуациях не позволяет определить и в полной мере оценить причинно-следственную связь в системе «профессиональная деятельность профессиональный риск» для ее предупреждения. Все дела, связанные с гибелью работников, тяжелыми и групповыми травмами, расследуются следственными органами, хотя они обладают большим объемом исчерпывающей информации о произошедших несчастных случаях, условиях и причинах их возникновения, по порядку 551 MAY ISSN:2349- 0012 Vol..2, Issue 5 расследования квалифицированных специалисты в области охраны труда несчастных случаев не имеют полномочий на проведение следственных данных для анализа причин, в результате чего данные в полной мере не расследуются и не обнародуются. Действующие формы статистической отчетности дают возможность вводить и систематизировать только ту информацию, которая необходима для полного анализа причин и условий производственного травматизма. К сожалению, он не подходит для сельскохозяйственных условий. Одним из произошедших способов оценки несчастных случаев. риска травм Конечно, является никто не оценка может гарантировать, что они не произойдут в будущем. Поэтому очень важно, чтобы работодатели, отвечающие за предотвращение несчастных случаев на таких производствах и внедрение эффективных профилактических мер и техники безопасности, имели доступ к источникам информации. Кроме того, работодатель должен убедиться, что предпринятые ранее превентивные меры достаточны для снижения рисков. Однако если на производстве произошел проанализировать несчастный причины, по случай, которым работодатель должен профилактические меры недостаточны для предотвращения несчастных случаев. На основании вышеизложенных фактов можно сделать следующий вывод. В сельском хозяйстве основная работа в хозяйстве носит сезонный характер, и многие работы выполняются с помощью наемного труда. С этой точки зрения, фермеры, дехканские хозяйства, кластеры, логистические, терминальные и другие субъекты, занятые в сельском хозяйстве, не имеют четкой государственной единицы, что требует обучения работников управлению охраной законодательством 552 труда, ознакомления руководящими с установленными принципами, периодический MAY ISSN:2349- 0012 Vol..2, Issue 5 медицинский вызывает ряд проблем, таких как осмотр. Большинство работников, работающих в сельском хозяйстве, являются сельскими жителями, а руководители также имеют среднее образование, поскольку до сих пор в сельскохозяйственных отраслях не было высшей оброзавательных курсов по управлению сельским хозяйством или по охране труда и технике безопасности. это касается и области охраны труда в сельском хозяйстве. Еще одной причиной появления вышеперечисленных объективных и субъективных причин, влияющих на обеспечение охраны труда и техники безопасности в сельском хозяйстве, является нехватка молодежи, способной работать в сельском хозяйстве, и негативные демографические тенденции. По этой причине задачи нашего правительства по разработке эффективных механизмов занятости молодежи в сельском хозяйстве и созданию безопасных условий труда остается одним из приоритетных направлений социально-экономического развития республики. Таким образом, основными направлениями совершенствования охраны труда в сельском хозяйстве являются: - непрерывное продолжение научно-исследовательской работы в области охраны труда работников сельскохозяйственного производства и перерабатывающей промышленности и совершенствование нормативных правовых документов; - активизация оценки труда в сельскохозяйственных секторах; -формирование знаний работодателей в области охраны труда путем повышения качества и количества подготовки руководителей хозяйств и специалистов по вопросам охраны труда; -контроль условий труда и охраны труда в сельскохозяйственных организациях, информирование работников о результатах мероприятий по улучшению условий труда; 553 MAY ISSN:2349- 0012 Vol..2, Issue 5 -совершенствование системы управления охраной труда в сельскохозяйственном производстве. -уделить внимание вопросам охраны труда путем привлечения научных работников образовательных учреждений, научно- исследовательских учреждений к организации научно-исследовательских работ по безопасности агротехносферы, безопасности жизнедеятельности и охраны труда; - обеспечить информирование работников фермерских хозяйств о рисках, связанных с выполнением трудовых обязанностей, сформировать приоритетное отношение к сохранению жизни и здоровья работников в процессе производства; - внедрение базы данных по изучению и анализу несчастных случаев, произошедших в органах местного самоуправления и органах государственного управления на основе законодательных документов и возможность создания «Единой базы данных» несчастных случаев, произошедших в сельском хозяйстве республики методом "передача снизувверх" этой информации, предсказывающей высокую частоту несчастных случаев. Список использованной литературы: 1. Конституция Республики Узбекистан - 1992 г. 2. Закон Республики Узбекистан «Об охране труда» - 2016 г. 3. Трудовой кодекс Республики Узбекистан-1996 г. 4. Постановление Кабинета Министров Республики Узбекистан от 31 декабря 2018 года за № 1066 «О мерах по совершенствованию деятельности Министерства занятости и трудовых отношений Республики Узбекистан». 5. Постановление Кабинета Министров Республики Узбекистан от 554 MAY ISSN:2349- 0012 Vol..2, Issue 5 6 июня 1997 года за № 286 «Об осмотре и учете несчастных случаев на производстве и иного вреда здоровью работников». 6. Типовое положение об организации обучения и проверки знаний по охране труда от 14 августа 1996 г. за № 272 7. Ибрагимов Э.И., Газиназарова С., Юлдашев О.Р. Спецкурс по охране труда. Учебник. -Т.:, Изд-во ТИМИ.. 2013. - 555 с. 8. Турсунов Ю. Обзор Трудового кодекса Республики Узбекистан. Т. "АДОЛАТ", 2015. -960с 9. Маннонов Ю.О., Мавлонов К.Г. Охрана труда. Методическое пособие. Издательство "ИЛМ ЗИЁ", 2015. 10. Узбекистан и международные договоры по правам человека Составители: /Мингбоев У., Пулатов Н.Х., Норбоев Ж.Б. Т, «Правосудие», 1998. 11. Рекомендации по организации работы по охране труда. Ташкент2016 12. Гусак-Катрич Ю.А. Охрана работает в сельском хозяйстве. Монография. М.: Высшая школа, 1968.-123с. 13. «Обеспечение макроэкономической устойчивости новой экономики Узбекистана: проблемы, анализ и результаты» сборник тезисов онлайн, дистанционной научной конференции.- 2020 г. Интернет-источники: 15. www.ziyo.edu.uz - сайт Министерства высшего и среднего специального образования. 16. www.agro.uz - сайт Министерства сельского и водного хозяйства. 17.www.mintrud.uz- сайт Министерства занятости и трудовых отношений. 555 MAY 

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ONCOLOGY REPORTS 37: 1477-1486, 2017 Characterization of the microRNA profile in early-stage cervical squamous cell carcinoma by next-generation sequencing Wansong Lin1,2, Mei Feng3, Guilin Chen3, Zhifeng Zhou1,2, Jieyu Li1,2 and Yunbin Ye1,2 1 Laboratory of Immuno-Oncology, Fujian Cancer Hospital and Fujian Medical University Cancer Hospital; 2 Fujian Key Laboratory of Translational Cancer Medicine; 3Department of Gynecologic Oncology, Fujian Cancer Hospital and Fujian Medical University Cancer Hospital, Fuzhou, Fujian 350014, P.R. China Received July 6, 2016; Accepted September 1, 2016 DOI: 10.3892/or.2017.5372 Abstract. Squamous cell carcinoma (SCC) is histologically the most prominent type of cervical cancer. There is accumulating evidence suggesting that microRNAs (miRNAs) play important regulatory roles in the biological processes of cervical squamous cell carcinoma (CSCC). Deciphering the miRNA regulatory network in CSCC could deepen our understanding at the molecular level of CSCC initiation and progression. In the present study, we performed next‑generation sequencing (NGS) to profile miRNA expression in 3 pairs of early-stage CSCC samples. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify primary findings in another 20 pairs of CSCC samples. We identified 37 known miRNAs that exhibited significant alterations in expression (2-fold change or greater), among which 8 miRNAs were upregulated and 29 miRNAs were downregulated. Nine of these miRNAs were selected for further qRT-PCR validation. A novel miRNA candidate was also reported for the first time in the present study to be upregulated. The Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that its target genes were involved in MAPK, calcium and adherent junction signaling pathways. The present study systematically characterized the miRNA expression variation in early-stage CSCC and provides novel biomarkers for diagnosis and treatment as well as an opportunity for further investigation of the molecular mechanisms underlying the pathogenesis and development of CSCC. Introduction In less developed countries, cervical cancer is the second most common cancer and the third leading cause of cancer-related Correspondence to: Dr Yunbin Ye, Laboratory of ImmunoOncology, Fujian Cancer Hospital and Fujian Medical University Cancer Hospital, 420 Fuma Road, Fuzhou, Fujian 350014, P.R. China E-mail: zjyunbin@sina.com Key words: microRNA, cervical cancer, squamous cell carcinoma, early-stage, next-generation sequencing deaths in females. There were an estimated 444,500 new cervical cancer cases and 230,200 deaths in these countries in 2012 (1). Almost 90% of the deaths occurred in developing countries and for these countries it is a huge economic health care burden. The highest cervical cancer death rates are in Asia, with an estimated 144,400 deaths occurring in 2012 (1). Squamous cell carcinoma (SCC) is the most common histological type of cervical cancer (~80% of cases) and is closely correlated with human papillomavirus (HPV) infection (2). Cervical screening can prevent SCC, as it allows for detection and treatment of precancerous lesions, and application of the HPV vaccine also reduces the risk of SCC (3). However, the high cost is a major barrier to making screening and vaccination widely available to all women in developing countries, including China. Cervical squamous cell carcinoma (CSCC) cases that are classified as International Federation of Gynecology and Obstetrics (FIGO) stages IB and IIA have a greater possibility of being cured and patients have better prognosis (4). Since early detection results in a better prognosis and cervical screening and vaccines are extremely expensive, there is an urgent need to identify more efficient early diagnostic biomarkers and therapeutic targets for CSCC. Therefore, it is necessary to augment our understanding of the molecular mechanisms underlying the pathogenesis and development of CSCC. Although previous studies have identified chromosomal variations, abnormal expression of oncogenes or tumorsuppressor genes and aberrant promoter methylation in CSCC, few are relevant to microRNAs (miRNAs). miRNAs are a class of small non-coding RNAs (ncRNAs) ~19-25 nucleotides (nts) in length that can regulate gene expression by known classic mechanisms such as binding to messenger RNA (mRNA) at the 3'-untranslated region (3'-UTR) to downregulate protein‑coding genes by increasing mRNA degradation (5). They also act through other newly found mechanisms such as binding to promoters, proteins, and by directly interacting with other ncRNAs (5,6). miRNAs play important regulatory roles in various biological processes e.g. differentiation, development, proliferation, apoptosis, cell cycle control and metabolism (7,8). Changes in miRNA expression have been identified in many diseases, such as cardiac and autoimmune disorders, schizophrenia and cancer (9). miRNAs have been found to be differentially expressed between almost all types of 1478 lin et al: MicroRNA PROFILING OF HUMAN CERVICAL SQUAMOUS CELL CARCINOMA analyzed benign and malignant tumors and non-tumor tissues, including CSCC (10). In addition, miRNAs participate in regulating a variety of neoplastic biological capabilities acquired during the multistep process of tumor development. Using miRNA microarray, Wilting et al observed that 46 miRNAs showed significantly differential expression between normal cervical squamous epithelium and CSCC (11). Both miR-19a and miR-19b were reported to promote cervical cancer cell proliferation (12), whereas miR-125b inhibited cervical cancer cell apoptosis (13); both processes are involved in cervical carcinogenesis. Other studies reported that miR-9 (14) and miR-135a (15) contributed to HPV-induced cervical cancer formation, and that miR-133b (16) and miR-10a (17) promoted the progression and development of cervical cancer by regulating tumor cell migration, invasion and metastasis. However, miR-424 (18), miR-124 (19) and miR-218 (20) have opposite roles. All of these cited studies analyzed the differences in expression of cervical cancer-associated miRNAs by reverse transcription-polymerase chain reaction (RT-PCR) and hybridization-based microarray that only measure known and relatively abundant miRNAs, but are unable to identify novel miRNAs. To date, no studies have focused on the miRNA profile in early-stage CSCC. Thus, investigating the miRNA profile in early-stage CSCC by high-throughput sequencing may facilitate the analysis of expression of all annotated miRNAs, detection of novel miRNAs and identification of candidate markers for early diagnosis, and treatment of CSCC. In the present study, we applied next-generation sequencing (NGS) to globally and systematically characterize miRNA alterations in early-stage CSCC by a comparison with paired normal samples. Furthermore, we validated these miRNAs by qRT-PCR and analyzed miRNA target genes and performed function annotation and pathway analysis. Materials and methods Clinical sample collection. For sequencing, 3 pairs of human CSCC tumor (T) and paired normal tissue (N) samples in FIGO stages I-II were obtained from patients undergoing surgery of the cervix. Additional, 20 pairs of CSCC and normal tissues were also collected for quantitative real-time polymerase chain reaction (qRT-PCR) validation. All tumor and paired normal tissues were examined by hematoxylin and eosin (H&E) staining, and all pathologic diagnosis were confirmed by 2 independent pathologists; tumor tissues containing >90% tumor cells were selected for further study (Fig. 1). The study protocol was approved by the Ethics Committees on Human Research of the Fujian Cancer Hospital. All patients agreed to join the study and signed a written informed consent. None of the patients had received preoperative adjuvant radiotherapy or chemotherapy. All samples were immediately snap-frozen in liquid nitrogen and then stored at -80˚C until RNA isolation. RNA isolation. Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations, and quantified using a ND-1000 spectrophotometer (NanoDrop, Rockland, DE, USA). The RNA quality was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and the 28S/18S ratio was determined on 1% agarose gel. Only RNA extracts with an RNA integrity number (RIN) value ≥7 and 28S/18S ratio >1.8 were used for further analysis. Deep sequencing and bioinformatic analysis. RNA sequencing and bioinformatic analysis were performed at the Beijing Genomics Institute (BGI; Shenzhen, China). In brief, total RNA was ligated to the 5'- and 3'-end adaptors, and sequentially amplified by an RT-PCR reaction. The fragments of ~62-75 bp (small RNA plus adaptors) were isolated from the agarose gel and then purified. These products were directly sequenced with Illumina HiSeq 2500 (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. After trimming adaptor sequences, removing contaminated reads and filtering low quality reads, the remaining clean reads were mapped to the human genome using short oligonucleotide alignment program (SOAP). The other small RNAs (rRNA, tRNA, snRNA, snoRNA and piRNA) were detected by screening against Rfam 10.1 and the GenBank database. The known miRNAs were identified by aligning them to the miRBase. The expression differences of the known miRNAs between the CSCCs and paired normal samples were evaluated by comparing the log2 ratio of the 2 groups. The miRNAs with at least a 2-fold-change of expression and a P-value <0.05 between the 2 groups in all three patients were selected for further study. The novel miRNAs were predicted using MIREAP software. Validation of mature miRNA expression by qRT-PCR. For validation of the selected miRNAs that showed differential expression and predicted novel miRNAs, qRT-PCR was applied using the Applied Biosystems 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. In brief, the miRNAs were reverse transcribed into cDNA using the miScript II RT kit (Qiagen, Valencia, CA, USA) with 5X miScript HiSpec buffer. The mixture was incubated at 37˚C for 60 min, and then at 95˚C for 5 min and then placed on ice. Quantitative PCR was performed using the miScript SYBR-Green PCR kit and 10X miScript Primer Assay (both from Qiagen). The reaction consisted of 1 cycle at 95˚C for 15 min, followed by 40 cycles at 94˚C for 15 sec and 55˚C for 30 sec and 70˚C for 30 sec. All reactions were repeated 3 times. The relative expression levels of miRNAs were calculated using the 2-ΔΔCt method with snRNA U6 as the internal reference. Quantitative PCR data were analyzed by SPSS statistics software (version 19.0; IBM, Armonk, NY, USA). A Student's t-test was used to compare the difference in expression between CSCC and normal tissues. P<0.05 was considered to indicate a statistically significant result. miRNA target prediction, functional annotation and pathway analysis. The target genes of the selected miRNAs with differential expression and novel miRNAs were predicted using RNAhybrid. For determination of the main biological functions of the candidate target genes, Gene Ontology (GO) enrichment analysis was applied. We mapped all candidates to GO terms in the database (http://www.geneontology. org/), using a hypergeometric test to find significantly enriched GO terms. The GO terms with corrected P-values with an ONCOLOGY REPORTS 37: 1477-1486, 2017 1479 Figure 1. H&E staining of CSCC and paired normal tissues. The H&E staining results of CSCC and paired normal tissues at a magnification of x40 under a microscope. Table I. Summary of the small RNA sequencing. Category Number -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------T-1 T-2 T-3 N-1 N-2 N-3 Total reads Clean reads % Unique sRNAs miRNAs Known miRNAs in miRBase miRNAs miRNA-5p miRNA-3p miRNA precursors Unannotated sRNAs Novel miRNA candidates 12,000,000 11,713,704 98.40 884,315 4,196 1,708 200 414 306 788 232,043 28 10,523,729 10,313,021 98.73 673,581 4,545 1,881 225 431 351 874 205,460 24 12,000,000 11,519,252 96.70 867,499 4,346 1,810 229 418 325 838 255,885 31 12,000,000 11,799,688 99.02 615,904 3,634 1,571 171 385 298 717 291,651 20 12,000,000 11,820,021 99.18 466,839 4,035 1,659 182 394 330 753 156,254 22 12,000,000 11,704,777 98.25 658,536 4,173 1,724 200 400 335 789 240,371 26 T, CSCC tumor tissue; N, paired normal tissue. enrichment ratio ≤0.05 were defined as significantly enriched target gene candidates. For identification of significantly enriched metabolic pathways or signal transduction pathways in target gene candidates, we mapped these candidates to the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases (21). The calculation method is the same as that in the GO analysis. Pathways with corrected P-values with an enrichment ratio ≤0.05 were considered as significantly enriched target gene candidates. Statistical analysis. All quantitative data are expressed as the mean ± standard deviation (mean ± SD) from at least 3 samples or experiments/data point. Significant differences were analyzed by the Student's t-test to compare 2 groups of independent samples. Results Overview of small RNA sequencing results. To determine the miRNA profile of early-stage CSCC, we performed high‑throughput NGS in 3 CSCC and their paired normal samples using Illumina HiSeq 2500. After removing adaptors, low quality tags and contaminants, an average of 11,478,411 clean reads (ranging from 10,313,021 to 11,820.021) remained (Table I). The majority of these clean reads were 18-28 nt in length, with 22 nt RNAs the most abundant. These clean reads were mapped to the Human Genome and rRNA, tRNA and snRNA were identified by the alignment to Rfam 10.1 and GenBank databases. Subsequently, an average of 1,726 mature miRNAs (ranging from 1,571 to 1,881) were successfully annotated in the miRBase, and thus identified 1480 lin et al: MicroRNA PROFILING OF HUMAN CERVICAL SQUAMOUS CELL CARCINOMA Figure 2. Scatter diagrams of miRNA expression between CSCC and normal tissues (x-axis, normal and y-axis, tumor). Red points represent miRNAs with ratio >2, blue points represent miRNAs with ratio >1/2 and ≤2 and green points represent miRNAs with ratio <1/2. Ratio, normalized expression in the tumor/normalized expression in the control tissue. Figure 3. Heatmap of miRNA expression varied significantly between CSCC and normal tissues in the 3 pairs of samples. as known miRNAs (Table I). The sequences which did not match with any database were considered as unannotated sRNAs (Table I). Identification and validation of differentially expressed known miRNAs between CSCC and normal tissues. To detect the known miRNAs which were differentially expressed between the CSCC and normal tissues, the expression level of each miRNA was first normalized to provide the expression of transcripts per million (TPM), and then the fold-change and P-values were calculated from the normalized expression. There were 424 differentially expressed miRNAs between T1 and N1, 480 between T2 and N2 and 440 between T3 and N3 (Fig. 2). Subsequently, on the basis of the cut-off criteria (fold-change >2 and p-value <0.05), 186, 149 and 126 miRNAs were selected from the data from each of the 3 pairs of patient samples. Finally, only 37 miRNAs exhibited consistent changes in all 3 pairs of samples; among these 37 differentially expressed miRNAs, 8 were significantly upregulated and 29 were significantly downregulated (Fig. 3; Table II). To further validate the deep sequencing results, qRT-PCR was applied to assess the significantly differentially expressed miRNAs in 3 pairs of samples used for sequencing and the additional 20 pairs of samples. Nine miRNAs were selected from the miRNAs with significantly modified expression and measured by qRT-PCR. All of the 9 miRNAs displayed the same expression pattern consistent with the sequencing data (Fig. 4). This indicated that our high through-put sequencing results were reliable. ONCOLOGY REPORTS 37: 1477-1486, 2017 1481 Table II. Summary of the significantly upregulated and downregulated miRNAs in CSCC. miRNA Fold-change (log2 tumor/normal) -----------------------------------------------------------------------------------------------------------------------------------------------------------------------T1 vs. N1 T2 vs. N2 T3 vs. N3 hsa-miR-934 hsa-miR-3927-3p hsa-miR-34b-5p hsa-miR-34c-5p hsa-miR-21-3p hsa-miR-7-5p hsa-miR-1277-3p hsa-miR-2355-5p hsa-miR-211-5p hsa-miR-204-5p hsa-miR-218-1-3p hsa-miR-202-5p hsa-miR-218-5p hsa-miR-1298-5p hsa-miR-204-3p hsa-miR-6510-3p hsa-miR-299-5p hsa-miR-1247-5p hsa-miR-206 hsa-let-7c-5p hsa-miR-99a-5p hsa-miR-487b-3p hsa-miR-154-5p hsa-miR-337-3p hsa-miR-495-3p hsa-miR-134-5p hsa-miR-125b-5p hsa-miR-376c-3p hsa-miR-195-5p hsa-miR-654-3p hsa-miR-127-3p hsa-miR-369-5p hsa-miR-139-5p hsa-miR-433-3p hsa-miR-329-3p hsa-miR-379-5p hsa-miR-887-3p 8.30318711 4.51831602 9.09373446 4.78205816 6.62522824 5.56817204 3.30345745 2.42581593 -7.08321336 -6.85930564 -7.33109609 -10.57502857 -7.941165 -5.92810297 -6.66817587 -3.36793893 -5.52895357 -7.39024513 -3.37648276 -6.26600709 -6.62465962 -5.00445576 -5.43273636 -2.21193294 -4.59233357 -3.89631011 -5.94077162 -3.55711379 -4.33482863 -4.63279944 -4.56838942 -5.41522226 -2.84273417 -4.37189471 -3.95914876 -3.57432673 -2.49199079 4.2842649 3.10372182 4.03811041 4.92246647 3.4080715 2.07123043 2.463592 3.65621776 -6.80271289 -9.83557679 -7.40258575 -4.68366909 -8.60425671 -4.54163652 -4.05060043 -2.73610137 -3.78516379 -4.67666656 -2.61057144 -2.93147207 -2.58200581 -3.79678741 -3.17579365 -4.38841479 -3.30958264 -3.08449386 -2.27910623 -2.91544225 -2.13708218 -2.26265207 -2.30793519 -2.08867671 -2.67661687 -2.16954783 -2.12515371 -2.49126887 -2.07624607 7.20535341 9.11787682 2.54681543 3.3850871 2.26011983 2.11570518 3.34499026 2.39229062 -11.34275801 -5.26254955 -6.9021942 -5.0642676 -2.97717439 -7.7387003 -7.09486942 -8.822762 -5.511115 -2.41593802 -7.18229503 -3.93093655 -3.74953697 -3.01875682 -2.93133666 -4.62100525 -3.28815361 -4.1468506 -2.73045243 -4.19191414 -4.13398935 -3.39215912 -3.28638674 -2.46223868 -4.37453647 -2.89020567 -3.0926029 -2.8998286 -2.66140231 Each miRNA is listed in the Table with P<0.01 in all 3 pairs of samples. CSCC, cervical squamous cell carcinoma. Target genes of the significantly altered miRNAs. To demonstrate the differences in gene expression associated with the miRNA profile in CSCC, the putative target genes of the miRNAs that were expected to be significantly differently expressed between CSCC and normal tissues were predicted using RNAhybrid. GO and KEGG pathway analyses were performed to enrich the main biological processes and pathways in which the target gene candidates were involved. However, the P-value of the enrichment ratio failed to achieve filter conditions and the analysis was not continued. Prediction of novel miRNAs, their target genes and pathways. To detect novel miRNAs, unannotated sRNAs were further processed using MIREAP. The number of novel miRNA candidates obtained from the 6 small RNA libraries ranged from 20 to 31 (Table I). There were 5 common novel miRNA 1482 lin et al: MicroRNA PROFILING OF HUMAN CERVICAL SQUAMOUS CELL CARCINOMA Figure 4. Validation of the significantly different miRNAs between CSCC and normal tissues by qRT-PCR in 20 pairs of samples (*P<0.05, **P<0.01, ***P<0.001). Figure 5. Prediction of novel miRNAs in CSCC. (A) Expression of novel_mir_7 by NGS. (B) Expression of novel_mir_7 by qRT-PCR validation in 20 cancer samples. (C) The sequence and the stem loop structure of novel_mir_7 (*P<0.05, **P<0.01). candidates in the 3 pairs of CSCC and normal samples. Among the 5 common miRNAs, only 1 miRNA, novel_miR_7, displayed a significant difference in expression (fold-change >1 and P-value <0.05) in all 3 pairs of samples: novel_miR_7 was selected as a putative novel miRNA and verified by qRT-PCR (Fig. 5). Its sequence and stem-loop secondary structure are shown in Fig. 5. To further understand the function of the novel miRNA, its target genes were predicted using RNAhybrid and also assessed by GO and KEGG pathway analyses. The GO analysis revealed the main biological processes in which the target genes are involved and is shown in Fig. 6. The KEGG pathway analysis indicated that 5 pathways were enriched; including the MAPK, calcium and adherent junction signaling pathways (Table III). ONCOLOGY REPORTS 37: 1477-1486, 2017 1483 Figure 6. Functional annotation for predicted target genes of the novel miRNA in CSCC. Table III. KEGG pathway analysis for predicted target genes of the novel miRNA in CSCC. KEGG no. KEGG pathway KO:04010 KO:04020 KO:04520 KO:05217 KO:04730 MAPK signaling pathway Calcium signaling pathway Adherent junction Basal cell carcinoma Long-term depression Significance a b b b b Q-value <0.05 in all 3 pairs of samples; bQ-value <0.01 in all 3 pairs of samples. Q-value, corrected P-value. KEGG, Kyoto Encyclopedia of Genes and Genomes. a Discussion Although CSCC caused by HPV infection can be prevented, it is still a common cancer in women. There is accumulating evidence to indicate that HPV infection alone is insufficient to cause the disease and miRNAs may play critical roles in carcinogenesis (22-24). Therefore, global analysis of miRNA expression comparing CSCC with normal tissue may aid in the better understanding of the function of miRNAs in the course of the disease as well as finding novel biomarkers for diagnosis, treatment and prognostic evaluation. To the best of our knowledge, this is the first study to profile miRNA expression in early-stage CSCC on the NGS platform. We identified 37 significantly differentially regulated known miRNAs in CSCC compared with normal tissue and confirmed a sample of 9 by qRT-PCR. Furthermore, a novel miRNA candidate, novel_miR_7, was identified for the first time and its target genes are involved in MAPK, calcium and adherent junction signaling pathways. miRNAs are rather stable, even in body fluids such as serum, plasma, urine and saliva (25). The expression of miRNAs is unique to tissues or organs and they can be easily detected (26). These characteristics make them ideal cancer biomarkers. Juan et al (27) performed deep sequencing on the serum pools of cervical cancer patients and healthy controls to identify putative novel miRNAs. They found that only 1 of 17 common novel miRNA candidates may distinguish cervical cancer patients from healthy controls. Wang et al (28) also constructed expression profiles of miRNAs in the serum of CSCC patients by microarray. They discovered that the levels of 291 of 338 detectable circulating miRNAs were changed >2-fold in serum samples from CSCC patients vs. controls. However, they did not validate these results through any other methodologies. Although detection of miRNAs in serum has been proposed to be a simpler and less invasive diagnostic method than sampling tissue, the consequential biological and technical variability, which is mainly assessed by a series of different normalization processes based on the expression of reference genes, is difficult to avoid. Currently, there is no known suitable extracellular reference RNA for a proper normalization, particularly in cell-free miRNA analysis (26). Commonly used references, including U6 small nuclear RNA (RNU6B) and 5S ribosomal RNA, may be degraded or expressed less stably than others in serum (29). Therefore, we decided to analyze miRNA expression in CSCC tissues. In previous studies, a microarray technique, which is biased 1484 lin et al: MicroRNA PROFILING OF HUMAN CERVICAL SQUAMOUS CELL CARCINOMA towards abundantly expressed known miRNAs, was the major platform for miRNA expression profiling (11,28). We chose a more sensitive and specific method, NGS, which enables highthroughput analysis and novel miRNA discovery, to measure miRNA expression. In the present study, we identified 9 miRNAs that exhibited differential expression in CSCC using deep sequencing and confirmed by qRT-PCR evaluation. Six were downregulated, including miR-211-5p, miR-204-5p/3p, miR-218-1-3p/5p and miR-202-5p, and 3 were upregulated, including miR-34b-5p, miR-34c-5p and miR-21-3p. Among these miRNAs, miR-218, miR-21 and miR-34b-5p have previously been discovered and their functions had been verified to be associated with cervical cancer. In comparison to normal cervical tissues the expression of miR-218 was reported to be lower in cervical cancer tissues (20), and that finding was concordant with our results. Its downregulation was significantly associated with worse overall survival, worse disease-free survival and pelvic/aortic lymph node recurrence. miR-218 was found to target survivin to inhibit cervical cancer progression by regulating clonogenicity, migration and invasion. Increased miR-21 expression was found to be correlated with poorer histological diagnosis in cervical cancer (30). Meanwhile, miR-21 was found to be mainly expressed in the tumor stromal microenvironment where it may be involved in cervical cancer progression. In addition, expression of miR-21 in HPV-positive samples and SCC was significantly higher than that noted in HPV-negative samples (31). Constitutive activation of aberrant STAT3 signaling via miR-21, which is regulated by the HPV oncoprotein E6, plays a pivotal role in the initiation and progression of HPV-induced cervical carcinogenesis (32,33). Our sequencing results found that miR-21 was upregulated in CSCC and it may act as an oncogene; which is consistent with previous studies (30,31). miR34b-5p was found to be downregulated in minimal deviation adenocarcinoma (MDA) compared with normal proliferative endocervical tissues (34), but in the present study, it was found to be upregulated in CSCC. The discrepancy suggests different expression patterns of miRNAs in different histological subtypes of tumors. The other differentially expressed miRNAs we described have seldom been reported in previous studies of cervical cancer, but they have been demonstrated to be correlated with other malignant tumors. Trends seen in the expression of certain miRNAs that were determined by sequencing were in accord with published functional studies. For example, miRNA-211 functions as a tumor-suppressor in melanoma (35,36) and glioma (37). It could block tumor invasion and migration by targeting NUAK1 (38), BRN2 (39), KCNMA1 (40) or MMP-9 (37). Both miR-204-5p/3p and miR-202 have tumor-suppressive function as well. Downregulation of miR-204-5p in colorectal cancer tissues was associated with poor prognosis: It suppresses colorectal cancer cell growth, migration, and invasion as well as promotes tumor sensitivity to chemotherapy by inhibiting RAB22A (41). In endometrial carcinoma, miR-204-5p exhibits a similar function through the TrkB-STAT3-miR‑204-5p regulatory pathways (42) and miR-204-3p inhibits the growth of hepatocellular carcinoma tumor endothelial cells by blocking the adhesion function of FN1 (43). miR-202 can suppress osteosarcoma cell proliferation and induce cell apoptosis by downregulating Gli2 expression (44). While miR-34c is considered to be a putative tumor suppressor in a majority of tumors (45-48), we found it to be upregulated in CSCC, which is in agreement with previous results that determined expression by miRNA microarray in cervical cancer (11,28). Thus, we hypothesized that miR-34c may play different roles in cervical cancer and other types of malignancy. NGS technology allows the rapid discovery of novel miRNAs, for some of which the biological function is unknown. We identified 5 common novel miRNA candidates with differential expression between CSCC and normal tissues. Our focus was narrowed to miRNAs that exhibited a significant change of expression in all 3 pairs of samples. We identified novel_miR_7 as a candidate novel miRNA and confirmed its presence. Functional annotation revealed that its predicted target genes are in the MAPK signaling pathway, which is closely related to cell proliferation, cell death, and the balance between them. Early in 2006, Engelbrecht et al indicated the involvement of MAPK in cervical cancer and demonstrated the correlation between the activity of MAPK and apoptosis in the disease process (49). The HPV 16 E2 protein may induce apoptosis by inhibiting MAPK signaling in CSCC (50). However, playing a role in cervical cancer carcinogenesis, the expression level of MAPK may serve as a marker for cervical cancer progression (51,52) and its inhibitors have shown the potential to be useful for cervical cancer therapy (53). Therefore, our results provide a solid basis for the characterization of the role of novel_miR_7 in the pathogenesis and development of CSCC and in the treatment of this disease. In summary, for the first time we identified known and novel miRNAs in early-stage CSCC by high-throughput NGS. Moreover, we validated these miRNAs in other independent samples by qRT-PCR. Our analysis of differentially expressed miRNAs not only support existing findings but also uncovered various differentially expressed miRNAs which had not previously been reported in CSCC, thus providing new molecular signatures of the disease. The prediction of novel miRNAs found that novel_miR_7 may be a promising biomarker for CSCC. 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FOCUS: ARCHITECTURE IN PHOTOGRAPHIC EXHIBITIONS 1858–1861 AN EXHIBITION PROPOSAL FOR THE MUSEUM OF FINE ARTS, HOUSTON By Natalie Banaszak BAH Honours, University of Guelph, Guelph, 2014 A thesis presented to Ryerson University In partial fulfillment for the degree of Master of Arts In the program of Film and Photography Preservation and Collections Management. Toronto, Ontario, Canada, and Houston, Texas, USA 2016 © Natalie Banaszak 2016 AUTHOR'S DECLARATION FOR ELECTRONIC SUBMISSION OF A THESIS 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 FOCUS: ARCHITECTURE IN PHOTOGRAPHIC EXHIBITIONS 1858–1861 — AN EXHIBITION PROPOSAL FOR THE MUSEUM OF FINE ARTS, HOUSTON Master of Arts, 2016, Natalie Banaszak, Film and Photography Preservation and Collections Management, Ryerson University. Abstract This thesis consists of a proposal for an exhibition titled Focus: Architecture in Photographic Exhibitions 1858–1861. Drawn from the collection of almost three hundred nineteenth-century architectural photographs at the Museum of Fine Arts, Houston, the exhibition explores the topic of architectural photographs exhibited between 1858 and 1861. This two-part thesis first approaches the exhibition from a practical side, outlining the steps in preparing the photographs for display. An essay follows, investigating the role of the Architectural Photographic Association in the context of the photographic market of this time. The thesis also includes appendices relating to various aspects of the exhibition design process, including an object checklist, matting and framing recommendations, condition reports, wall labels, wall elevations, and an informational brochure. iii Acknowledgements First and foremost, this project would not be possible without the help of Thierry Gervais, Malcolm Daniel, and Marta Braun. The guidance I received from all of you was invaluable to me. Thierry, thank you for your patience and for always pushing me to be better. Malcolm, thank you for welcoming me into the department, and always entertaining my ideas and helping me refine them. Marta, I appreciate you always supporting me, and am grateful for your suggestions towards this thesis as its second reader. I cannot begin thank those who have read this thesis, edited it, and provided suggestions. Adrian, thank you for your never-ending support; from listening to long rants and making those into real thoughts, withstanding last minute editing requests, and cheering me on. You are a true gem. Jamie, thanks for keeping me sane in the in-between parts. To my Firecrackers, thank you all for the support, the critical eyes, and your firm grips on the English language. Congratulations to my fellow classmates; thank you for always being an inspiration and a soundboard for ideas. Finally, my thanks go out to the staff at the Museum of Fine Arts, Houston, for taking time out of their busy schedules to accommodate me, particularly: Selina Lamberti for your genuine friendship, and the countless hours of advice; Jason Dibley, for the frequent hospitality in your workspace, and for teaching me so much; Toshi Koseki and August DiStefano for indulging my conservation and preparatory requests; and Allison Pappas and Adrian Alejo for your willingness to go above and beyond to help me with anything and everything in between. iv Table of Contents Declaration Abstract Acknowledgements List of Figures List of Appendices ii iii iv vi vii Introduction Survey of the Available Literature 1 5 Part One: Preparing an Exhibition Curatorial statement Exhibition timeline Checklist and rationale List of Photographs Cataloguing and preparing wall labels Preservation considerations Matting and framing Wall elevations and layout Preparing an exhibition pamphlet 13 13 14 15 17 23 26 27 28 29 Part Two: The Exhibitions of the Architectural Photographic Association (1858-1861) The Association’s First Exhibition The 1859 Exhibition The Association’s Downfall Conclusion 33 34 38 41 44 Appendices References 46 71 v List of Figures Figure 1: Fratelli Alinari, Lorenzo Ghiberti’s Bronze Door, 1852–1858, albumen silver print from glass negative, 42.8 x 31.7 cm, The Museum of Fine Arts, Houston, 2004.211. 17 Figure 2: Fratelli Alinari, Campanile for Duomo of Santa Maria del Fiore, Florence, 1852–1858, albumen silver print from glass negative, 44.4 x 29.5 cm, The Museum of Fine Arts, Houston, 2004.212. 17 Figure 3: Édouard Baldus, Pavillon Denon, Louvre, 1855–1856, salted paper print from glass negative, 44 x 33.8 cm, The Museum of Fine Arts, Houston, 2004.249. 18 Figure 4: Bisson Frères, Moissac Abbey Cloister, 1857, albumen silver print from glass negative, 35.6 x 45 cm, The Museum of Fine Arts, Houston, 2004.313. 18 Figure 5: Charles Clifford, [Main Door, Salmanca Cathedral], before 1858, albumen silver print from glass negative, 43 x 32.4 cm, The Museum of Fine Arts, Houston, 2004.350. 19 Figure 6: Charles Clifford, Salamanca. Puerta del Convento de las Duenas, 1853, albumen silver print from glass negative, 39.7 x 27.3 cm, The Museum of Fine Arts, Houston, 2004.351. 19 Figure 7: Tommaso Cuccioni, View of the Roman Forum towards the Capitoline Hill, Looking West, 1855, albumen silver print from glass negative, 27.3 x 38.1 cm, The Museum of Fine Arts, Houston, 2004.369. 20 Figure 8: Roger Fenton, printed by Francis Frith & Co., Lichfield Cathedral, Central Doorway, West Porch, 1858 printed 1863–1865, albumen silver print from glass negative, 41.9 x 34.9 cm, The Museum of Fine Arts, Houston, 2014.89. 20 Figure 9: Francis Frith & Co., Columns, Pharaoh's Bed, 1856–1861, albumen silver print from glass negative, 20.4 x 16.4 cm, The Museum of Fine Arts, Houston, 88.233. 21 Figure 10: Robert Macpherson, View of Rome from the French Academy, Monte Pincio, 21 1851–1858, albumen silver print from glass negative, 20.4 x 16.4 cm, The Museum of Fine Arts, Houston, 2004.222. Figure 11: Carlo Naya, Piazzetta of San Marco looking towards the Island of St. George, 22 1857–1859, albumen silver print from glass negative, 42.4 x 53.5 cm, The Museum of Fine Arts, Houston, 2004.619. vi Figure 12: Robertson and Beato, Tophane Fountain, 1854–1858, albumen silver print from glass negative, 26.5 x 31.2 cm, The Museum of Fine Arts, Houston, 2000.255. Figure 13: Digital image taken around 2004 for Tommaso Cuccioni, View of the Roman Forum towards the Capitoline Hill, Looking West, 1855, albumen silver print from glass negative, 27.3 x 38.1 cm, The Museum of Fine Arts, Houston, 2004.369. 22 30 List of Appendices Appendix A: Wall Labels Appendix B: Condition Reports Appendix C: Matting and Framing Swatches and Worksheets Appendix D: Wall Elevation Appendix E: Exhibition Brochure vii 46 52 54 68 69 Introduction The Department of Photographs at the Museum of Fine Arts, Houston has a fixed exhibition space along a hallway in the lower level of the Audrey Jones Beck Building. Located there is a permanent display titled The History of Photography: Selections from the Museum’s Collection, in which the photographs rotate every four months, and the curators alternate in sharing the responsibility of selecting the group of objects. The wall directly opposite is a rotating Focus display, which includes prints by one photographer or a theme from the Museum’s collection of over thirty thousand photographs. For my thesis, I proposed an exhibition of nineteenth-century architectural photographs for this space. This selection will present a history of exhibitions of architectural photography with a focus on the Architectural Photographic Association, in order to expand the public’s understanding of the demand for architectural photographs in the 1850s to 1860s. The exhibition would accompany any History of Photography rotation, which “present the medium's history in a slightly different light. In this way, an increasingly complex picture of photographic history emerges for those who move slowly, look closely, and return for more.”1 My exhibition reflects the Museum’s collection mandate by starting a conversation about how photographs are displayed as art, how they were used by architects, and how they were distributed by photographic societies and studios in the nineteenth century. The exhibition at the Crystal Palace in London in 1851 was, for the majority of those in attendance, a preliminary exposure to photographs.2 By the mid-1850s, photographic exhibitions became more common throughout Europe, and particularly in Britain and France, intrinsically 1 "A History of Photography: Selections from the Museum’s Collection." The Museum of Fine Arts, Houston. 2016. Accessed April 21, 2016. https://www.mfah.org/history-photography-selections-museums-collection/. 2 John Hannavy, "Exhibitions of Photography," in Encyclopedia of Nineteenth-century Photography, New York: Routledge, Taylor & Francis Group, 2008: 508. 1 linked with the rise of photographic societies.3 The Photographic Society of London, now the Royal Photographic Society, was founded in 1853, and the following year, the Société Française de Photographie was formed in Paris. These societies helped foster an international community of photographers, working to validate their work as art, and to make technical advances in the medium or profit off of the photographs. Members of photographic societies, like the Société Française de Photographie, or the Photographic Society of London, or the Glasgow Photographic Society, were photographers and chemists. They would exchange prints with each other for the purposes of refining their own techniques and building their personal collections. These societies held annual exhibitions, in which the photographs were selected by juries to ensure that they showcased only the finest examples of photographic practices and inventions of the time. The 1850s also saw a rise in the number of photographers travelling abroad, particularly to Italy and the Near East.4 Photography was seen as a more reliable and practical medium for the documentation of encounters overseas.5 As a result, the number of commercial photographic studios across Europe and Asia increased. Scholar Anne McCauley argues that beginning in the 1850s, commercial photographers defined public perception of photography.6 In 1848, there were thirteen photographic studios in Paris, and by 1878, that number rose to almost four hundred.7 McCauley argues that the years following 1855 were the period of greatest growth of the photographic industry.8 In the 1850s and 1860s, many photographers bought existing studios to avoid the cost of starting up their own businesses. Most of these studios only survived because 3 Anne M. Lyden, A Royal Passion: Queen Victoria and Photography, Los Angeles: J. Paul Getty Museum, 2014. Sylvie Aubenas and Jacques Lacarrière, Voyage En Orient, Paris: Hazan, 1999. 5 Risto Sarvas and David M. Frohlich, From Snapshots to Social Media: The Changing Picture of Domestic Photography, New York: Springer, 2011: 31. 6 McCauley 8. 7 Ibid 8 Ibid 31. 4 2 of funding from family members, or external benefactors.9 According to McCauley, the market of the 1850s and 1860s consisted of art reproductions,10 photographs of new construction, and portraiture. Advances in photographic technology at the time aided in the success of such studios, and enabled photographers to travel. Specifically, the invention of wet collodion glass plate negatives made it easier to print a high quantity of sharp images upon a photographer's return home from their travels. This allowed for a larger-scaled production and distribution of photographic prints than ever before.11 The increased success and popularity of photographic societies, commercial studios, and wet collodion glass plate negatives amalgamated to create an unprecedented international community of photography. This was the climate in which the Architectural Photographic Association began, holding its inaugural meeting in London in 1858. The Association reveled in its immediate popularity and success. Architects belonging to the Association would travel locally and abroad, purchasing prints by well-established photographers. These photographs would then be assembled into an annual exhibition in London, where a subscription fee of one guinea or upwards per annum would grant access and allow patrons to come and purchase copies of photographs that were on display.12 For reference, a guinea in 1858 was equivalent to 21 shillings, and in today’s market, that has a relative purchasing power of 171.93 Canadian Dollars.13 By 1861, the Association struggled with overwhelming demand and short finances and so was disbanded. Due to its inability to send out photographs to subscribers fast enough, it was overcome by its own business model in just four years. My thesis is therefore guided by the following question: how does the 9 Ibid., 60. Ibid., 266. 11 Sarvas and Frohlich, 31. 12 The Literary Gazette, 43. 13 Relative purchasing power calculated by: https://www.measuringworth.com/calculators/ukcompare/r elativevalue.php; calculated April 21 2016. 10 3 work of the Association reveal the existence of a demand and a market for architectural photography? For the exhibition, I chose twelve architectural photographs from the Museum’s collection, eight of which are copies of prints that were included in the exhibitions of the Architectural Photographic Association between 1858 and 1861. The remaining four photographs were exhibited at either the Société Française de Photographie, the Photographic Society of London, or the Glasgow Photographic Society during that same time. My thesis consists of an outline for an exhibition proposal titled Focus: Architecture in Photographic Exhibitions 1858–1861 and is structured into two parts. The first part of my thesis includes practical considerations from the start to the finish of the exhibition process, all of which can be done in-house at the Museum. The tentative exhibition date falls out of the timeline of this thesis, so I will describe everything short of physically mounting the display. The second part features an essay analyzing the photographs that were exhibited at the Architectural Photographic Association and investigating how the exhibitions were a catalyst for the market of architectural photography between 1858 and 1861. 4 Literature Survey My literature survey consists of three main components: photographic collection management and exhibition preparation, the history of exhibitions by photographic societies from 1858 to 1861, and the role of architectural photography in the nineteenth-century. The first section contains the practical literature which I consulted in creating the exhibition, in order to ensure the photographs were handled safely and that public access to these photographs would continue beyond the exhibition. Secondly, scholarship on the exhibitions of photographic societies from 1858 to 1861 gives necessary context into the history of photographic exhibitions and insight into the unique model of the Architectural Photographic Society. Finally, a gap exists between scholarship about architectural photography and that of the photographic market: establishing the context for the creation and uses of these images, but failing to discuss their dissemination. Photographic Care and Collections Management Resources on photographic care and museum management lay the foundation for all of the work done in preparation for the exhibition, specifically with regard to cataloguing for wall labels and writing preservation considerations. Integral to any exhibition, beyond preservation and management, is visitor interpretation; therefore, the section closes with a discussion of one comprehensive text on how best to plan exhibitions with the visitor experience in mind. Rebecca A. Buck and Jean Allman Gilmore run their own artifact and museum management firm, Buck & Gilmore LLC. Their textbook, MRM5: Museum Registration 5 Methods14 was published in 2010 by the American Library Association, and proposes a thorough introduction to every aspect of art and artifact registration in a museum environment. Since it is not focused on photography, I treated the sections on “In House Exhibitions” and “Digital Asset Management” as reference texts for general best practices. After this broad approach, I consulted specific texts regarding the care, management, and exhibition of photographs. The publication by founding curator of the Image Permanence Institute, James M. Reilly, titled Care and Identification of Nineteenth Century Photographic Prints15 provides useful information about how best to treat nineteenth-century photographs, including specific matting and framing suggestions, and how to provide safe, long-term access through digitization. In order to gain necessary techniques on cataloguing, digitizing, and exhibiting the photographs, I consulted Bertrand Lavédrine’s 2009 book titled Photographs of the Past: Process and Preservation.16 Lavédrine draws from his experience as director of the Centre de Recherche sur la Conservation des Collections (CRCC) and conservation science professor at the Muséum National d'Histoire Naturelle in Paris to give recommendations on best practices for matting and framing, exhibiting, digitizing, and cataloguing photographs. Finally, I cross-referenced the above information with a series of PDF documents published by the Library of Congress, found online in their “Cataloguing and Digitizing Toolbox.”17 These tip sheets are constantly being updated, and referencing them ensures that the information found in the aforementioned, older publications is still relevant. 14 Rebecca A. Buck and Jean Allman Gilmore, MRM5: Museum Registration Methods, 5th ed., American Association of Museums: AAM Press, 2010. 15 James M. Reilly, Care and Identification of 19th Century Photographic Prints, Rochester: Eastman Kodak Company, 1986. 16 Bertrand Lavédrine, Photographs of the Past: Process and Preservation, Los Angeles: Getty Conservation Institute, 2009. 17 The Library of Congress, "Cataloging & Digitizing Toolbox," Prints and Photographs Reading Room, Library of Congress, October 22, 2010, http://www.loc.gov/rr/print/cataloging.html. 6 I also consulted manuals regarding writing exhibition labels, such as the 2015 republication of renowned museum consultant Beverly Serrell’s Exhibit Labels: An Interpretive Approach.18 This manual for preparing an exhibition gives guidance on writing and designing visitor-friendly exhibition spaces, including considerations for different types of learning styles, and remedies for accessibility concerns. Her advice on word count when writing different types of labels, including exhibition titles, introductory labels, group labels, and caption labels guided my editing process.19 Serrell places a large emphasis on logical flow throughout the exhibition design process, which helps visitors identify the objectives of the exhibition more clearly. These texts are outstanding resources out of a vast selection available to guide curators and collections managers to assist with the process of physically preparing exhibitions and ensuring photographs are safely stored and displayed. The basis of all interpretive text is thorough, accurate cataloguing, for without proper management, storage, and care, photographs would not survive the harsh conditions of exhibition. Exhibitions of Photographic Societies from 1858 to 1861 The foundation of my essay, cataloguing, and wall labels involved diligent research about the photographic exhibitions of 1858 to 1861, in order to ensure that proper historical context was maintained. A study about the Architectural Photographic Association is paired with writings surrounding the work of the Photographic Society of London or the Société Française de Photographie, in order to provide a cultural context for the exhibitions during this time period. Most surviving primary sources on the foundation of the Architectural Photographic Association are managed by the Royal Institute of British Architects (RIBA), held at the British 18 19 Beverly Serrell, Exhibit Labels: An Interpretive Approach, 2nd ed. Maryland: Rowman & Littlefield, 2015. Serrell, 33–36. 7 Architectural Library in London. Robert Elwall was the founding curator of photographs at RIBA. His 1985 article titled “The Foe-to-Graphic Art: The Rise and Fall of the Architectural Photographic Association”20, published in The Photographic Collector, is the only existing historical analysis of the Architectural Photographic Association. Elwall produced a thorough account of the rise and subsequent downfall of the Association, discussing the events surrounding the institution’s creation, exhibitions, and dissolution. It is from this article that I learned about the methods by which the Association was collecting and exhibiting prints, the fact that architects had decisive authority, and the specific details regarding each of the four exhibitions. What was particularly important to me was Elwall’s conclusion, in which he states that the Association fell apart by 1861 because the demand on the market exceeded the means of the Association. The Association was a commercial success in the limited time it existed, proving that there was a demand for architectural photography in the market of the 1850s to 1860s. This led me to investigate whether or not other photographic societies shared the same mandates or successes. The work of scholars such as Roger Taylor and Paul-Louis Roubert reveals that photographic societies were generating income in a much different way from the Architectural Photographic Association. Roubert, associate professor in the history of photography and the current president of the Société Française de Photographie, has published several articles about exhibitions in Études Photographique. In his “1859, Exposer La Photographie”21 published in 2000, Roubert explained that the Société Française de Photographie was run according to a system akin to that of Parisian Salons: only the work selected by a jury would be presented. 20 Robert Elwall, ""The Foe-to-Graphic Art": The Rise and Fall of the Architectural Photographic Association," The Photographic Collector 5.2 (1985): 142–56. 21 Paul-Louis Roubert, "1859, Exposer La Photographie," Études Photographiques 8 (November 2000): 4–21. 8 While the exhibitions featured a wide range of subject matter, jurors favoured portraiture and art reproductions. Taylor’s 2002 publication Photographs Exhibited in Britain, 1839–1865: A Compendium of Photographers and Their Works22 reveals similar preferences. Taylor’s book is a compilation of exhibition catalogues from British photographic exhibitions. Unlike Roubert, Taylor does not analyze the contents or intentions of the exhibitions; he simply lists the exhibition date, name, and location, and the title and maker of each photograph included. Taylor’s book is a useful tool for comparing exhibitions to one another, as it lists the maker and exhibition title for each photograph exhibited in Britain between 1858 and 1861. Scholars writing about the exhibitions of photographic societies do not discuss that these exhibitions also served to help photographers sell prints. This scholarship was useful when contextualizing the Architectural Photographic Association’s commercial, utilitarian approach to exhibition, and highlighted ways in which it differentiated them from other photographic societies. The Role of Architectural Photographs in the Nineteenth-Century The final section of this literature survey provides context regarding the utilitarian role, political goals, and commerce of architectural photography in the 1850s and 1860s. Scholars like Claude Baillargeon and Donald English identify architectural photography as a promotion of the enterprises of political entities, while scholars like Franca Malservisi, Maria Rosaria Vitale and Christine M. Boyer discuss architectural photography as a tool for cultural conservators, artists, and architects. Photography and art historian Anne McCauley addresses and studies the distribution and sale of photographs at this time. 22 Roger Taylor, Photographs Exhibited in Britain, 1839–1865: A Compendium of Photographers and Their Works, Ottawa: National Gallery of Canada, Library and Archives, 2002. 9 The first major role of architectural photography to be recognized throughout the 1850s and onward is in the political undertakings of city and country leaders. Claude Baillargeon, associate professor of art history at Oakland University, focuses on how photographs of the construction of French monuments like the Sacré-Cœur Basilica in the nineteenth-century were a way for the nation to demonstrate its power and prowess. His article “Construction Photography in the Service of International Public Relations: The French Connections” was published as a chapter in the 2013 Ashgate publication Nineteenth-Century Photographs and Architecture: Documenting History, Charting Progress, and Exploring the World, edited by Micheline Nilsen.23 The book itself is also politically charged, exploring how architectural photography aided in political ventures of industrialization, colonization, and national hubris. Many other scholars approach architectural photography from a political standpoint, but most relevant to this study is American scholar Donald E. English’s Political Uses of Photography in the Third French Republic, 1871–1914.24 This text focuses on how photography evolved into a medium capable of reaching the masses, which was then manipulated by the political Right. He identifies the direction which the photographic market of the 1860s was heading towards, making it an important tool when contextualizing the distribution of photographs from 1858 to 1861. Beyond political interpretations, the role of architectural photography has been long established as utilitarian, as it was valued for its efficiency and detail. Christine M. Boyer, professor and urban historian at the Princeton University School of Architecture, argues that the photographs of the Mission Héliographique of 1851 were crucial to establishing a collective 23 Claude Baillargeon, “Construction Photography in the Service of International Public Relations: The French Connections,” in Nineteenth-Century Photographs and Architecture: Documenting History, Charting Progress, and Exploring the World, ed. Micheline Nilsen, Surrey, UK: Ashgate, 2013, 139–53. 24 Donald E. English, Political Uses of Photography in the Third French Republic, 1871–1914, Ann Arbor, MI: UMI Research Press, 1984. 10 memory and building nationhood in France.25 This study presents the motivation behind architectural photographs, arguing that they were intended for preservation and restoration projects. The photographs of the Mission were never distributed, and remain in an archive to this day. French architectural historian Franca Malservisi and Spanish architectural historian Maria Rosaria Vitale presented a study in 2013 on the restoration of the Gros-Horloge in Rouen, arguing that commissioning photographers to document the process was instrumental in establishing the role of architectural photography in cultural restorations and in preserving cultural heritage.26 Their analysis shows how professionals such as conservators and architects used photography to aid in restoration, yet does not discuss whether or not these photographs were disseminated. Both of these cases demonstrate how scholars approach architectural photography, placing historical significance in the creation of the photographs and their use, but not their distribution. The leading resource on the distribution of photography in the 1850s and 1860s was written by Elizabeth Anne McCauley, professor of the history of photography and modern art at Princeton University. Industrial Madness: Commercial Photography in Paris, 1848–187127 is a comprehensive review of the photographic market in France. McCauley argues that the demand at the time was for portraiture and art reproductions, in the form of cartes-des-visites and cased images. She contextualizes the rise of the photographic market within the Second Empire, establishing the role of the commercial photographer as aspiring bourgeois, seller of bohemia, or 25 Christine M. Boyer, "La Mission Héliographique: Architectural Photography, Collective Memory and the Patrimony of France, 1851," In Picturing Place: Photography and the Geographical Imagination, edited by Joan M. Schwartz and James R. Ryan, London: I.B. Tauris, 2003: 21–54. 26 Franca Malservisi and Maria Rosaria Vitale, "Photography versus the Historical Record: The Role of Photography in Rouen's Gros-Horloge Restoration," Future Anterior 10 no.2 (2013): 49–65. 27 Elizabeth Anne McCauley, Industrial Madness: Commercial Photography in Paris, 1848–1871. New Haven: Yale UP, 1994. 11 art copyist. She intentionally omits architectural photography from her book,28 claiming that other scholars already focus on the individual studios, businesses, and careers of major architectural photographers of the time like Édouard Baldus29 or Roger Fenton.30 The literature analyzed in these three sections gave me the necessary background to address my thesis subject. First, the practical literature lays groundwork for appropriate and safe treatment of photographs, thorough cataloguing, safe and optimal digitizing, and proper interpretation. Second, literature concerning the photographic exhibitions of other societies proves that the Architectural Photographic Association has yet to be situated within the discourse of the history of photography. Finally, most scholars of architectural photography focus on its creation, use, or political aspects, yet omit a discussion of how the images were distributed and sold; meanwhile, scholars studying the market of photography do not include architectural photography in their discussions. This thesis will demonstrate how the Architectural Photographic Association balances the creation and distribution of architectural photographs, and how it differs from other photographic societies of the time. 28 Ibid., 8. See: Malcolm R. Daniel and Barry Bergdoll, The Photographs of Édouard Baldus, New York: Metropolitan Museum of Art, 1994. 30 See: Gordon Baldwin, Malcolm R. Daniel, and Sarah Greenough, All the Mighty World: The Photographs of Roger Fenton, 1852–1860, New Haven: Yale University Press, 2004. 29 12 Part One: Preparing an Exhibition Curatorial Statement Focus: Architecture in Photographic Exhibitions 1858–1861 explores the role that exhibitions of photographic societies played in the distribution and sale of architectural photography in the late 1850s to early 1860s. The Great Exhibition of 1851 in London, held at the Crystal Palace, was much of the general public’s first exposure to photographs. By the mid1850s, photographic exhibitions became more common throughout Europe, particularly in Britain and France, and are intrinsically linked with the rise of photographic societies. The Photographic Society of London, now the Royal Photographic Society, was founded in 1853, and the Société Française de Photographie was formed in Paris a year later. The focus of this exhibition, the Architectural Photographic Society, was founded in 1858. This exhibition features photographs shown at the major exhibitions of photographic societies in France and Britain, to illustrate the significance of the unique business model of the Architectural Photographic Association. Photographs exhibited at societies like the Société Française de Photographie and the Photographic Society of London are paired with those exhibited at the Architectural Photographic Association, in order to demonstrate how each of these societies responded to the demand for architectural subjects in the European photography market. This exhibition is the culmination of a Master of Arts thesis in Film and Photography Preservation and Collections Management by Natalie Banaszak of Ryerson University in Toronto, Canada. Drawn from the collections of the Museum of Fine Arts, Houston, these photographs give a glimpse into a time period where the relationship between photography and architecture was still in its infancy. The photographs vary in subject matter, place depicted, and 13 modes of representation, demonstrating that there was no prescribed way to photograph architecture. Some works were commissioned by governments and political powers; while some were made to be sold to everyone from tradesmen, like architects or artists, to tourists, whether they had the means to actually travel or just dreamed of seeing distant cities. Preparing an Exhibition: Timeline I worked with a timeline of approximately six months, specifically from January 11th to June 30th 2016, coinciding with the dates of my placement at the Museum of Fine Arts, Houston. By January 30th: ● Survey architectural photographs, to establish the strengths and weaknesses of the collection By February 29th: ● Define exhibition ambition ● Begin background research ● Receive approval from department head (Malcolm Daniel, Gus and Lyndall Wortham Curator of Photography) By March 30th: ● Obtain contact information for representatives in the conservation department and the matting and framing department ● Finalize checklist, inform collections manager and department head of choices ● Verify cataloguing information in the database and cross-check with research ● Write curatorial statement By mid-April: ● Finish digitizing the photographs ● Complete matting and framing recommendations, including: colour of mat, size of mat, size of frame, colour of frame 14 By April 30th: ● Design brochure to accompany the exhibition By mid-May: ● Complete wall labels, including the introductory panel, section texts, and object labels ● Enter label text into the database By May 30th: ● Review all curatorial writing ● Complete wall elevations June 30th: ● Final day to complete any outstanding work Checklist and Rationale As per the aforementioned timeline, the first step was to survey the collection of nineteenth-century architectural photography in order to establish its strengths and weaknesses. The Museum of Fine Arts, Houston, has over thirty thousand photographs, making it the museum’s largest collection. Searching the database for photographs dated before 1861 yields 2401 results. 1,500 are by the Fratelli Alinari, in the form of twenty albums donated by the estate of Mary Lynch Kurtz in the year 2000. Aside from the Alinari albums, there remain 875 photographs created before the year 1861. 284 of these are architectural photographs, and 181 of these came into the museum through the acquisition of Manfred Heiting’s personal collection between 2002 and 2004. The Heiting encyclopedic collection is made up of over 4000 photographs that he collected over a span of thirty years. Most photographs chosen for this exhibition come from the Manfred Heiting Collection. Upon completing the survey, I made a preliminary selection of photographs which included work by photographers that exhibited their work in Britain or France from 1858 to 15 1861. I chose this particular time period as it is contemporaneous with the existence of the Architectural Photographic Association. Of this list of over one hundred images, I flagged the photographs that were featured in the exhibitions of the Architectural Photographic Association, the Société Française de Photographie, the Photographic Society of Glasgow, and the Photographic Society of London. My goal was to allocate twelve to fifteen prints in total, a majority of them copies of prints exhibited at the Architectural Photographic Society. I ended up with a final checklist of twelve photographs, eight of which were displayed at the Architectural Photographic Association, while the other four were featured at exhibitions of other photographic societies at the time. This set of photographs was chosen in order to visually present the scope of the Association’s work, and to contextualize it within the models established by other photographic exhibitions and societies. The full checklist with images, in alphabetical order by photographer’s last name, follows. 16 List of Photographs Figure 1: Fratelli Alinari, Lorenzo Ghiberti’s Bronze Door, 1852–1858, albumen silver print from glass negative, 42.8 x 31.7 cm, The Museum of Fine Arts, Houston, 2004.211. Figure 2: Fratelli Alinari, Campanile for Duomo of Santa Maria del Fiore, Florence, 1852–1858, albumen silver print from glass negative, 44.4 x 29.5 cm, The Museum of Fine Arts, Houston, 2004.212. 17 Figure 3: Édouard Baldus, Pavillon Denon, Louvre, 1855–1856, salted paper print from glass negative, 44 x 33.8 cm, The Museum of Fine Arts, Houston, 2004.249. Figure 4: Bisson Frères, Moissac Abbey Cloister, 1857, albumen silver print from glass negative, 35.6 x 45 cm, The Museum of Fine Arts, Houston, 2004.313. 18 Figure 5: Charles Clifford, [Main Door, Salmanca Cathedral], before 1858, albumen silver print from glass negative, 43 x 32.4 cm, The Museum of Fine Arts, Houston, 2004.350. Figure 6: Charles Clifford, Salamanca. Puerta del Convento de las Duenas, 1853, albumen silver print from glass negative, 39.7 x 27.3 cm, The Museum of Fine Arts, Houston, 2004.351. 19 Figure 7: Tommaso Cuccioni, View of the Roman Forum towards the Capitoline Hill, Looking West, 1855, albumen silver print from glass negative, 27.3 x 38.1 cm, The Museum of Fine Arts, Houston, 2004.369. Figure 8: Roger Fenton, printed by Francis Frith & Co., Lichfield Cathedral, Central Doorway, West Porch, 1858 printed 1863–1865, albumen silver print from glass negative, 41.9 x 34.9 cm, The Museum of Fine Arts, Houston, 2014.89. 20 Figure 9: Francis Frith & Co., Columns, Pharaoh's Bed, 1856–1861, albumen silver print from glass negative, 20.4 x 16.4 cm, The Museum of Fine Arts, Houston, 88.233. Figure 10: Robert Macpherson, View of Rome from the French Academy, Monte Pincio, 1851–1858, albumen silver print from glass negative, 20.4 x 16.4 cm, The Museum of Fine Arts, Houston, 2004.222. 21 Figure 11: Carlo Naya, Piazzetta of San Marco looking towards the Island of St. George, 1857–1859, albumen silver print from glass negative, 42.4 x 53.5 cm, The Museum of Fine Arts, Houston, 2004.619. Figure 12: Robertson and Beato, Tophane Fountain, 1854–1858, albumen silver print from glass negative, 26.5 x 31.2 cm, The Museum of Fine Arts, Houston, 2000.255. 22 Cataloguing and preparing wall labels Research was a necessary precursive step to completing wall labels for the exhibition, in order to ensure cataloguing information was correct. The Fratelli Alinari photographs only needed a change to the constituent record. Before the change, all Alinari photographs had three constituents: Giuseppe Alinari, Romualdo Alinari, and Leopoldo Alinari. This approach did not leave room for their corporate name, Fratelli Alinari, and made searching more difficult. One had to assume that searching for any Alinari brother would yield all of the Alinari records. After consulting with data standards specialist Merrianne Timko, collections manager Jason Dibley, and photography cataloguer Selina Lamberti, we decided to change all of the Alinari records to have a primary display constituent of Fratelli Alinari, as this more accurately reflects the shared attribution between all three brothers. Most of the database records had accurate titles, credit lines, and dates. There were, however, two records that needed particular attention: 2004.350, or Charles Clifford’s [Main Door, Salamanca Cathedral]; and 2000.255, and Robertson and Beato’s Tophane Fountain. The Clifford record was easy to fix, I replaced an unclear title with a concise, descriptive one. The previous title read “Salamanca, Nord Thor, Gallerie Sud,” which was based on a modern pencil inscription on the mount. Searching for that title in other collections like the Metropolitan Museum of Art, the J. Paul Getty Museum, and the Victoria and Albert Museum yielded no results, and the translation did not correspond to what was depicted in the photograph. Museum policy dictates that it is better to have an accurate, descriptive title as the primary display than an inaccurately attributed title, so I moved the old title lower in the hierarchy and removed it from display. 23 The virtual record for Robertson and Beato’s Tophane Fountain demanded more attention. First of all, the photograph was incorrectly attributed to the Fratelli Alinari, despite an inscription in the negative bearing the signature “Robertson & Beato photog.” Previous cataloguers had omitted this inscription, which I added into the appropriate field. I first deleted the Fratelli Alinari from the record, and added James Robertson and Felice Beato, after consulting sources to be certain it was Felice and not Antonio Beato working with James Robertson at the time. Most scholars believe that James Robertson worked with Felice Beato under the corporate body name of Robertson & Beato, and that the corporate body Robertson & Beato & Co. was used when Antonio Beato joined them.31 Besides the constituent problems, this record also had a date of c.1860 attributed. I can say for certain that it is taken before 1858, as it was exhibited at the Architectural Photographic Association that year under the name “Constantinople: Fountain at Tophanne.”32 Robertson only opened his studio in Constantinople in 1854 or 1855.33 By 1857, Robertson and Beato departed for a Grand Tour of Jerusalem, and by 1858, Beato was spending the majority of his time in India.34 Therefore, a likely date range for the photograph falls between 1854 and 1857. Museum policy is to indicate this range as a display date, with an en dash (–) in between, like so: “1854–1857.” The start and end dates then have to be accordingly assigned as 1854 and 1857. The final issue to resolve was the title of the photograph. The inscription in the negative reads “La fontaine de Xophinna”, so this was the only title on the record before I catalogued it. Instead, I assigned the English language 31 Anne Lacoste, Felice Beato: A Photographer on the Eastern Road, Los Angeles: J. Paul Getty Museum, 2010: 184. 32 Taylor, 240. 33 Richard Pare, Photography and Architecture, 1839–1939, Montreal: Canadian Centre for Architecture, 1982: 245. 34 Lacoste, 184. 24 translation, Tophane Fountain, as the primary title. This record was the most involved cataloguing I had to do for the exhibition. The first step in writing label text was to identify my exhibition’s “big idea,”35 to which all labels would refer back to. Beverly Serrell indicates that big ideas “define or describe the content of the exhibition,”36 and “provide a thread of meaning, coherence, and weight.”37 Though the big idea can take the form of different sentence structures, mine became the following question: where could architects obtain photographs in the 1850s and 1860s, and how did they use them? After I wrote my big idea I shifted focus to the wall labels, for which I followed Serrell’s “Ten Commandments:”38 “1. Labels should begin with concrete, visual references to the objects they interpret to bring them to life. 2. Labels should relate to the big idea of the exhibit, not ramble without focus or objectives or contain sub-sub-subtopics. 3. Labels should emphasize interpretation (offering provocation) over instruction (presenting information). 4. Labels should know their audience and address visitors’ prior knowledge, interests, and/or misconceptions. 5. Labels that ask questions should be visitors’ questions 6. Label design should reflect the label’s content or context and have a recognizable system of organization of label types. 35 Beverly Serrell, Exhibit Labels: An Interpretive Approach, second ed., Lanham: Rowman & Littlefield, 2015: 7. Serrell, 9. 37 Ibid., 12. 38 Ibid., 2. 36 25 7. Labels should be written with a vocabulary that is within reach of the majority of visitors. 8. Labels should be short and concise, more like a tweet than a tome. 9. Labels for interactives should have instructions or interpretations integrated in logical, intuitive ways. 10. The typography (typefaces, sizes, design, colors, lighting, materials, and placement) should make them legible and easy on the eyes, not busy or distracting.”39 The completed wall labels can be seen in Appendix A. Preservation considerations When works are selected for exhibition at the Museum of Fine Arts, Houston, they are sent to the conservation department before framing. The conservators complete a condition report on all of the photographs, and, upon establishing that the photographs are safe for exhibition, execute any necessary treatment. With nineteenth-century material, this usually involves a surface clean or the painting-over of a blemish. These particular photographs were all in stable condition for exhibition. The completed condition reports and specific recommendations for each photograph can be found in Appendix B, while this section will focus on general guidelines. Calculating the estimated lux-hours is important with any exhibition in order to ensure the photographs are not under duress. Bertrand Lavédrine recommends a maximum light dose of 12000 lux-hours per year for nineteenth-century photographs, identifying them as highly 39 Serrell, 2–3. 26 sensitive to light.40 The Conservation Centre for Art and Historical Artifacts in Philadelphia, however, labels albumen and salted paper prints as only moderately sensitive to light, and recommends a much higher maximum light dose of 50,000 lux-hours per year.41 The formula they prescribe to calculate total lux-hours is: the average lux, multiplied by the exhibition hours per day, multiplied by the number of days in exhibition.42 The Museum is open for an average of 8.33 hours a day.43 I proposed a four-month exhibition for these photographs, or 108 days. This total excludes every Monday, since the Museum is closed and the exhibition lights stay off. Therefore, I calculated that the average lux for each photograph should not exceed 55.57, for a four-month exhibition display, at 8.33 hours per day. This will yield 50,000 lux-hours per year, falling within the exposure range recommended by the Conservation Centre for Art and Historical Artifacts in Philadelphia. Finally, dark time for the photographs should ideally be three times longer than the exhibition, so I recommended a twelve-month rest period for these objects. Matting and Framing After taking necessary precautions to maintain a safe gallery space for the photographs, I ensured to do the same with each object's microenvironment. This section features matting and framing suggestions for each photograph, including a description of the current housing situation. I examined each photograph, and proposed an appropriate mat size and colour for each. 40 Bertrand Lavédrine, Photographs of the Past: Process and Preservation, Los Angeles: Getty Conservation Institute, 2009: 297. 41 Sarah Wagner, Connie McCabe, and Barbara Lemmen, Guidelines for Exhibition Light Levels Fir Photographic Materials, Report, May 2007, Accessed April 20, 2016, http://www.ccaha.org/publications/paper: 3. 42 The Conservation Centre for Art and Historical Artifacts, Light Exposure of Artifacts on Exhibition: CCAHA Guidelines, Web Publication, http://www.ccaha.org/uploads/media_items/light-exposure-of-artifacts-onexhibition.original.pdf 43 Calculated on 29 May 2016, based on the opening hours available at: http://www.mfah.org/visit/hours-andadmissions/. 27 In tandem with this, I selected frame sizes and colour. As with the condition reports, this section focuses on general recommendations for all of the photographs, while more detailed suggestions for each photograph can be found in Appendix C. All of the photographs are currently in a 4-ply mat, save for one: Roger Fenton’s Lichfield Cathedral, which has a 4-ply back and an 8-ply front. I recommend all of the photographs be re-matted to have 8-ply mats, as it provides the most support for the photographs. Though they are all mounted on a secondary support, a mat made completely out of 8-ply board will help prevent the photographs from warping, and separates the photographs from the glazing when framed.44 The photographs should all be mounted to the mat using folded photo corners, made out of conservation-quality, silversafe paper. The folded corners protect the object from damage due to harmful adhesives or residue,45 and can be easily opened to remove the object from the mat at any time, should anyone have to inspect the verso, digitize the photograph, or perform conservation treatments and exams. Frames for the photographs should be made out of wood, with an acrylic glazing. Glass glazing can cause problems since it is fragile, and heavy.46 I recommended an ultra-violet (UV) filtering acrylic for these photographs, as it is lighter, and serves to protect the objects from damaging UV radiation.47 The Museum of Fine Arts, Houston, uses Artique 8-ply, solid colour core, alpha-cellulose conservation-quality mat boards, available for purchase from Larson-Juhl. I recommended this mat board for all of the photographs. Depending on what suited the tonality of each object, I suggested one of two shades of a tan colour for the mat board. The first shade, ‘Thatch,’ is slightly lighter than the second shade, ‘Birch.’ I chose shades that are not too different from each 44 Jill Snyder and Maria Reidelbach. Caring for Your Art. New York, NY: Allworth Press, 2001: 59. Lavédrine, 298. 46 Ibid., 299. 47 Ibid. 45 28 other, to ensure that the photographs maintain an even hierarchy. I assigned “Thatch”, the lighter shade, to the following photographs: Charles Clifford’s [Main Door, Salamanca Cathedral], the Robertson and Beato, the Édouard Baldus, Fratelli Alinari’s Campanile for Duomo of Santa Maria del Fiore, Florence, the Tomasso Cuccioni, the Roger Fenton, and the Carlo Naya. I recommended “Birch” for the remaining photographs: Charles Clifford’s Puerto del Convento de las Duenas, Fratelli Alinari’s [Ghiberti’s Baptistery Doors, Florence], the Francis Frith, the Bisson Frères, and the Robert Macpherson. Wall Elevations and Layout The exhibition wall is 13.7 meters (45 feet) long and 2.9 meters (9.5 feet) tall. The photographs are to be hung with their center point at 152.4 centimeters (60 inches) from the floor, as this matches and complements the History of Photography design. Once I became familiar with the gallery space, I created a vector drawing set of the space in a complementary program, Inkscape. The elevation drawings, which include each photograph in its frame and corresponding wall label, are attached as Appendix D. The drawing allowed me to visually confirm there was an adequate amount of space between the photographs and permanent wall features, such as doors, signs, and electrical outlets, so the exhibition could be mounted with ease. Preparing an exhibition pamphlet Much of the Manfred Heiting Collection was photographed when it came into the Museum’s collection in 2004. However, a large portion of the photography done between 2000 and 2004 has a very skewed white balance. Some appear overwhelmingly orange or yellow in 29 tone, while others have a green or blue hue. Eight out of the twelve photographs in my exhibition had this problem, and I needed to photograph them again before including them in my thesis and in the exhibition pamphlet. The following section describes how I digitized these photographs. Figure 13: Digital image taken around 2004 for Tommaso Cuccioni, View of the Roman Forum towards the Capitoline Hill, Looking West, 1855, albumen silver print from glass negative, 27.3 x 38.1 cm, The Museum of Fine Arts, Houston, 2004.369. First, I transported the photographs from their home vault to an interim art storage with a large copy stand that is always set up for digitization. Handling the photographs is easier and safer with two people, so I sought help from Photography Collections Cataloguer Selina Lamberti. The copy stand consists of a Nikon D100 camera on a “Super Repro” copy stand made by an Italian manufacturer, Industria Fototecnica Firenze. Attached are two lights, powered by Hikari Halogen JDD bulbs, at a 45 degree angle towards the centre of the copy stand. Bertrand Lavédrine identifies the preservation of digital files to be a problem, since technology is changing so rapidly.48 The Nikon D100 has the capability to shoot in the Tag Image File Format 48 Lavédrine, 310. 30 (TIFF), which are useful to keep in case the edited images, in JPG format, should ever become obsolete or corrupt in the process of conversion. To begin, I placed a fresh piece of white mat board onto the copy stand, to guarantee a clean surface for the photographs. Next, I placed the photographs one by one on the mat board. I lay a colourchecker underneath all of the photographs, ensuring that the tonality could be corrected in Adobe Photoshop when processing the digital files. I also included a label underneath each photograph, with the accession number and title of the photograph. Doing this made naming the files and distinguishing nondescript versos of the photographs much simpler. For each photograph, I set the camera settings according to the Museum standards, which is an aperture of f/9.5 and a focal length of 35mm. Initially, the photographs were either out of focus or blurry, because the camera was at the top of the copy stand and vibrating when I pressed the shutter. I set the camera to a ten second self-timer, which reduced the shake. After the photographs were digitized, I edited them in Photoshop. For the most part, the white balance was correct and the colours did not need manipulating. In cases where the tonality had shifted, I used the colourchecker to set the white balance and correct the digital file. I also straightened and cropped all of the images, but did not make any modifications beyond that, as per Lavédrine’s recommendations to keep all photographs as unedited as possible.49 Finally, I saved two versions of each file: one version cropped to the photograph, and one with the secondary support included. I uploaded three versions to The Museum System (TMS), the aforementioned two versions of the recto, and one photograph of the verso. I then selected the two images of the recto as ‘approved for web’, to appear on the Online Collection Module, which will reduce the need for handling the physical object. 49 Lavédrine, 306. 31 Digitizing the prints was an essential first component in the design of the brochure. I chose the form of a brochure for a publication to accompany the exhibition due to its short and concise, yet informative and visually appealing format. The brochure features the curatorial statement, information on the Architectural Photographic Association, and reproductions of all of the images included in the exhibition. Just as in the text panels and label text, I followed Museum standards and chose the EB Garamond font. I selected colours that were complementary to the photographs themselves. The text for the brochure is a condensed version of Part Two of this thesis, and a template I drafted for the brochure is attached as Appendix E. 32 Part Two: The Exhibitions of the Architectural Photographic Association (1858–1861) The Architectural Photographic Association was formed in May of 1857,50 and officially inaugurated at a meeting on the night of Thursday, January 7th 1858.51 The president of the Association was Charles Robert Cockerell (1788–1863), British architect and professor of architecture at the Royal Academy in London from 1839 to 1859.52 The architects involved with the Association travelled and purchased prints from photographers’ studios at the destinations of these travels. They assembled these prints into an exhibition in London, where a subscription fee of one guinea or upwards per annum would grant access for patrons to come and purchase copies of photographs that were on display. After the exhibitions, the Association would order the subscribers’ choices of prints from each photographer.53 At the inaugural meeting, Cockerell announced that the number of subscribers had already reached 750, and that the Association had managed to assemble 360 photographs from various European cities, with plans to soon expand to the Middle East and Asia.54 The organization saw immediate success upon its first exhibition, revealing a high demand for architectural photographs, and the opportunity for the existence of a market in 1858. The Association held four annual exhibitions from 1858 until its dissolution in 1861. This essay postulates that the growth, critical and public reception, and ultimate downfall of the Architectural Photographic Association all demonstrate the demand for photographs as source material for architects in the 1850s and 1860s. 50 “Société Photographique d’Architecture,” Bulletin de Société Français de Photographie (1858): 48. "Architectural Photographic Association," The Literary Gazette: Journal of Belles Lettres, Science, and Art, no. 2138 (January 9, 1858): 42. 52 See “C.R. Cockerell, R.A.,” Royal Academy of Arts, Collections Online: http://www.racollection.org.uk/ixbin/indexplus?_IXACTION_=file&_IXFILE_=templates/full/person.html&person =5580 53 The Literary Gazette, 43. 54 Ibid. 51 33 The Association’s First Exhibition: 1858 The first exhibition of the Architectural Photographic Association took place from January to February of 1858. To guarantee the sale of a certain number of prints by each photographer, subscribers could only choose one subject from each of the screens into which the exhibition was divided. This prevented subscribers from “assembling a portfolio of photographs documenting one building.”55 Photographers exhibiting in 1858 included Fratelli Alinari, Édouard Baldus, Francis Bedford, Bisson Frères, Charles Clifford, Roger Fenton, Robertson & Beato, Captain Inglefield, William Lyndon Smith, Robert Cade, Royal Engineers, John Wheeley Gough Gutch, and Reverend Joseph Lawson Sisson.56 This relatively small group of photographers nevertheless represents the broad geographical scope of the Association right from the outset: Charles Clifford was photographing Spain, Robertson and Beato in Constantinople, the Fratelli Alinari were in Italy, Baldus and the Bisson Frères were photographing French buildings and monuments, and Fenton and Bedford were in the English countryside. Amongst the 364 photographs featured in this exhibition were Robertson and Beato’s Tophane Fountain, Fratelli Alinari’s Campanile for Duomo of Santa Maria del Fiore, Florence, and Lorenzo Ghiberti's Bronze Door, Gates of Paradise, Baptistery of San Giovanni, Florence, Édouard Baldus’ Pavillon Denon, Louvre, and Charles Clifford’s Salamanca: Puerto del Convento de las Duenas and [Main Door, Salamanca Cathedral]. These photographs are all either albumen or salted paper prints from wet collodion glass plate negatives, a medium which was favoured for its sharpness — a result of the transparency of the glass. They are all large 55 Elwall, 146. Roger Taylor, Photographs Exhibited in Britain, 1839–1865: A Compendium of Photographers and Their Works. Ottawa: National Gallery of Canada, Library and Archives, 2002. 56 34 prints for the time, the smallest being Robertson & Beato’s, measuring 26.5 by 31.2 centimeters, while the rest are approximately 40 by 30 centimeters. The size of the photographs allows them to either depict an entire building, or enhance specific architectural details. For instance, Baldus’ Pavillon Denon, Louvre, Alinari’s Campanile, and Robertson and Beato’s Tophane Fountain portrayed entire buildings. Conversely, doors were a specific architectural detail that was the focus of Clifford’s [Main Door, Salamanca Cathedral], the Fratelli Alinari’s Lorenzo Ghiberti’s Bronze Door or Clifford’s Salamanca: Puerto del Convento de las Duenas. While the specific locations and subjects of these prints vary, they demonstrate the Association’s preference for larger images with sharp detail which were found useful by architects as source material. The notion of using photography as source material was still fairly new, developed chiefly by the 1851 Mission Héliographique. Anne de Mondenard argues that the Mission was the first joint public commission in the history of photography.57 The Mission commissioned five photographers to travel throughout France, creating photographs of a list of buildings and monuments. These photographs were instantly archived for use as visual documents for preservation and restoration, and ironically undiscovered until the late twentieth-century.58 Though the Mission created photographs, architects did not immediately view them as useful resources. By 1857, the representation of architecture through photography had become a more established tradition.59 Henry Cole, director of the South Kensington Museum in London, now the Victoria and Albert Museum, was very active in the 1857 to 1865 construction of the 57 Anne de Mondenard, La Mission Héliographique: Cinq Photographes Parcourent la France en 1851, Paris: Centre des Monuments Nationaux, 2002: 12. 58 de Mondenard, 12–13. 59 Martin Barnes and Christopher Whitehead, "The 'Suggestiveness' of Roman Architecture: Henry Cole and Pietro Dovizielli's Photographic Survey of 1859," Architectural History 41 (1998): 197. 35 Museum’s building and grounds.60 While visiting the Vatican Museum in 1858, Cole wrote: “the first step to be taken, to turn it to profitable account for [South] Kensington, would be to have the whole photographed: not the separate objects, but views on each side, in as many portions as necessary.”61 He wanted the Museum’s architect, Captain Fowke, to travel to Rome himself, but Fowke did not travel to Italy until 1863. Barnes and Whitehead argue that Cole’s failure to get Fowke to Italy added a sense of urgency to Cole’s collection of photographic records of the buildings in Rome.62 In 1859, Cole commissioned Italian photographer Pietro Dovizielli to take 49 photographs of buildings he was particularly fascinated with. Dovizielli’s photographs focused on architectural details with a “didactic and anti-picturesque bias.”63 While the Association’s first exhibition received excellent reviews and was deemed a success, certain architects complained about the subject matter of the photographs, claiming that there were not enough photographs of architectural details and sculptures. This was due to the fact that the Association was not commissioning photographs, but purchasing examples of prints which photographers had already taken. Unlike Henry Cole, the Association did not have the power to dictate what was photographed; the architects only chose which photographs to collect and how to best arrange them in the exhibitions. The photographs in the first exhibition of the Association demonstrate the varied approaches photographers took when creating visual records of buildings. There are no photographs of architectural details, save for the few views of doors and entryways. Though the photographs were executed with “minimal artistic input from the photographer,”64 as Cole preferred, the exhibition of the Architectural Photographic Association 60 Barnes and Whitehead, 193. Ibid., 196. 62 Ibid. 63 Ibid., 198. 64 Ibid., 197. 61 36 in 1858 would not have provided him with sufficient visual documentation to create the South Kensington plans, lacking crucial detail elements such as colonnades, steps, obelisks, and fountains.65 Despite the lack of specific subject matter, the Civil Engineers and Architect’s Journal praised the exhibition and photographs, emphasizing how useful this endeavour would be for architects.66 The utilitarian nature of the exhibitions was immediately recognized amongst architects and engineers, who constituted the predominant readership of this journal. Architects, who could not attend the exhibitions of the Association could read the reviews published in professional journals, such as the Civil Engineers and Architect’s Journal or Builder. This way, they could stay current with popular architectural sites, and become acquainted with the names of photographers should they decide to purchase prints of certain buildings for their own inspiration. News of the exhibition exceeded the professional realm, and journals which appealed to a more general public featured news of the Association. The Literary Gazette writes that “wide as will be the amount of information spread by this Association, and powerful as is the feeling of interest it inspires, these are nothing compared with the advantages that will be reaped from it by architectural students and professors.”67 This review was also translated into French and published in La Lumière a few weeks later,68 indicating that the Association’s first exhibition had a widespread impact. The review itself confirms public interest in the subject matter, while also outlining the photographs’ potential as valuable source material for architects. A hopeful sentiment for the Association’s future success was echoed by the reviews of its first annual 65 Ibid. “Architectural Photographic Association,” The Civil Engineers and Architect’s Journal, no. 21 (1858): 62. 67 Ibid. 68 "Association Photographique D'Architecture," La Lumière, no. 4 (January 23, 1858): 14. 66 37 exhibition, many of which were included not only in profession-specific journals but in popular weekly newspapers and journals as well.69 It was not uncommon for photographic journals and bulletins of the time to review international exhibitions, but the reviews never before placed a focus on architectural subject matter alone. The publication of the exhibition reviews in popular weeklies such as The Spectator suggests that the Association reached a wider audience than just architectural or photographic professionals. In reading reviews of the exhibitions of the Architectural Photographic Association, the general public could refine their affinity for the subject matter, and develop a discerning taste for architectural photographs. The reviews served as an invaluable network, and, even today, allow a glimpse into the production of major photographers from Europe and Asia. The 1859 Exhibition Despite criticisms and shortcomings, the Association decisively maintained the same model for its second exhibition, which took place from December 1858 to February 1859. It featured 383 photographs, including works by: Robert Macpherson, Messrs Cimetta, Arthur James Melhuish, Robert Cade, Robertson and Beato, Reverend Percy Lousada, Archibald Lewis Cocke, Édouard Baldus, Francis Frith, William Lyndon Smith, John Wheeley Gough Gutch, the Royal Engineers, Francis Bedford, and Carlo Ponti.70 Robert Macpherson exhibited his entire oeuvre of one hundred and twenty photographs of Rome, the largest body of work by a single photographer. In this group was his photograph titled The Garden-Front of the Villa Medici on the Pincian Hill. The foreground of this photograph is the fountain of the Villa Medici, while a panoramic view of Rome unfolds in the background. The photograph is neither focusing on the 69 70 See also “Architectural Photographic Association,” The Spectator, no. 1579 (October 2, 1858): 30–31. Taylor, 333. 38 Villa, which did not even make it into the frame, its garden, which is barely visible, nor the full panorama of Rome, which is shielded by trees. It is instead a marriage of the three in which Macpherson plays into the viewers’ imaginations by including architecture in the context of its surroundings, allowing them to situate themselves within the scene. This particular image marks a shift from the previous exhibition of the Association, as the previously mentioned photographs all showcase entire buildings or specific architectural components, including as little of the surroundings in the frame as possible. This photograph is a different type of source material for architects; it conveys the general mood and feel of the environment surrounding the Villa, not the structure itself. Again in this case, Cole would not have been satisfied with this image. He referenced the Villa in his journal, and wrote: “The garden front of the house is ascribed to M. Angelo. It has been arranged so as to receive bas reliefs of antiquity and they are very happily brought in. We should have a photograph of it and also of the Colonnade at the side. This is about 25 feet high and I think shows that that height would hardly be sufficient for our own Colonnade at Kensington.”71 Even though Macpherson’s framing leaves out all of these details, his entire body of work, which accounted for almost one third of the total photographs in the exhibition, was highly praised and hailed as the highlight of the exhibition.72 Also in 1859, the Société Française de Photographie held its third annual photographic exhibition in what is now the Grand Palais73 in Paris. There are differences between the two exhibitions which help provide context for the workings of the Architectural Photographic Association. The Association was the only organization that had a specific focus on architectural 71 Barnes and Whitehead, 196. Elwall, 149. 73 The exhibition catalogue refers to the location as the southwest pavilion of the Palais de Champs-Élysees. This was actually the Palais d’Industrie, an exhibition space built for the 1855 Paris World Fair, and demolished in 1897 to make way for the Grand Palais, erected in 1900. 72 39 photography. Exhibitions of other photographic societies, like the Société Française de Photographie or the Photographic Society of London, did not place restrictions on subject matter. However, research shows that they both predominantly featured art reproductions, portraiture, and landscapes.74 The Association was filling a void in terms of the availability of architectural photography. Gustave Le Gray only exhibited a single photograph of architecture in 1859 at the Société Française de Photographie. In contrast, and reverting to the previous example, Robert Macpherson had one hundred and twenty photographs from Rome in the Architectural Photographic Association’s 1859 exhibition. Architects looking to draw source material had much more to choose from at the Association. In addition, the Société Française de Photographie had a jury system in place in order to decide which photographs would be on display.75 In that way, it was run akin to Parisian Salons, thereby attributing the photographs with a status of art objects. Only what were defined as the best examples of photography would be considered. The jury was made up of a variety of professionals: photographers, including Hippolyte Bayard, Gustave Le Gray, and Louis-Rémy Robert, who was also the head of painting at the Sèvres porcelain manufactory (then called the Royal Porcelain Manufactory); theoretician Léon Foucault, then a physicien (physicist) at the Imperial Observatory; and deputy of the National Assembly Adolphe Moreau, to name a few. The heterogeneous jury displayed the society’s diverse interests and offered differing perspectives on photography. In contrast, the Architectural Photographic Association was run solely by professionals in the field of architecture, and the 74 This can be deduced from perusing the exhibition catalogues, either the SFP catalogue which can be found at ‘Société Française De Photographie, Catalogue De La Troisième Exposition De La Société Française De Photographie, Paris: Renou Et Maulde, 1859, doi:http://www.e-rara.ch/doi/10.3931/e-rara-15868,’ or the catalogue for the 1859 exhibition of the Photographic Society of London, found in Roger Taylor, Photographs Exhibited in Britain, 1839–1865: A Compendium of Photographers and Their Works. Ottawa: National Gallery of Canada, Library and Archives, 2002. 75 Société Française De Photographie, Catalogue De La Troisième Exposition De La Société Française De Photographie, Paris: Renou et Maulde, 1859. doi:http://www.e-rara.ch/doi/10.3931/e-rara-15868: 2. 40 chosen photographs were specifically intended to serve architects and please subscribers. As architects were recognizing the uses for these photographs, they validated the work of the Association. The priorities of the Association were simultaneously utilitarian and commercial: the aim was not only to show triumphs of photography as art, but to distribute these architectural photographs as visual records and profit from the subscription service. In all of these ways, the Architectural Photographic Association’s exhibition of 1859 was unprecedented and helped solidify the Association’s reputation amongst architects. The third and largest exhibition opened in 1860, and was made up of 510 photographs.76 Reviews indicate that architects were impressed with photography’s ability to capture details that escaped the eyes of draughtsmen or sketch artists.77 This exhibition is strangely excluded from Roger Taylor’s compendium of British photographic exhibition catalogues, making it difficult to access information regarding which photographers were featured and what types of photographs they included. The Association’s Downfall The year 1861 marked the height of the Association’s activities, and that year’s exhibition was also its last. Besides the exhibition itself, the Association held a series of lectures, during which the attendees debated the use of photography versus architectural drawings.78 This final exhibition was the Association’s most diverse moment, including works by: the Fratelli Alinari, Thomas Annan, Captain Austin, Édouard Baldus, Thomas J. Barnes, Francis Bedford, Bisson Frères, William Jr. Church, Archibald Lewis Cocke, Cundall & Downes, Captain 76 Taylor, 309. Elwall, 149. 78 Ibid 153. 77 41 Dawson, Philip Henry Delamotte, Captain Henry Dixon, Dolamore & Bullock, R. Eaton, Francis Charles Earl, Francis Frith, W. T. Frost, Captain Robert Gordon, John Wheeley Gough Gutch, Gustave Le Gray, Dr. William Despard Hemphill, Robert Macpherson, Arthur James Melhuish, W. J. C. Moens, James Mudd, Dr. John Murray, W. H. Nichols, Carlo Ponti, Rev. Joseph Lawson Sisson, Thomas Sutton, Stephen Thompson, and Thomas Tyley.79 These photographers represent the broad scope of the Association and its growth since 1858. Included in this exhibition was Francis Frith’s photograph from Egypt, Columns, Pharaoh’s Bed.80 The photographer also exhibited his photographs of Egypt at the Association’s second annual exhibition in 1859. Columns, Pharaoh’s Bed, Philae is smaller than the other photographs discussed in this essay, measuring at 20.4 centimeters tall by 16.4 centimeters wide. Despite its size, this photograph of the Greek-style temple’s columns is so sharp that names and years, carved into the stone columns, are visible to the naked eye. It embodies what both the Association and architects valued as useful: a sharp image depicting a particular architectural style or detail. Trouble began in 1859, after just two exhibitions. The Association could not distribute the photographs to subscribers fast enough, and the print quality was often inferior to the prints in the exhibitions. Critics also regarded the elaborate system for purchasing and distributing photographs as too commercial, saying: “photographs are put down at a price, so that a more commercial aspect is given to it than is desirable.”81A motion was put forth to dissolve the organization altogether but the Association was not disbanded, much to the relief of the Building News who argued that “without the APA… the supply of photographs to the architect would be 79 Taylor 309–310. Taylor 333. 81 Elwall, 146. 80 42 threatened — it would be unwise to rely on the output of amateurs who would probably forsake photography when the novelty had worn off.”82 Instead, members called for a change of leadership and elected Sir William Tite as president.83 Tite was a British architect, Member of Parliament from 1855 until his death in 1871, and president of the Royal Institute of British Architects from 1861 to 1863 and 1867 to 1870.84 Despite these changes, the Association was in deep trouble by 1861: the exhibition costs exceeded any profits. This time, the Association was dissolved, and the majority of its collection of prints sold in order to pay off debts.85 It was reconstituted once more, with an altered collection mandate to commission photographers, not architects, to travel abroad. The photographers captured negatives that the Association collected and printed from, sending proofs to subscribers each year. In this way, the Association and its photographers had more control over the subject matter and style. In 1862, photographers Cundall and Downes took thirty-six views of Wells Cathedral, and each subscriber was sent eleven of these. In 1863, the Association produced twenty-two photographs of French architecture, and journeyed again to France and to Germany in 1864, 1866, and 1868. According to Elwall, by 1868 the need for the Association diminished as photographs became cheaper and more widely available on the popular market.86 The Association’s ultimate demise was not due to its inability to adapt to the changing market. The fact that the Association was disbanded after four years indicates that its focus was not purely commercial, but instead on supplying photographs to architects. If the Association’s goals were purely commercial, its downfall would have been avoided at all costs. Like many 82 Elwall 149. Elwall, 148. 84 Campbell Dodgson, Dictionary of National Biography, 1885–1900. Vol. 56. London: Smith, Elder, 1898: 418. 85 Elwall, 154. 86 Ibid. 83 43 private, commercial, photographic studios, it would have sought investors to stay afloat. The financial troubles of the Association serve as a portrait of the climate of architectural photography on the commercial market, since it was overwhelmed by demand after just four years of exhibitions. The Association became a victim of its own success, ultimately thwarted by its inability to keep up with the popularity of its unique business model. Conclusion Many questions regarding the Architectural Photographic Association still remain. Who were the Association’s subscribers? How many subscriptions were sold to architects, and how many to members of the general public? Were there recreational enthusiasts of architectural photography, and did they subscribe? These questions would help identify how the Association evolved over time, and how it changed the role of architectural photography in society. This essay concludes that the Association acted as a catalyst for architectural photography, making it accessible to architects and the general public alike through exhibitions and their reviews. Its unique business model and initial success demonstrate the demand for architectural photography in the 1850s, while its brief history and downfall serve as an indication of how that demand rapidly evolved into a potential market that could not be served. The Association made the subject more accessible, and hosted discussions and debates on the benefits of photography as a draughtsman’s tool. The exhibitions of the Architectural Photographic Association validated architecture as a more popular subject matter in photography than described in the literature, presenting many of its uses by the end of the 1850s. The fact that the Association was led by architects, not photographers, distinguishes it from other societies and certainly helped to solidify its reputation. The widespread reviews of the Association, published in popular weeklies like The 44 Spectator and specialized photographic and architectural journals alike, demonstrate the immediate impact the Association had on the public. Finally, the downfall of the Association proves that not only was there a demand for architectural photography, but that it grew to exceed the means of the Association in just four years. Overall, this essay serves as a call to action for a shift in scholar’s emphasis from how photographs were created to how they were distributed. In writing future histories of architectural photography, the Architectural Photographic Association should not be ignored, deepening the current understanding of the photographic market from 1858 to 1861. 45 Appendix A: Wall Labels Francis Frith & Co. (British, founded 1859) Columns, Pharaoh's Bed 1856–1861 Albumen silver print from glass negative Museum purchase funded by Mr. and Mrs. Alexander McLanahan in honor of Edward B. Mayo 88.233 A copy of this photograph was on display at the Architectural Photographic Association’s largest and final exhibition in 1861. Critics of the Association’s exhibitions always commented on the lack of photographs of architectural details, and Frith certainly caters to this need with this particular image. The photograph is sharp, bright, and packed with details: even though it is the smallest in this exhibition, you can read the names and dates travellers carved into the columns with your naked eye. Francis Frith made his first of three tours to Egypt and Lebanon in 1856. He worked systematically to provide pictorial evidence of Biblical sites, publishing his work for a British Victorian audience upon his return. Frith’s photographs provide a sense of romantic meandering from one Middle Eastern site to another, without ever having to leave your armchair. James Robertson (British, 1813–1888) Felice Beato (British, born Italy, 1832–1909) Tophane Fountain 1854–1858 Albumen silver print from glass negative Gift of Mrs. Efrem Kurtz 2000.255 James Robertson opened a commercial photographic studio in Constantinople (now Istanbul) in 1854, and soon after, Felice Beato joined his practice. They signed their negatives with the corporate name “Robertson & Beato photog.,” as visible in the lower right of this photograph. Here, they depict the eighteenth-century Tophane Fountain, with a ladder leaning up against the side of the wall serving as a measuring tool for viewers to grasp the scale of the structure. They still capture some of the movement of the busy market square on the right side of the fountain, giving us a sense of the busy city functioning around this building. However, this does not interfere with the sharpness of the building details, a style which was favoured amongst 46 architects who found source material amongst the photographs exhibited by the Architectural Photographic Association in 1858. Fratelli Alinari (Italian, 1852–) Gates of Paradise, Baptistery of San Giovanni, Florence 1852–1858 Albumen silver print from glass negative Museum purchase funded by Alice C. Simkins in honor of Isabel B. Wilson, The Manfred Heiting Collection 2004.211 Fratelli Alinari (Italian, 1852–) Campanile for the Cathedral of Santa Maria del Fiore, Florence 1852–1858 Albumen silver print from glass negative Museum purchase funded by Craig Lidji in memory of Lee Hage Jamail, The Manfred Heiting Collection 2004.212 The Alinari Brothers first established an international reputation for themselves based on their photographs of Florence when they exhibited at the Paris Exposition of 1855. This image captures the entire length of Giotto’s bell tower from a high vantage point, likely the roof of a nearby building. The façade of the Cathedral, partially obstructed by the Baptistry, is bare; it was redone with the same red, green, and white marble as the campanile between 1867 and 1887. Though the photographs cannot capture the colour, the intricacies in the construction and stonework are still visible. The photographs have a flat focus, with universally sharp details from the foreground to the background. This allowed architects who saw the print at the Architectural Photographic Association’s 1858 exhibition to study not only the bell tower and baptistry doors, but to imagine how they all harmoniously make up the Cathedral complex. Robert Macpherson (English, 1811–1872) View of Rome from the French Academy, Monte Pincio c. 1863 Albumen silver print from glass negative Museum purchase funded by Alice C. Simkins in memory of W. Stewart Simkins, The Manfred Heiting Collection 2004.222 47 This particular image marks a shift from the previous exhibition of the Association, in which the photographs all showcase entire buildings or specific architectural components with as little of the surroundings in the frame as possible. Here, Macpherson is neither focusing on the Villa Medici (the French Academy), which is not even in the frame, its garden, which is barely visible, or the full panorama of Rome, framed by trees in the background. By marrying these three, Macpherson instead engages the viewers’ imaginations. Including architecture in the context of its surroundings allows anyone to situate themselves within the scene. It was among the 120 views of Rome exhibited by Macpherson at the Architectural Photographic Association’s 1859 exhibition. His body of work, which accounted for almost one third of the total photographs on view, was hailed as the highlight of this exhibition. It is a different type of source material for architects; it captures the general mood and feel of the environment surrounding the Villa, not the structure itself. Édouard Baldus (French, 1813–1889) Pavillon Denon, Louvre 1855–1856 Salted paper print from glass negative Gift of Manfred Heiting, The Manfred Heiting Collection 2004.249 In 1855, Baldus received a commission to photograph the construction of the New Louvre. Here, he subtly captures this state of flux; though scaffolding is visible, the construction itself is not the focus of his photograph. He instead celebrates the grandeur of the almost-complete building and its decoration. Even light rakes across the entire façade of the Pavillon, illuminating the ornamental details. By the the end of his commission in 1857, Baldus created more than 1200 photographs of the Louvre. His images, a triumph of nineteenth-century photography, were so popular that he continued to photograph the Louvre for the government and his own personal business until the mid-1860s. Only through photography could he compile such a vast archive as an art form while simultaneously making his work available to architects as source material. He exhibited this and other work from the commission at the Architectural Photographic Association’s first exhibition in 1858. Louis-Auguste Bisson (French, 1814–1876) Moissac Cloister 1857 Albumen silver print from glass negative Gift of Manfred Heiting, The Manfred Heiting Collection 2004.313 This photograph is rich in subtle devices which make it very useful as source material for architects, but outstanding as an artwork as well. Architects tended to prefer close-up detailed views of single building components, or views of entire building façades as source material. Yet 48 from this vantage point, the Bisson brothers simultaneously present the sculptural intricacies of the columns, the wooden beams of the roof, and how the constructed building functions and interacts within the natural landscape in one single image. It pulls us into the dark, mysterious corner, and leads us back into the light of the perpendicular hallway and garden. This image was widely exhibited at photographic societies throughout Europe between 1858 and 1860. Charles Clifford (English, 1819–1863) [Main Door, Salamanca Cathedral] 1855–1860 Albumen silver print from glass negative Gift of Manfred Heiting, The Manfred Heiting Collection 2004.350 Charles Clifford (English, 1819–1863) Salamanca, Puerta del Convento de las Dueñas 1853 Albumen silver print from glass negative Gift of Manfred Heiting, The Manfred Heiting Collection 2004.351 Though convents and cathedrals were popular subject matter for British and French architectural photographers, the sculptural ornamentation on either building seen in Clifford’s photographs is not an architectural style readily found in either Britain or France. Diplomatic relations between Britain and Spain were not formed until 1850, allowing curious travellers to venture there for the first time. In keeping with the tradition of the Grand Tour, these photographs embody British society’s craving for travel and foreign places, but they also merge architectural photography with nineteenth-century Europe’s fascination with orientalism. These photographs straddle a fine line between the East and West, the familiar and unfamiliar, and served to bridged the gap between representations of familiar European architecture and those of Eastern architecture at the Architectural Photographic Society’s first exhibition. Tommaso Cuccioni (Italian, c. 1790–1864) View of the Roman Forum towards the Capitoline Hill, Looking West 1855 Albumen silver print from glass negative Museum purchase funded by the Brown Foundation Accessions Endowment Fund, The Manfred Heiting Collection 2004.369 From the mid-eighteenth century, it had become customary for young, British aristocrats to travel to Italy. This coach trip, called the Grand Tour, was initially considered an essential part of their education, but turned into a trendy pastime. The ruins of the Roman Forum were a popular stop for those undertaking their Grand Tour, and the subject of many drawings, paintings, and, 49 after the 1840s, photographs. In fact, The Museum of Fine Arts, Houston, has three almost identical photographs taken from the same viewpoint between 1853 and 1860. This print, a copy of which was exhibited at the 1858 exhibition of the Photographic Society of Scotland, is crisp and large, with rich, eggplant tones and bright highlights. It is very classical but marvelously executed, and provided architects and members of the public alike with a chance to study the Roman Forum without having to leave Scotland. Carlo Naya (Italian, 1816–1882) Piazzetta of San Marco looking towards the Island of St. George 1857–1859 Albumen silver print from glass negative Museum purchase funded by Cathy and Giorgio Borlenghi, The Manfred Heiting Collection 2004.619 Carlo Naya exhibited a copy of this print at the Société Française de Photographie’s 1859 exhibition, just two years after opening his photographic studio in Venice. Unlike most photographs selected by the Architectural Photographic Association, this image does not focus on any one particular building, making its usefulness as source material questionable. Instead, it captures characteristics of Venice: the water surrounding it, the ornamental, almost-Eastern architecture, and the hot sun. The dramatic use of shadows on the right, within which three figures take shelter, contrasted with the bright white highlights on the Doge’s Palace on the left leave viewers with a sense of the heat of the Venetian afternoon. Roger Fenton (English, 1819–1869) Printed by Francis Frith & Co. (British, founded 1859) Lichfield Cathedral, Central Doorway, West Porch 1858 Albumen silver print from glass negative, printed 1863–1865 by Francis Frith & Co. Museum purchase funded by James Edward Maloney 2014.89 Fenton worked with large-format glass plate negatives, excelling in many genres of photography: sweeping and soft English landscapes, monumental architectural views, reportage of the Crimean War, and portraits of the Royal Family. Here, the priest and parishioner in the doorway serve to give a sense of scale, while the simultaneously helping the composition of the photograph, inviting viewers in through the open door. The dark shadows contrast the light stonework, allowing architects and other viewers to study the intricacies of the doorway. With this image, included in the 1859 exhibition of the Société Française de Photographie, Fenton shows us his mastery of the medium, marrying perfect lighting, technique, and Romantic spirit. The Architectural Photographic Association The 1850s also saw a rise in the number of photographers travelling abroad, particularly to Italy and the Near East. Photography was seen as a more reliable and practical medium for the 50 documentation of overseas encounters. As a result, the number of commercial photographic studios across Europe and Asia rose by the mid-1850s. Beginning in the 1850s, commercial photographers defined public perceptions of photography. The demand in the 1850s and 1860s was for art reproductions, photographs of new construction, and portraiture. Advances in photographic technology at the time aided in the success of photographic studios, and enabled photographers to travel. Wet collodion glass plate negatives made it possible to print a high quantity of sharp images upon a photographer's return home from their travels, which resulted in a larger-scaled production and distribution of photographic prints than ever before. The increased success and popularity of photographic societies, commercial studios, and wet collodion glass plate negatives converged to create an unprecedented international community of photography. Architecture from around the world became a popular photographic subject in the 1850s, and photographers like Édouard Baldus, Charles Clifford, or Francis Frith devoted the majority of their practice to the subject. The Architectural Photographic Association was formed in May of 1857, and officially inaugurated at a meeting on the night of Thursday, January 7th 1858. The architects belonging to the Association would travel, purchase prints from photographers’ studios at their destinations, and then assemble these prints into an exhibition in London. Subscribers could come to the exhibitions and purchase copies of photographs that were on display. After the exhibitions, the Association would order the chosen prints from each photographer, and send them to the subscribers. The Association held four annual exhibitions from 1858 until 1861, when it was dissolved due to its inability to keep up with an overwhelming demand. Photographic Exhibitions from 1858 to 1861 The exhibition at the Crystal Palace in London in 1851 was, for the majority of those in attendance, a preliminary exposure to photographs. By the mid-1850s, photographic exhibitions became more common throughout Europe, and particularly in Britain and France, intrinsically linked with the rise of photographic societies. The Photographic Society of London, now the Royal Photographic Society, was founded in 1853, and a year later, the Société Française de Photographie was formed in Paris. These societies helped foster an international community of photographers, working to validate their work as art, and to make technical advances in the medium or profit off of the sale of photographs. Members of photographic societies, like the Société Française de Photographie, the Photographic Society of London, or the Glasgow Photographic Society, were mostly photographers and chemists. They would exchange prints with each other for the purposes of refining their own techniques and building their personal collections. These societies held annual exhibitions, curated and selected by juries to ensure that the exhibitions included only the finest examples of photographic practices of the time. 51 Appendix B: Condition Reports (Arranged by accession number, in ascending order) Accession Number Condition of Primary Support/Object Condition of Secondary Support Overall Condition, Suggestions/Concerns 88.233 Francis Frith & Co. Columns, Pharaoh's Bed Microcracks throughout the emulsion. 2 mm diameter foxing on photograph in bottom left. Small (3mm) tear along bottom edge. Yellowing of highlights and slight fading Slight yellowing, slight warping, but overall good condition Good condition 2000.255 Robertson & Beato Tophane Fountain Small losses along left edge. Microcracks and abrasions throughout. Overall very little yellowing/fading/ Good condition Great 2004.211 Fratelli Alinari Gates of Paradise, Baptistery of San Giovanni, Florence Losses/discoloration (possible retouching?) along bottom edge, especially in bottom left corner. “159” is inscribed in the negative, bottom right corner. Surface abrasion in shape of a semi-circle, open right, on the bottom left. Small losses and microabrasions throughout. Ink inscription reads: “Firenze / Porta principale del Battistero.” Blind stamp is embossed below. Foxing throughout, increases in frequency closer to the bottom edge. Surface dirt throughout, mostly in bottom left corner and along the bottom of the mount. Handling creases along left and right sides. Good condition. Stable. Could use a surface clean. 2004.212 Fratelli Alinari Campanile for the Cathedral of Santa Maria del Fiore, Florence Retouching in bottom right corner in the form of a 2 inch long curved line. 2 tears along the bottom edge: one in the centre (1 cm long, vertical orientation) and one near the right corner (5 mm). Small surface stains and losses throughout. Slight yellowing overall. Silvering very lightly along the bottom right and bottom left edges in Dmax areas. Dirt throughout, but more near the right, top, and bottom edges. Foxing throughout. Graphite “X” in bottom right of recto. Good condition. Eventually the tears could be mended, but too small to intervene right now. 2004.222 Robert Macpherson Fading and yellowing along left, bottom, and top edges. Small (<1 mm diameter) Trace of paper manufacturer's stamp along the right edge, whole length. Slight surface Great condition. Negative is in worse shape, makes the print 52 View of Rome from the French Academy, Monte Pincio losses and micro abrasions throughout. 3 mm tear along right edge. Evidence of retouching: an oblong circle along right edge, with a lengthwise diameter of 3mm, and a small circle near bottom edge center, with roughly a 1.5 mm diameter. stains throughout. Adhesive/linen residue along top edge, fabric fraying. look like it has losses and cracks in places it doesn’t. 2004.249 Édouard Baldus Pavillon Denon, Louvre Evidence of retouching bottom left and center right. Some foxing top left and top right. Microscope inspection confirms medium as salted paper print. Slight yellowing throughout. Surface dirt near bottom left near the edge, bottom right corner, and centre right near the edge. Small stain in the bottom right approx. 2 inches above the bottom edge. Good condition. Could benefit from a surface clean, but not essential. 2004.313 Bisson Frères Moissac Cloister Losses, filled in with ink along top and right edge, and more near the top right corner. Microcracks and small losses/surface dirt throughout. Slight silvering in Dmax areas along top and right edge, more concentrated in top right corner. Foxing very heavy throughout. Large tear along left edge, horizontal orientation, just under 5 cm long. Circular wear (abrasion) on bottom edge, less than 1 mm in diameter. Surface dirt heavy throughout, more concentrated near the edges (especially the bottom). Graphite inscriptions and artist’s facsimile signature stamp (ink) in bottom right corner. Embossed blind stamp in bottom centre. Overall fair condition. Mount shows more wear than the photograph itself, which is in great condition. 2004.350 Charles Clifford [Main Door, Salamanca Cathedral] Blind stamp embossed (upside down) in the bottom left corner: “CLIFFORD / PHOTOGRAPHER.” 2 cm abrasion in bottom right, runs diagonally. Micro abrasions throughout. Small losses in top right (less than 1 mm) and evidence of retouching. Loss along left edge, near top left corner; and in bottom right corner along right edge. Adhesive or label residue in top left corner. Graphite inscription bottom left corner reads “48” and “Salamanca / Nord Thor Gallery / Sud.” Slight foxing throughout. Surface dirt throughout, more buildup along 4 edges. Slightly warped/buckling. Top centre there is a graphite (3 mm) mark parallel to and just above the top edge of the photograph. Top right corner missing 1 layer of paper. Good condition, minor surface clean could be useful in future. Graphite marks can be erased. Photograph should not be in humid environment, as the paper will continue to dangerously buckle. 2004.351 Charles Clifford Salamanca, Puerta del Convento de Minimal damage. Microscratches throughout, mostly visible to naked eye in bottom left. Slight tear and loss in bottom left corner, in the tip of the corner itself. Dealer inscription in graphite in bottom right centre and corner. Slight warping/buckling throughout. Yellowing and dirt along all four edges. Handling damage Great condition, mostly stable, if exposed to excess humidity the secondary support will warp more which could be unfavourable for the 53 las Duenas There is a “43” inscribed in red ink in the bottom right corner, and a “55” in the bottom left corner of the negative. in bottom left corner (small loss, layers of paper are separating) photographic emulsion. 2004.369 Tommaso Cuccioni View of the Roman Forum towards the Capitoline Hill, Looking West Loss/retouching in upper left, near top left corner. Slight foxing in highlights of print. Cracks in the negative (scratches?) near left edge, run diagonally towards centre, were painted in (in the negative?). Slight yellowing of highlights. Paper manufacturing stamp visible along entire length of the right edge (similar to the Macpherson). Surface dirt throught. Sparse foxing spots to the left of the image and surrounding embossed blind stamp, which is in the lower centre. Graphite inscription reads “Rome / Forum” in bottom right corner. Slight warping/buckling. Great. Exposure to humidity would be detrimental and cause the photograph to buckle more. 2004.619 Carlo Naya Piazzetta of San Marco looking towards the Island of St. George, “109” is inscribed in the negative in the bottom right corner. Small groups of abrasions and scratches along the top edge of the image. Slight yellowing of highlights. Microcracks throughout. Surface stain near top right corner. Loss/stain near left edge, around 2 mm in diameter. Surface stains along the edges especially. Foxing slightly. Yellowing on the edges, especially the bottom, right, and left. Great condition. Microscopic inspection would reveal the intricacies of the stains, and determine whether or not they can be removed. 2014.89 Roger Fenton, printed by Francis Frith & Co. Lichfield Cathedral, Central Doorway, West Porch Yellowing and fading around all four edges, could be overmatted. Overall in good condition. Slight yellowing of highlights throughout. Small microcracks and abrasions throughout. Good condition The object is in good, stable condition. 54 Appendix C: Matting and Framing Swatches and Worksheets Artique Matboard: Thatch (A4937) Artique Matboard: Birch (A4837) 55 56 57 58 59 60 61 62 63 64 65 66 67 68 Appendix D: Wall Elevation With Sightlines: Without Sightlines: 69 Appendix E: Exhibition Brochure 70 71 References Ackerman, James S. "CCA Mellon Lectures: On the Origins of Architectural Photography." 4 Dec. 2001. Aubenas, Sylvie, and Jacques Lacarrière. Voyage en Orient. Paris: Hazan, 1999. Baillargeon, Claude. "Construction Photography in the Service of International Public Relations: The French Connections."Nineteenth-century Photographs and Architecture: Documenting History, Charting Progress, and Exploring the World. Ed. Micheline Nilsen. Burlington: Ashgate, 2013. 139–54. Baldwin, Gordon. Architecture in Photographs. Los Angeles: J. Paul Getty Museum, 2013. Baldwin, Gordon, Malcolm R. Daniel, Sarah Greenough. All the Mighty World: The Photographs of Roger Fenton, 1852–1860. New Haven: Yale University Press, 2004. Boyer, Christine M. "La Mission Héliographique: Architectural Photography, Collective Memory and the Patrimony of France, 1851." Picturing Place: Photography and the Geographical Imagination, edited by Joan M. Schwartz and James R. Ryan, London: I.B. Tauris, 2003: 21–54. Buck, Rebecca A., and Jean Allman Gilmore. MRM5: Museum Registration Methods. 5th ed. American Association of Museums: AAM, 2010. Chlumsky, Milan, and Bernard Marbot. Les Frères Bisson Photographes De Flèche En Cime 1840–1870. Paris: Bibliothèque Nationale De France, 1999. Daniel, Malcolm R., and Barry Bergdoll. The Photographs of Édouard Baldus. New York: Metropolitan Museum of Art, 1994. Dodgson, Campbell. Dictionary of National Biography, 1885–1900. Vol. 56. London: Smith, Elder, 1898. 72 English, Donald E. Political Uses of Photography in the Third French Republic, 1871–1914. Ann Arbor: UMI Research, 1984. Hannavy, John. "Exhibitions of Photography." In Encyclopedia of Nineteenth-century Photography. New York: Routledge, Taylor & Francis Group, 2008: 508–510. Lacoste, Anne. Felice Beato: A Photographer on the Eastern Road. Los Angeles: J. Paul Getty Museum, 2010. Lavédrine, Bertrand. Photographs of the Past: Process and Preservation. Los Angeles: Getty Conservation Institute, 2009. Lyden, Anne M., Sophie Gordon, and Jennifer Green-Lewis. A Royal Passion: Queen Victoria and Photography. Los Angeles: J. Paul Getty Trust, 2014. Malservisi Franca, and Maria Rosaria Vitale. "Photography versus the Historical Record: The Role of Photography in Rouen's Gros-Horloge Restoration." Future Anterior 10 no.2 (2013): 49–65. McCauley, Elizabeth Anne. Industrial Madness: Commercial Photography in Paris, 1848–1871. New Haven: Yale UP, 1994. de Mondenard, Anne. La Mission Héliographique: Cinq Photographes Parcourent la France en 1851. Paris: Centre des Monuments Nationaux, 2002. Pare, Richard. Photography and Architecture 1839–1939. Montreal: Canadian Centre for Architecture, 1982. Reilly, James M. Care and Identification of 19th Century Photographic Prints. Rochester: Eastman Kodak, 1986. Roubert, Paul-Louis. "Funding a Photographic Elite: Nineteenth-century Photographic Societies and the Financing of Photography as a Mark of Social Distinction." Trans. James Gussen. 73 Études Photographiques 24 (November 2009): 18–41. Roubert, Paul-Louis. "1859, Exposer La Photographie." Études Photographiques 8 (November 2000): 4–21. Serrell, Beverly. Exhibit Labels: An Interpretive Approach. Second ed. Lanham: Rowman & Littlefield, 2015. Snyder, Jill, and Maria Reidelbach. Caring for Your Art. New York: Allworth, 2001. Société Française De Photographie. Catalogue De La Troisième Exposition De La Société Française De Photographie. Paris: Renou Et Maulde, 1859. Taylor, Roger. Photographs Exhibited in Britain, 1839–1865: A Compendium of Photographers and Their Works. Ottawa: National Gallery of Canada, Library and Archives, 2002. Taylor, Roger, and Larry J. Schaaf. Impressed by Light: British Photographs from Paper Negatives, 1840–1860. New York: Metropolitan Museum of Art, 2007. The Conservation Centre for Art and Historical Artifacts, Light Exposure of Artifacts on Exhibition: CCAHA Guidelines, Web Publication, http://www.ccaha.org/uploads/media _items/light-exposure-of-artifacts-on-exhibition.original.pdf The Library of Congress. "Cataloging & Digitizing Toolbox." Prints and Photographs Reading Room, Library of Congress. 22 Oct. 2010. Web. 11 Mar. 2016. Wagner, Sarah, Connie McCabe, and Barbara Lemmen. Guidelines for Exhibition Light Levels for Photographic Materials. Report. May 2007. Accessed April 20, 2016. http://www.ccaha.org/publications/paper. 74 

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Psychiatr Psychol Klin 2015, 15 (1), p. 4–12 Carlo Garofalo , Patrizia Velotti , Giulio Cesare Zavattini , Marco Tommasi3, Roberta Romanelli3, Helena Espírito Santo4, Aristide Saggino3 1 2 1 Received: 27.03.2015 Accepted: 15.04.2015 Published: 30.04.2015 On the factor structure of the Dissociative Experiences Scale: contribution with an Italian version of the DES-II Struktura czynnikowa Skali Przeżyć Dysocjacyjnych: włoska wersja skali DES-II Department of Dynamic and Clinical Psychology, Sapienza University of Rome, Italy Department of Education Sciences, University of Genoa, Italy 3 Department of Psychological, Humanistic and Territorial Sciences, University of Chieti-Pescara, Italy 4 Department of Psychology, Miguel Torga Institute, Coimbra, Portugal Correspondence: Carlo Garofalo, Department of Dynamic and Clinical Psychology, Sapienza – University of Rome, Via degli Apuli, 1, 00185, Rome, Italy, tel.: +393283898543, fax: +39 010 20953728, e-mail: cgarofalo24@gmail.com, carlo.garofalo@uniroma1.it 1 2 Abstract Aim of the study: Notwithstanding its clinical and empirical relevance, there is no consensus on how to conceptualize dissociation. This may be partly due to the conflicting results yielded on the factor structure of the gold-standard selfreport measure of dissociation (the Dissociative Experiences Scale-Revised; DES-II, Carlson and Putnam, 1993). In an attempt to advance research on this topic, we sought to explore the factorial structure of an Italian version of the DES-II. Material and methods: A sample of 320 subjects (122 inmates and 198 community participants) was administered the Italian version of the DES-II. Results: The Italian version of the DES-II showed good psychometric properties and replicated a two-factor structure. Items content seemed to support the distinction into two qualitatively different forms of dissociative experiences, described as detachment and compartmentalization phenomena. In line with the expectations, participants in the inmate sample reported higher rates of dissociative experiences than community participants, on both dimensions. Conclusions: This study provides further support for the validity of the Italian version of the DES-II for use with community and inmate samples. Furthermore, we corroborated previous evidence on a two-factor structure of the DES-II, which is consistent with theoretical assumptions describing two distinct, albeit overlapping, dissociative dimensions (i.e., detachment and compartmentalization). Key words: dissociation, community sample, inmates, detachment, compartmentalization Streszczenie Cel badania: Niezależnie od wartości klinicznej i empirycznej pojęcia, jakim jest dysocjacja, nie ma zgody co do właściwego sposobu jego konceptualizacji. Częściowo może to być spowodowane sprzecznymi wynikami analizy struktury czynnikowej złotego standardu wśród narzędzi do samooceny zjawisk dysocjacyjnych, jakim jest Skala Przeżyć Dysocjacyjnych (Dissociative Experiences Scale-Revised; DES-II, Carlson i Putnam, 1993). Mając na celu pogłębienie badań na ten temat, autorzy podjęli się analizy struktury czynnikowej włoskiej wersji skali DES-II. Materiał i metody: Badanie obejmowało próbę 320 osób (122 więźniów i 198 osób należących do populacji ogólnej). Zastosowano w nim włoską wersję skali DES-II. Wyniki: Włoska wersja skali DES-II wykazała dobre właściwości psychometryczne i została w niej zreplikowana struktura dwuczynnikowa. Treść pozycji na skali wydawała się potwierdzać istnienie podziału na dwie jakościowo inne formy przeżyć dysocjacyjnych, ujmowane jako oddzielenie (ang. detachment) i szufladkowanie (ang. compartmentalization). Zgodnie z oczekiwaniami więźniowie częściej zgłaszali występowanie przeżyć dysocjacyjnych niż osoby należące do populacji ogólnej, na obu wymiarach. Wnioski: Niniejsze badanie dostarcza kolejnych dowodów na zasadność stosowania włoskiej wersji skali DES-II w badaniach na populacji ogólnej oraz więziennej. Potwierdzono również poprzednie dane świadczące o dwuczynnikowej strukturze skali DES-II, co jest zgodne z założeniami teoretycznymi opisującymi dwa odrębne, choć nakładające się na siebie wymiary dysocjacji (tj. oddzielenie i szufladkowanie). Słowa kluczowe: dysocjacja, populacja ogólna, więźniowie, oddzielenie, szufladkowanie 4 © Medical Communications Sp. z o.o. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (CC BY-NC-ND). Reproduction is permitted for personal, educational, non-commercial use, provided that the original article is in whole, unmodified, and properly cited. On the factor structure of the Dissociative Experiences Scale: contribution with an Italian version of the DES-II Struktura czynnikowa Skali Przeżyć Dysocjacyjnych: włoska wersja skali DES-II INTRODUCTION D issociation encompasses those processes involving a “lack of normal integration of thoughts, feelings, and experiences into the flow of consciousness and memory” (p. 727; Bernstein and Putnam, 1986). Although they may occasionally occur in the daily life of healthy people, pathological levels of dissociative experiences have been reported as characteristic or co-occurring across diverse psychiatric conditions (Dell and O’Neil, 2009; Sar et al., 2007; Schäfer et al., 2010), with the strongest association usually reported between dissociation and vulnerability to psychotic symptoms (Barker-Collo, 2001; Simões et al., 2014). Recent evidence also supported the presence of dissociation in individuals who display violent behaviour (Moskowitz, 2004; Ruiz et al., 2008). Notwithstanding the clinical relevance of dissociation (Holmes et al., 2005; Liotti, 2006), there is a need for a clearer conceptualization of the dissociation construct (Dell and O’Neil, 2009). According to the DSM-5 (American Psychiatric Association, 2013), dissociative disorders (DD) are defined into three broad categories, namely: dissociative identity disorder, dissociative amnesia, and depersonalization/derealization disorder. In the DES literature, dissociation has historically been described with a three-factor model, respectively encompassing absorption, depersonalization/derealization, and amnesia experiences (e.g., Carlson and Putnam, 1993). Notably, in this framework identity alteration would not be distinguished from other experiences. Another conceptualization which is worth mentioning has been derived from clinical description of dissociation (Allen, 2001; Cardeña, 1994) that subsequently yielded convergent empirical findings (Brown, 2002; Holmes et al., 2005). According to this model (Allen, 2001), two distinct qualitative forms of dissociation were described, namely: detachment and compartmentalization. Detachment has been depicted as the most pervasive form of dissociative disturbance, which encompasses depersonalization, derealization, and similar phenomena such as out-of-body experiences. On the other hand, compartmentalization incorporated the more dramatic and perplexing of dissociative phenomena: amnesia, fugues, and episodes of dissociative identity disorder (Allen, 2001; Holmes et al., 2005). Finally, another way to conceptualize dissociation is to distinguish between pathological and non-pathological dissociative experiences (e.g., Waller et al., 1996; Watson, 2003). As a result, some authors recently argued that to date there is no agreement on how to conceptualize dissociation (Dell and O’Neil, 2009). Notably, factor analytic studies with mainstream measures of dissociation could help in reaching a clearer picture. If the Structured Clinical Interview for DSM-IV Dissociative Disorders (SCID-D; Steinberg, 1993) is regarded as the best diagnostic assessment tool for DD, the Dissociative Experience Scale (DES; Bernstein and Putnam, 1986) is considered the gold-standard instrument to © PSYCHIATR PSYCHOL KLIN 2015, 15 (1), p. 4–12 quantify the frequency of self-reported dissociative experiences (van IJzendoorn and Schuengel, 1996). It has been translated into more than 20 languages and it has mostly been used in its revised version (DES-II; Carlson and Putnam, 1993). The DES can be used as a screening instrument for DD and for determining the contribution of dissociation to other psychiatric syndromes as well as with nonclinical samples (Carlson and Putnam, 1993; Espírito Santo and Abreu, 2009; van IJzendoorn and Schuengel, 1996). Unfortunately, studies exploring the factor structure of the DES-II have to date been unsupportive in deciding among different conceptual models, yielding conflicting results. The original three-factor model proposed by Carlson and Putnam (1993) was confirmed across several studies (Carlson et al., 1993; Fabbri Bombi et al., 1996; Ross et al., 1991; Ruiz et al., 2008; Stockdale et al., 2002). Nevertheless, other authors found different factorial patterns for the DES, ranging from one- (Bernstein et al., 2001; Lipsanen et al., 2003; Mazzotti and Cirrincione, 2001) to seven-factor models (Ray et al., 1992). Moreover, Amdur and Liberzon’s (1996), and Espírito Santo and Abreu’s (2009) findings supported a factorial structure composed by four factors. Also, a two-factor structure first emerged in the study by Waller et al. (1996), who adopted a typological model of dissociation. However, Waller et al. (1996) referred to these factors as respectively resembling pathological (Factor 1) and non-pathological (Factor 2) dissociation. Notably, a categorical distinction between pathological and non-pathological dissociation has failed to prove its utility (Leavitt, 1999) and the dimensional conceptualization of dissociation as a continuum ranging from normal to pathological forms is preferred both in clinical and research settings (Bernstein and Putnam, 1986; Bernstein et al., 2001; Carlson et al., 1993; Holmes et al., 2005; Ruiz et al., 2008). Recently, a French version of the DES (Larøi et al., 2013) replicated the two-factor structure, although proposing a different interpretation. Indeed, Larøi et al. (2013) described two forms of dissociative experiences. The first one included both dissociative amnesia episodes and depersonalization/derealization experiences and was described as resembling dissociation episodes, which may act as a defensive mechanism. The second factor was depicted as encompassing dissociative episodes associated with different forms of “cognitive failures,” often concerning memory or attention. They referred to this type as automatic pilot-related dissociation episodes. Notwithstanding the possible clinical relevance of such an explanation, to the best of our knowledge this interpretation of the two factors does not seem consistent with any other empirical evidence (Brown, 2002), nor with mainstream clinical theories (Dell and O’Neil, 2009; Holmes et al., 2005). Such inconsistency in the factor structure of the DES fosters at least one risk, which is the use of the original subscales reported by Carlson and Putnam (1993) without testing the factorial structure and the item loadings of the DES with the target DOI: 10.15557/PiPK.2015.0001 5 Carlo Garofalo, Patrizia Velotti, Giulio Cesare Zavattini, Marco Tommasi, Roberta Romanelli, Helena Espírito Santo, Aristide Saggino sample, which in turn can lead to wrong-headed theoretical inferences on the dissociation construct. In the present study, we sought to explore the factorial structure of an Italian version of the DES-II, using a combined sample of inmates and community-dwelling participants. It is noteworthy that to date there have been no studies that have investigated the psychometric properties and the factorial structure of the DES in Italian samples, let alone used it with Italian offender samples, even though the Italian translation has been available and widely used for almost 20 years (Barbasio and Granieri, 2013; Conti, 2000; Fabbri Bombi et al., 1996). per week). The DES-II total score is then computed averaging the score on each of the 28 items. Participants were instructed to rate the extent to which they experienced dissociative symptoms, if any, without being under the effects of alcohol or drugs. The DES-II showed high reliability in its original version (test-retest = .79 < r < .84; split-half = .83 < r < .93; Cronbach’s α = .95; Carlson and Putnam, 1993) as well as in its Italian translation (Cronbach’s α = .91; split-half: r = .92; Fabbri Bombi et al., 1996). In the present study, we used the Italian translation reported by Conti (2000), which in our study showed an excellent internal consistency (Cronbach’s α = .96). MATERIAL AND METHODS Statistical analyses Participants We chose natural logarithmic transformations as the best technique to reduce the skewness in our raw data (Roberts, 2008). A first way to determine the number of factors to retain was a parallel analysis (Watkins, 2000) using a Monte Carlo PCA, because it is a way of calculating the average eigenvalues for 100 sets of random data of the same size as our data (28 variables × 320 participants). After doing that, each eigenvalue obtained in SPSS was compared with the corresponding value from the random results generated by parallel analysis. Then, we performed an exploratory factor analysis with Maximum Likelihood (ML) estimator, because it allows the estimation of indices of model fit and permits the computation of model parameter standard errors, confidence intervals, and significance tests. The Root Means Squared Error of Approximation (RMSEA) is particularly indicated for testing model fit. According to Browne and Cudeck (1993), a good model should have a RMSEA value lower than .08, while Hu and Bentler (1999) said that it should be lower than .06. MacCallum et al. (1996) defined the following criteria: a model has a close fit if RMSEA < .05; a fair fit if .05 < RMSEA < .08; a mediocre fit if .08 < RMSEA < .10; a poor fit if RMSEA > .10. The appropriate number of factors is determined by examining the RMSEA values for the sequence of models. The sequence starts with a monofactorial model and a new factor is added step by step until a model with an RMSEA lower than .05 (ideally) is reached (Fabrigar and Wegener, 2012). The RMSEA difference (∆RMSEA) between two models is also important. According to Fabrigar and Wegener (2012), any difference of .02 or greater can be considered a substantial difference in fit; differences between .01 and .019 can be considered marginal differences; differences less than .01 can be considered not meaningful. Therefore, if adding a new factor, ∆RMSEA is lower than .01 (in absolute values), then the new factor can be considered not meaningful and the previous model determines the appropriate number of factors. After calculating descriptive statistics for both samples, we tested for significant differences between inmates and controls, by carrying out multiple t-tests for independent samples. The sample was composed of 320 subjects: 122 inmates and 198 community participants. Inmates had an average age of 39.97 years (SD = 11.76) and community participants had an average age of 32.51 years (SD = 10.30); 98% of inmates and 58.6% of individuals from the community were males. All participants were Caucasian, and all inmates were convicted of violent offenses (i.e., armed robbery, assault, sexual offenses or abuse, murder or attempted murder). Exclusion criteria for both groups were the presence of cognitive disability or a diagnosed psychiatric disorder. Procedures Participants in the community sample were enrolled using the snowball sampling technique. The inmate sample was recruited from different jails and prisons located in or around Rome, as part of a larger study on inmates’ psychopathology. Participants completed the measures anonymously, individually or in small-group sessions settled in the prison library in the presence of a licensed psychologist. Participants from both groups provided written informed consent to voluntarily take part in the study. The Italian Ministry of Justice and the Sapienza University of Rome Research Ethics Board approved the whole procedure, which conformed to the principles included in the Declaration of Helsinki. Measure 6 The Dissociative Experiences Scale-II (DES-II; Carlson and Putnam, 1993) consists of 28 statements describing various dissociative experiences, some common (e.g., missing part of a conversation) and others much more unusual (e.g., standing in front of a mirror without recognizing). Participants had to rate the percentage of occurrence of dissociative experiences using a scale from 0% to 100% (by 10s, resulting in an 11-point scale ranging from 0 to 100, with 0% meaning never and 100% meaning at least once DOI: 10.15557/PiPK.2015.0001 © PSYCHIATR PSYCHOL KLIN 2015, 15 (1), p. 4–12 On the factor structure of the Dissociative Experiences Scale: contribution with an Italian version of the DES-II Struktura czynnikowa Skali Przeżyć Dysocjacyjnych: włoska wersja skali DES-II # factors χ2 df p RMSEA 90% RMSEA ∆RMSEA 1 873.51 350 <.001 .0705 .0627–.0741 2 597.35 323 <.001 .0537 .0450–.0579 –.0168 3 476.72 297 <.001 .0458 .0361–.0506 –.0079 Tab. 1. Goodness of fit indexes of factorial models tested in exploratory factor analysis with ML estimation. ∆RMSEA lower than the critical value of .01 is in boldface RESULTS The Kaiser-Meyer-Olkin Measure of Sampling Adequacy was .96 (should be ≥ .60) and Bartlett’s test of sphericity value was significant (χ2 = 4398.643, df = 378, p < .001), indicating that the data was adequate for factor analysis. According to the screeplot, an evident leveling off in correspondence of the third factor emerged. Screeplots are criticized because it is often difficult to see a clear drop in the curve. The first four eigenvalues were: 11.13; 1.15; .61; .49. Even if in our case the drop is clearly evident, there was a strong difference between the first and the second eigenvalue. Yet the second eigenvalue is smaller than the criterion value from parallel analysis, which showed only one component exceeding the corresponding criterion values for a randomly generated data matrix of the same size (eigenvalue #2 = 1.50; SD = .04). Therefore, we carried out an analysis for testing differences in RMSEA (∆RMSEA) between models with different number of factors. We limited our analyses to a model composed by one, two, or three factors. Tab. 1 shows the goodness of fit indexes (χ2, RMSEA, 90% C.I. of RMSEA and ∆RMSEA) of factorial models. DES-II items Factor 1 Factor 2 h2 1. Driving a car and realizing one doesn’t remember part of the trip .428 .025 .199 2. Missing part of a conversation .557 –.099 .241 3. Finding oneself in a place but unaware of how one got there .332 .455 .531 4. Finding oneself dressed in clothes one doesn’t remember putting on –.007 .752 .558 5. Finding unfamiliar things among one’s belongings .352 .417 .507 6. Being called with a different name by people one doesn’t know .447 .250 .421 7. Seeing oneself as if looking at another person .176 .626 .580 8. Not recognizing friends or family members –.164 .828 .520 9. Not remembering important events in one’s life .366 .294 .373 10. Being accused of lying when one is telling the truth .511 .137 .380 11. Not recognizing one’s reflection in a mirror .049 .709 .554 12. Other people and objects do not seem real .155 .549 .447 13. Feeling as though one’s body is not one’s own .039 .694 .522 14. Remembering past so vividly one seems to be reliving it .715 –.075 .440 15. Not sure if remembered event happened or was a dream .584 .079 .413 16. Being in a familiar place but finding it unfamiliar .505 .214 .454 17. Absorption in television program or movie .532 .061 .333 18. So involved in fantasy that it seems real .492 .204 .426 19. Able to ignore pain .384 .317 .420 20. Staring into space .712 .027 .535 21. Talking out loud to oneself when alone .392 .103 .222 22. Feeling as though one were two different people .471 .255 .457 23.Usually difficult things can be done with ease and spontaneity .700 –.095 .404 24. Not sure whether one has done something or only thought about it .743 –.120 .440 25. Finding evidence of having done things one can’t remember doing .641 .072 .481 26. Finding notes or drawings that one must have done but doesn’t remember doing .488 .150 .365 27. Hearing voices inside one’s head .038 .722 .562 28. Looking at the world through a fog .079 .641 .488 % explained var. 23.73 20.10 Cronbach’s α .936 .934 Note. DES-II: Dissociative Experiences Scale-II; h2: communalities. Loadings greater than .30 (in absolute value) are bolded. Tab. 2. Items content and factor loadings of the DES-II obtained with oblimin rotation (ML estimation), corresponding percentages of variance explained by each factor and internal consistency coefficients (Cronbach’s α) for items grouped in each factor © PSYCHIATR PSYCHOL KLIN 2015, 15 (1), p. 4–12 DOI: 10.15557/PiPK.2015.0001 7 Carlo Garofalo, Patrizia Velotti, Giulio Cesare Zavattini, Marco Tommasi, Roberta Romanelli, Helena Espírito Santo, Aristide Saggino DES-II items 1. Driving a car and realizing one doesn’t remember part of the trip 2. Missing part of a conversation 3. Finding oneself in a place but unaware of how one got there 4. Finding oneself dressed in clothes one doesn’t remember putting on 5. Finding unfamiliar things among one’s belongings 6. Being called with a different name by people one doesn’t know 7. Seeing oneself as if looking at another person 8. Not recognizing friends or family members 9. Not remembering important events in one’s life 10. Being accused of lying when one is telling the truth 11. Not recognizing one’s reflection in a mirror 12. Other people and objects do not seem real 13. Feeling as though one’s body is not one’s own 14. Remembering past so vividly one seems to be reliving it 15. Not sure if remembered event happened or was a dream 16. Being in a familiar place but finding it unfamiliar 17. Absorption in television program or movie 18. So involved in fantasy that it seems real 19. Able to ignore pain 20. Staring into space 21. Talking out loud to oneself when alone 22. Feeling as though one were two different people 23. Usually difficult things can be done with ease and spontaneity 24. Not sure whether one has done something or only thought about it 25. Finding evidence of having done things one can’t remember doing 26. Finding notes or drawings that one must have done but doesn’t remember doing 27. Hearing voices inside one’s head 28. Looking at the world through a fog Note. DES-II: Dissociative Experiences Scale-II. Tab. 3. Structure matrix of DES-II obtained with oblimin rotation (ML estimation) 8 The analysis showed that ∆RMSEA was under the critical value of .01 (Fabrigar and Wegener, 2012) between the model with two and three factors. Therefore, the addition of a third factor is not meaningful and we can conclude that DES measures two latent factors. On the basis of item content, items which were grouped in Factor 1 seemed to incorporate both amnesia and absorption experiences. Hence, this factor seemed to resemble compartmentalization, according to Allen’s (2001) model and Holmes et al.’s (2005) model, whereas items grouped into Factor 2 encompassed depersonalization and derealization (i.e., detachment). Tab. 2 shows the factor loadings of each item after oblimin rotation. Items 3, 5, and 19 loaded on both factors. However, according to their content and in order to preserve the theoretical coherence of the factors’ composition, items 3 and 5 seemed more consistent with Factor 2, whereas item 19 with Factor 1. Correlation between factors was high (r = .776). Tab. 3 shows the structure matrix of the factor analysis. Tab. 4 shows the descriptive statistics (means and standard deviations) for both inmates and community participants and the multiple t-tests for the DES-II total score and subscale scores. DOI: 10.15557/PiPK.2015.0001 Factor 1 .446 .486 .655 .528 .649 .625 .621 .425 .575 .609 .553 .546 .533 .661 .640 .657 .575 .637 .609 .731 .465 .652 .632 .658 .692 .595 .552 .534 Factor 2 .329 .297 .691 .747 .668 .568 .751 .711 .554 .501 .744 .660 .722 .433 .494 .573 .439 .554 .590 .534 .382 .590 .403 .409 .528 .497 .749 .696 Significant differences emerged in all three scores. Interestingly, within both samples, compartmentalization experiences (Factor 1) yielded a relatively higher score than detachment experiences (Factor 2). DISCUSSION As a whole, our results provide further support for the use of the Italian version of the DES-II as the gold-standard self-report measure to assess dissociation in nonclinical Inmates Community (n = 122) (n = 198) Variables Mean SD Mean SD t p DES-II (F1) 22.01 16.12 14.71 14.41 4.094 <.001 DES-II (F2) 11.03 16.33 5.59 12.16 3.177 <.01 DES-II 18.08 15.61 11.45 13.10 3.917 <.001 Note. DES-II (F1): Dissociative Experiences Scale-II Compartmentalization score; DES-II (F2): Dissociative Experiences Scale-II Detachment score; DES-II: Dissociative Experiences Scale-II total score. Tab. 4. Means, standard deviations (SD) and multiple t-test comparisons for the DES-II total score and subscales between Italian inmates and community participants © PSYCHIATR PSYCHOL KLIN 2015, 15 (1), p. 4–12 On the factor structure of the Dissociative Experiences Scale: contribution with an Italian version of the DES-II Struktura czynnikowa Skali Przeżyć Dysocjacyjnych: włoska wersja skali DES-II individuals, extending its reliability and validity to the use with incarcerated offenders. Regarding the factorial structure of the DES-II, our contribution joins the complicated picture reported in the literature. Although we replicated a two-factor structure, our item loadings differed from those reported by Waller et al. (1996)*, with both a clinical (DD) and a nonclinical sample, and Larøi et al. (2013) in a community sample. Interestingly, the factor structures reported in these two studies were also different from each other. Furthermore, both Waller et al. (1996) and Larøi et al. (2013) incorporated in a common factor items referring to depersonalization and amnesia, and interpreted the two factors as representing different degrees of severity. This approach is inconsistent with clinical essays (Allen, 2001; Cardeña, 1994) which have historically considered depersonalization and amnesia as separate forms of dissociation. Moreover, to our knowledge, all previous factor analyses showed that they were always reported in separate factors (e.g., Holmes et al., 2005), and that items representing different degrees of severity were equally distributed across factors (e.g., Carlson and Putnam, 1993). In the current Italian version of the DES-II, all the items reported by Carlson and Putnam (1993) as describing depersonalization or derealization loaded on Factor 2. Similarly, all the items referring to amnesia and absorption forms of dissociative experiences loaded on a separate factor, being grouped in Factor 1**. Notably, even though the primary use of a measure such as the DES is to screen individuals with clinically relevant or empirically significant levels of dissociation, rather than attempting to characterize the nature of dissociation, our factorial structure seems partly consistent with a qualitative distinction between two types of dissociative experiences, being compartmentalization (here, Factor 1), and detachment (here, Factor 2). On the one hand, states involving a compartmentalization involve both amnesia and other phenomena broadly defined as somatoform dissociation, which are not included within the DES-II items (Brown, 2002; Cardeña, 1994; Holmes et al., 2005; Nijenhuis et al., 1996). According to this framework, the distinctive feature of compartmentalization is represented by an inability to intentionally * It is worth noting that Waller et al. (1996) used a taxometric approach in an effort to develop a short measure for the screening of pathological vs. non-pathological dissociation, rather than conducting a factor analytic study per se. ** It should be acknowledged that there are two exceptions. In fact, item 4 and item 8 loaded on our Factor 2 (i.e., depersonalization), whereas they are usually considered as part of the amnesia domain (Carlson and Putnam, 1993; Ross et al., 1991). However, item 4 had already been reported as part of the depersonalization construct (Carlson et al., 1993), suggesting that its loading may depend on the interpretation people give to its content (i.e., people “having the experience of finding themselves dressed in clothes that they don’t remember putting on,” which may also derive from a depersonalization episode while getting dressed). Similarly, we propose that item 8 (i.e., people “being told that they sometimes do not recognize friends or family members”) may be interpreted as a derealization experience (e.g., “those do not seem my real friends, or relatives”) rather than an actual amnesic fragmentation, especially in nonclinical individuals. © PSYCHIATR PSYCHOL KLIN 2015, 15 (1), p. 4–12 control processes or actions that people are normally able to deliberately control (Brown, 2002). On the other hand, following Holmes et al.’s suggestions (2005), we propose that subjective experiences of an altered state of consciousness, accompanied by a sense of separation from some aspects of everyday experience are characteristic of the detachment type of dissociation. Consistent with a dimensional conceptualization of dissociation, it has been argued that states of detachment lie on a continuum ranging from temporary experiences in the everyday life of healthy individuals, to persistent and/or acute conditions associated with a psychiatric disorder (Holmes et al., 2005). In between these two extremes, there would certainly be other forms of detachment states with associated degrees of severity and levels of functional impairment (Holmes et al., 2005). The principal distinction between compartmentalization and detachment states is thought to be the preservation of apparently disrupted functions, which occurs in the context of compartmentalization phenomena (Holmes et al., 2005). Indeed, even if the affected functions (e.g., the ability to bring usually accessible information into conscious awareness in the case of dissociative amnesia) are no longer amenable to intentional control, these compartmentalized processes keep operating normally, being in turn able to influence feelings, thoughts, and actions (Brown, 2002; Cardeña, 1994). In other words, even though they are reversible in principle (Cardeña, 1994) they work separately so that this compartmentalization cannot be reversed by a simple act of will (Holmes et al., 2005). Notably, such distinction has been corroborated by neurobiological evidence showing different dissociative experiences belonging to each component share common underlying mechanisms in the brain (Brown, 2002; Simeon et al., 2003). Moreover, the existence of a distinction between the two components seems to be useful in clinical settings, since these two forms of dissociation are relevant in the context of different psychopathological syndromes (Allen, 2001). However, less attention has been paid to the role of phenomena of absorption, defined as the experience of disconnecting oneself from the surroundings and getting involved in one’s thoughts or other imaginative processes (Waller et al., 1996), which loaded on the same factor as dissociative amnesia in the present study. According to our results, we suggest that states of absorption resemble more a mild form of compartmentalization than one of detachment, somehow involving a loss of volitional control (e.g., people “finding that sometimes they are listening to someone talk and they suddenly realize that they did not hear part or all of what was said” could be thought of as an inability to maintain attentional control, or simply distractibility). Moreover, another item regards people “having the experience of sometimes remembering a past event so vividly that they feel as if they were reliving that event,” partially resembling a sensorial alteration which is typical of somatoform dissociation (Nijenhuis et al., 1996), whose kind of experiences are not explicitly reported DOI: 10.15557/PiPK.2015.0001 9 Carlo Garofalo, Patrizia Velotti, Giulio Cesare Zavattini, Marco Tommasi, Roberta Romanelli, Helena Espírito Santo, Aristide Saggino 10 in the DES-II, yet are thought to be part of the compartmentalization type of dissociation (Holmes et al., 2005). Supporting our view of absorption being a mild form of compartmentalization, Bernstein et al. (2001) noticed that absorption items were the more endorsed ones in nonclinical subjects, compared to those regarding both depersonalization and amnesia, and other authors agreed in considering absorption less pathological than both amnesia and depersonalization (Waller et al., 1996). However, future studies are needed given that Allen (2001) included absorption at the low end of the continuum in the detachment domain. It should also be noted that previous works (e.g. Allen et al., 1999) described a second possible form of dissociative amnesia which was different from the one associated with compartmentalization, being an encoding problem triggered by extreme detachment. To further corroborate the proposed distinction between detachment and compartmentalization phenomena, some authors have reported findings attesting that people can experience detachment states unrelated to and without also experiencing compartmentalization episodes (Baker et al., 2003; Simeon et al., 2003), and also the opposite pattern has been reported (Brown et al., 2005; Holmes et al., 2005). Accordingly, in both samples considered here, a relatively higher degree of compartmentalization (as opposed to detachment) experiences emerged. However, the proportion of variance shared by the two factors was approximately 60%. Notwithstanding the potentially relevant clinical impact of a qualitative distinction between detachment and compartmentalization type of dissociation, further convergent evidence is needed to support a bi-dimensional conceptualization of dissociation, the latter being the assumption that detachment and compartmentalization states are on two separate continuums (Allen, 2001; Holmes et al., 2005). For instance, further studies are warranted in an attempt to replicate the two-factor solution of the DES-II reported here, both with other Italian independent samples and in other countries, as well as in clinical populations. To date, the inconsistency in its factor structure across studies and samples, as well as the high degree of shared variance among the factors, lead us to support the use of the DES-II as measuring a uni-dimensional construct (Bernstein et al., 2001; Holmes et al., 2005; Ruiz et al., 2008; van IJzendoorn and Schuengel, 1996). This is especially true for research purposes (i.e., allowing a comparison of results from a different sample and corresponding factor structure), whereas in clinical practice it could be useful to disentangle different dimensions of dissociative experience, yet taking into account the overall degree of severity. As a whole, our samples’ scores were in line with normative data reported in the literature worldwide with regard to the presence and severity of dissociation in both community (Carlson and Putnam, 1993; Carlson et al., 1993; Espírito Santo and Abreu, 2009; Spitzer et al., 2006; Stockdale et al., 2002) and inmate samples (Becker-Blease and DOI: 10.15557/PiPK.2015.0001 Freyd, 2007; Espirito-Santo and Costa, 2013; van IJzendoorn and Schuengel, 1996; Moskowitz et al., 2005; Ruiz et al., 2008; Snow et al., 1996). As expected, inmates reported higher rates of dissociative experiences when compared with community-dwelling individuals. This finding suggests that dissociation may characterize the mental functioning of incarcerated individuals. Indeed, their rates of dissociative symptoms seemed comparable with those of patients with psychiatric disorders (e.g., van IJzendoorn and Schuengel, 1996). Thus, mental health services in prison should include an evaluation of dissociative symptoms in their standard assessment for newly incarcerated individuals as well as during the period of incarceration. Our findings should be considered in light of their limitations, which also represent directions for future research. First, we did not assess the presence of psychotic symptoms in our inmate sample (although we excluded those diagnosed with psychiatric disorders, we cannot rule out the possible presence of transient psychotic episodes), and this could have influenced the higher degree of dissociative experiences that we found in the inmate sample. However, the presence of inmates with psychotic symptoms is likely to be constant across other inmate populations (Ruiz et al., 2008). Then, we only used self-report measure, whereas a multi-method assessment (e.g., an expert-rated measure for assessing psychiatric symptoms, or an interview to investigate dissociative experiences) could limit the measurement bias that may affect questionnaires, even though both measures we used are widely accepted as valid and reliable instruments. Finally, we did not include a sample of psychiatric patients, nor a sample of patients with DD, thus our findings should not be generalized to those populations prior further explorations. CONCLUSIONS With these cautions in mind, this study provides further evidence to the reliability and validity of the DES-II as a measure to assess dissociation in nonclinical and inmate populations, representing the first Italian validation of the scale. Our results also highlight the alarming prevalence of dissociation in prisoners, calling for the inclusion of a proper psychiatric assessment in prison, which should include an evaluation of dissociative symptoms. Finally, the two-factor structure of the Italian version of the DES-II emerged in the present study, representing the first empirical support deriving from self-report assessment of dissociation to the dichotomy between detachment and compartmentalization types of dissociation (Allen, 2001; Holmes et al., 2005). Nevertheless, further replications are necessary, possibly with clinical samples and with patients suffering from DD. © PSYCHIATR PSYCHOL KLIN 2015, 15 (1), p. 4–12 On the factor structure of the Dissociative Experiences Scale: contribution with an Italian version of the DES-II Struktura czynnikowa Skali Przeżyć Dysocjacyjnych: włoska wersja skali DES-II Conflict of interest The authors do not report any financial or personal connections with other persons or organizations which might negatively affect the content of this publication and/or claim authorship rights to this publication. Acknowledgment We are indebted to Prof. Jon Allen for his invaluable comments on an earlier version of the manuscript. PIŚMIENNICTWO: REFERENCES: Allen JG: Traumatic Relationships and Serious Mental Disorders. John Wiley & Sons Ltd, Chichester 2001. Allen JG, Console DA, Lewis L: Dissociative detachment and memory impairment: reversible amnesia or encoding failure? Compr Psychiatry 1999; 40: 160–171. 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Hindawi BioMed Research International Volume 2017, Article ID 8317357, 9 pages https://doi.org/10.1155/2017/8317357 Research Article Emotion Recognition from EEG Signals Using Multidimensional Information in EMD Domain Ning Zhuang,1 Ying Zeng,1,2 Li Tong,1 Chi Zhang,1 Hanming Zhang,1 and Bin Yan1 1 China National Digital Switching System Engineering and Technological Research Center, Zhengzhou 450002, China Key Laboratory for NeuroInformation of Ministry of Education, School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, China 2 Correspondence should be addressed to Bin Yan; ybspace@hotmail.com Received 27 March 2017; Revised 21 June 2017; Accepted 16 July 2017; Published 16 August 2017 Academic Editor: Robertas Damaševičius Copyright © 2017 Ning Zhuang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper introduces a method for feature extraction and emotion recognition based on empirical mode decomposition (EMD). By using EMD, EEG signals are decomposed into Intrinsic Mode Functions (IMFs) automatically. Multidimensional information of IMF is utilized as features, the first difference of time series, the first difference of phase, and the normalized energy. The performance of the proposed method is verified on a publicly available emotional database. The results show that the three features are effective for emotion recognition. The role of each IMF is inquired and we find that high frequency component IMF1 has significant effect on different emotional states detection. The informative electrodes based on EMD strategy are analyzed. In addition, the classification accuracy of the proposed method is compared with several classical techniques, including fractal dimension (FD), sample entropy, differential entropy, and discrete wavelet transform (DWT). Experiment results on DEAP datasets demonstrate that our method can improve emotion recognition performance. 1. Introduction Emotion plays an important role in our daily life and work. Real-time assessment and regulation of emotion will improve people’s life and make it better. For example, in the communication of human-machine-interaction, emotion recognition will make the process more easy and natural. Another example, in the treatment of patients, especially those with expression problems, the real emotion state of patients will help doctors to provide more appropriate medical care. In recent years, emotion recognition from EEG has gained mass attention. Also it is a very important factor in brain computer interface (BCI) systems, which will effectively improve the communication between human and machines [1]. Various features and extraction methods have been proposed for emotion recognition from EEG signals, including time domain techniques, frequency domain techniques, joint time-frequency analysis techniques, and other strategies. Statistics of EEG series, that is, first and second difference, mean value, and power are usually used in time domain [2]. Nonlinear features, including fractal dimension (FD) [3, 4], sample entropy [5], and nonstationary index [6], are utilized for emotion recognition. Hjorth features [7] had also been used in EEG studies [8, 9]. Petrantonakis and Hadjileontiadis introduced higher order crossings (HOC) features to capture the oscillatory pattern of EEG [10]. Wang et al. extracted frequency domain features for classification [11]. Time-frequency analysis is based on the spectrum of EEG signals; then the energy, power, power spectral density (PSD), and differential entropy [12] of certain subband are usually utilized as features. Short-time Fourier transform (STFT) [13, 14], Hilbert-Huang transform (HHT) [15, 16], and discrete wavelet transform (DWT) [17–19] are the most commonly used techniques for spectrum calculating. It has been commonly tested and verified that higher frequency subband such as Beta (16–32 Hz) and Gamma (32–64 Hz) bands outperforms lower subband for emotion recognition [20, 21]. Other features extracted from combination of electrode are utilized too, such as coherence and asymmetry of electrodes in different brain regions [22–24] and graph-theoretic features [25]. Jenke et al. had done a research comparing the 2 BioMed Research International IMF1 IMF2 EEG signals (5 s) EMD IMF3 Feature extraction SVM classifier Recognition results IMF4 IMF5 Figure 1: Block diagram of the proposed method. performance of different features mentioned above and got a guiding rule for feature extraction and selection [26]. Some other strategies such as utilizing deep network to improve the classification performance have also been researched. Zheng and Lu used deep neural network to investigate critical frequency bands and channels for emotion recognition [27]. Yang et al. used hierarchical network with subnetwork nodes for emotion recognition [28]. EMD is proposed by Huang et al. in 1998 [29]. Unlike DWT, which needs to predetermine transform base function and decomposition level, EMD can decompose signals into IMF automatically. These IMFs represent different frequency components of original signals, with band-limited characteristic. By applying Hilbert transform to IMF, we can get instantaneous phase information of IMF. So EMD is suitable for analysis of nonlinear and nonstationary sequence, such as neural signals. EMD is a good choice for EEG signals and we utilize it for emotion recognition from EEG data. Which feature is effective for emotion recognition in EMD domain? Which IMF component is best for classification? Is the performance based on EMD strategy better compared to time domain method and time-frequency method or not? All these have not been researched yet and we investigate them in our research. EMD has been widely used for seizure prediction and detection, but for emotion recognition based on EMD, there is not so much research. Higher order statistics of IMFs [30], geometrical properties of the decomposed IMF in complex plane [31], and the variation and fluctuation of IMF [32] are used as features for seizure prediction and detection. For emotion recognition, Mert and Akan extracted entropy, power, power spectral density, correlation, and asymmetry of IMF as features and then utilized independent component analysis (ICA) to reduce dimension of the feature set [33]. The classification accuracy is computed with all the subjects mixed together. In this paper, we present an emotion recognition method based on EMD. We utilize the first difference of IMF time series, the first difference of the IMF’s phase, and the normalized energy of IMF as features. The motivation of using these three features is that they depict the characteristics of IMF in time, frequency, and energy domain, providing multidimensional information. The first difference of time series depicts the intensity of signal change in time domain. The first difference of phase measures the change intensity in phase and normalized energy describes the weight of current oscillation component. The three features constitute a feature vector, which is fed into SVM classifier for emotional state detection. The proposed method is studied on a publicly available emotional database DEAP [20]. The effectiveness of the three features is investigated. IMF reduction and channel reduction for feature extraction are both discussed, which aim at improving the classification accuracy with less computation complexity. The performance is compared with some other techniques, including fractal dimension (FD), sample entropy, differential entropy, and time-frequency analysis DWT. 2. Method To realize emotional state recognition, the EEG signals are decomposed into IMFs by EMD. Three features of IMFs, the fluctuation of the phase, the fluctuation of the time series, and the normalized energy, are formed as a feature vector, which is fed into SVM for classification. The whole process of the algorithm is shown in Figure 1. 2.1. Data and Materials. DEAP is a publicly available dataset for emotion analysis, which recorded EEG and peripheral physiological signals of 32 participants as they watched 40 music videos. All the music video clips last for 1 minute, representing different emotion visual stimuli, with grade from 1 to 9. Among the 40 music videos, 20 are high valence visual stimuli and 20 are low valence visual stimuli. The situation is exactly the same for arousal dimension. After watching the music video, participants performed a self-assessment of their levels on arousal, valence, liking, dominance, and familiarity, with ratings from 1 to 9. EEG was recorded with 32 electrodes, placing according to the international 10-20 system. Each electrode recorded 63 s EEG signal, with 3 s baseline signal before the trial. In this paper, we used the preprocessed EEG data for study, with sample rate 128 Hz and band range 4–45 Hz. EOG artefacts were removed as method in [20]. The data was segmented into 60-second trials and a 3-second pretrial baseline removed. The binary classifications of valence and arousal dimension are considered. We utilized the participants’ selfassessment as label. If the participant’s rating was <5, the label BioMed Research International 3 of valence/arousal is low and if the rating was ≥5, the label of valence/arousal is high. Each music video lasts for 1 minute, and 5 s EEG signals are extracted as a sample. So for each subject who watched 40 music videos, we acquire 480 labeled samples. 2.2. Empirical Mode Decomposition. EMD decomposes EEG signals into a set of IMFs by an automatic shifting process. Each IMF represents different frequency components of original signals and should satisfy two conditions: (1) during the whole data set, the number of extreme points and the number of zero crossings must be either equal or differ at most by one; (2) at each point, the mean value calculated from the upper and lower envelope must be zero [29]. For input signal 𝑥(𝑡), the process of EMD is as follows: (1) Set ℎ(𝑡) = 𝑥(𝑡) and ℎold (𝑡) = ℎ(𝑡). (2) Get local maximum and minimum of ℎold (𝑡). (3) Interpolate the local maximum and minimum with cubic spline function and get the upper envelope 𝑒max (𝑡) and lower envelope 𝑒min (𝑡). (4) Calculate the mean value of the upper and lower envelope as 𝑚 (𝑡) = (𝑒min (𝑡) + 𝑒max (𝑡)) . 2 (1) (5) Subtract ℎold (𝑡) with 𝑚(𝑡): ℎnew (𝑡) = ℎold (𝑡) − 𝑚 (𝑡) . (3) (6) If imf𝑛 is gotten, set ℎold (𝑡) as ℎold (𝑡) = ℎold (𝑡) − imf𝑛 . (4) Go to step (2) and repeat steps (2)–(5) to get imf𝑛+1 . By the iterative process described above, 𝑥(𝑡) can be finally expressed as 𝐿 𝑥 (𝑡) = ∑ imf𝑛 + 𝑟. 2.3.1. First Difference of IMF Time Series. The first difference of times series 𝐷𝑡 depicts the intensity of signal change in time domain. Previous research has revealed that the variation of EEG time series can reflect different emotion states [2]. For an IMF component with 𝑁 points, IMF{imf1 , imf2 , . . . , imf𝑁}, the definition of 𝐷𝑡 is 𝐷𝑡 = 1 𝑁−1 ∑ |imf (𝑛 + 1) − imf (𝑛)| . 𝑁 − 1 𝑛=1 (5) 𝑧 (𝑛) = 𝑥 (𝑛) + 𝑗𝑦 (𝑛) . (7) The analytic signal can be further expressed as follows: 𝑧 (𝑛) = 𝐴 (𝑛) 𝑒𝑗𝜑(𝑛) , (8) where 𝐴(𝑛) = √𝑥(𝑛)2 + 𝑦(𝑛)2 is the amplitude of 𝑧(𝑛) and 𝜑(𝑛) = arctan(𝑦(𝑛)/𝑥(𝑛)) is the instantaneous phase. First difference of phase 𝐷𝑝 is defined as 𝐷𝑝 = 1 𝑁−1 󵄨󵄨 󵄨 ∑ 󵄨𝜑 (𝑛 + 1) − 𝜑 (𝑛)󵄨󵄨󵄨 𝑁 − 1 𝑛=1 󵄨 (9) which measures the change intensity in phase and represents the physical meaning of instantaneous frequency. 2.3.3. Normalized Energy of IMF. For an 𝑁-point IMF, IMF{imf1 , imf2 , . . . , imf𝑁}, the normalized energy 𝐸norm is defined as follows: 𝑛=1 It is a linear combination of IMF components and the residual part 𝑟. Figure 2 shows a segment of original EEG signals corresponding to the first five decomposed IMFs. EMD works like an adaptive high pass filter. It shifts out the fastest changing component first and as the level of IMF increases, the oscillation of IMF becomes smoother. Each component is band-limited, which can reflect the characteristic of instantaneous frequency. (6) 2.3.2. First Difference of IMF’s Phase. Based on EMD, EEG is decomposed into multilevel IMFs, each IMF being bandlimited and representing an oscillation component of original EEG signals. For an 𝑁-point IMF, IMF{imf1 , imf2 , . . . , imf𝑁}, Hilbert transform is applied to it, obtaining an analytic signal 𝑧(𝑛) (2) If ℎnew (𝑡) satisfies the two conditions of IMF, then the first IMF component imf1 is gotten; otherwise, set ℎold (𝑡) = ℎnew (𝑡) and go to step (2), repeating steps (2)–(5) until ℎnew (𝑡) satisfies the two conditions of IMF. Finally imf1 is gotten as imf1 = ℎnew (𝑡) . 2.3. Feature Extraction. In this paper, three features of IMF are utilized for emotion recognition, the first difference of time series, the first difference of phase, and the normalized energy. The first difference of time series depicts the intensity of signal change in time domain. The first difference of phase reveals the change intensity of phase, representing the physical meaning of instantaneous frequency. Normalized energy describes the weight of current oscillation component. The motivation of using these three features is that they depict the characteristics of IMF in time, frequency, and energy domain, utilizing multidimensional information. 𝐸norm = 2 ∑𝑁 𝑛=1 imf (𝑛) 2 ∑𝑁 𝑛=1 𝑠 (𝑛) , (10) where 𝑠(𝑛) is the original EEG signal points. So the numerator is the energy of IMF and the denominator represents the energy of original EEG data set. The normalized energy describes the weight of current oscillation component. When fed into the classifier, log(𝐸norm ) is taken as an element of the feature vector according to [26]. 4 BioMed Research International EEG 1 0 −1 100 200 300 400 500 600 700 800 900 1000 100 200 300 400 500 600 700 800 900 1000 100 200 300 400 500 600 700 800 900 1000 100 200 300 400 500 600 700 800 900 1000 100 200 300 400 500 600 700 800 900 1000 100 200 300 400 500 600 700 800 900 1000 IMF1 0.5 0 −0.5 IMF2 0.5 0 −0.5 IMF3 0.5 0 −0.5 IMF4 0.5 0 −0.5 IMF5 0.5 0 −0.5 Points Figure 2: EEG signals and the corresponding first five IMFs. 2.4. SVM Classifier. The extracted features are fed into SVM for classification. SVM is widely used for emotion recognition [34, 35], which has promising property in many fields. In our study, LIBSVM is implemented for SVM classifier with radial basis kernel function and default parameters setting [36]. 3. Performance Verification In the following subsections, we test our method on DEAP emotional dataset. Training and classifying tasks were conducted for each subject independently and we utilized leaveone-trail-out validation to evaluate the classification performance. Each subject watched 40 music video clips, and every video clips lasted 1 minute. In our experiment, we utilized the participants’ self-assessment as label. Every 5 s EEG signals are extracted as a sample, so for each subject we acquire 480 labeled samples. In leave-one-trail-out validation, for each subject, 468 samples extracted from 39 trails were assigned to training set, and 12 samples extracted from the remaining one trail were assigned to test set. So there was no correlation between samples in the training set and the test set. Among the total 40 trails of one subject, each trail will be assigned to the test set once as the validation data. The 40 results from the 40 test trails then can be averaged to produce a general estimation for each subject. The final mean accuracy is computed among all the subjects. 3.1. Effectiveness of the Features for Emotion Recognition. In order to evaluate the effectiveness of the three features for emotion recognition, we first use only one single feature for classification each time. All the experiments in this subsection are under the condition that the first five IMF components and total 32 electrodes are utilized for feature extraction. The training and classifying for each subject were conducted, respectively, and the mean accuracy was computed among all the subjects. The mean classification accuracies of three features are given in Figure 3. It shows that all the three features can distinguish high level from low level on both valence and arousal dimension, higher than random probability of 50%. For valence dimension, the classification accuracy yields 68.27%, 64.46%, and 61.07% with features 𝐷𝑡 , 𝐷𝑝 , and 𝐸norm , respectively. For arousal dimension, the classification accuracy yields 69.89%, 67.56%, and 63.76% with features 𝐷𝑡 , 𝐷𝑝 , and 𝐸norm , respectively. 3.2. IMF Reduction for Feature Extraction. In this subsection, we did two experiments to investigate the role of different IMF components in emotion recognition. In the BioMed Research International 5 Table 1: Comparison of performance for different IMFs selected for feature extraction (32 channels) (standard deviation shown in parentheses). Valence Component IMF1 IMF2 IMF3 IMF4 IMF5 IMF1-2 IMF1–3 IMF1–4 IMF1–5 Arousal 𝑡-test (IMF1) 𝑝=1 𝑝 = 0.0002 𝑝=0 𝑝=0 𝑝=0 𝑝 = 0.4399 𝑝 = 0.2705 𝑝 = 0.1688 𝑝 = 0.1086 Accuracy (%) 70.41 (7.05) 63.47 (7.10) 61.45 (8.57) 59.55 (8.56) 55.74 (9.20) 69.02 (7.00) 68.47 (6.69) 67.99 (6.58) 67.59 (6.58) 80 Table 2: Performance of 8 channels selected for feature extraction (Fp1, Fp2, F7, F8, T7, T8, P7, and P8) (standard deviation shown in parentheses). 70 60 Accuracy (%) 𝑡-test (IMF1) 𝑝=1 𝑝 = 0.0032 𝑝 = 0.0019 𝑝 = 0.0012 𝑝=0 𝑝 = 0.1940 𝑝 = 0.3116 𝑝 = 0.2107 𝑝 = 0.1293 Accuracy (%) 72.10 (7.51) 66.58 (9.36) 64.56 (10.52) 63.99 (10.96) 62.38 (12.23) 70.47 (8.29) 70.08 (8.10) 69.60 (8.08) 69.00 (8.37) Label 50 Predict 40 30 20 10 0 Valence Arousal Dt Dp EＨＩＬＧ Figure 3: Classification accuracies of three single features. For each subject, one single feature was extracted from the first five IMF components. “𝐷𝑡 ,” “𝐷𝑝 ,” and “𝐸norm ” in the figure are corresponding to the three single features, respectively. The mean accuracies for all circumstances were computed among all the subjects. Error bars show the standard deviation of the mean accuracies across all subjects. first experiment, each time only one IMF component was utilized for feature extraction and we analyzed which IMF is effective for emotion recognition. In the second experiment, we further verified whether the combination of multi-IMFs would improve the accuracy. Table 2 gives all the results in detail. Standard deviation of the mean accuracies across all subjects is shown in parenthesis. “IMF1,” “IMF2,” “IMF3,” “IMF4,” and “IMF5” are corresponding to single IMF component. “IMF1–3” in the table represents the first three IMFs, corresponding to IMF1, IMF2, and IMF3. Similarly, “IMF1–4” and “IMF1–5” are corresponding to the first four IMFs and the first five IMFs, respectively. It shows that IMF1 yields the best performance, 70.41% for valence and 72.10% for arousal. As the level increases, the performance decreases sharply. The performance of IMF5 is only 55.74% for valence and 62.38% for arousal. We applied 𝑡-test (𝛼 < 0.05) to examine the performance between only High Low 𝐹1 score Accuracy (%) Valence High 6664 2024 Arousal Low 2723 3949 0.7374 69.10 (6.95) High 7493 1555 Low 2748 3564 0.7769 71.99 (7.77) IMF1 utilized for feature extraction and other circumstances. The null hypothesis is “the performance is similar” and if 𝑝 value is larger than 𝛼, the null hypothesis is accepted. The results of 𝑡-test in Table 1 show that the performance of IMF1 is more splendid than other single components, IMF2, IMF3, IM4, and IMF5, with 𝑝 far less than 0.05. It also shows that performance of multi-IMF combinations is similar to only IMF1 utilized for feature extraction, with 𝑝 larger than 0.05. IMF1 represents the fastest changing component of EEG signals, with the highest frequency characteristic. As the level increases, the oscillation becomes smoother with frequency becoming lower and lower. So we infer that the valence and arousal of emotion relate more tightly to high frequency. It is also coincided with the finding in [26] that Beta (16–32 Hz) and Gamma (32–64 Hz) bands are successfully selected more often than other bands. These two bands are higher frequency subbands of EEG signals. So combining the results of classification accuracy and 𝑡test, in practical use, we just need to extract features from IMF1, which will save vast time and relieve computation burden because only one level of EMD decomposition needed to be done. 3.3. Channel Reduction for Feature Extraction. Form verification in Section 3.2, we know that using component IMF1 will achieve good performance. In this subsection, we will investigate which electrodes are informative based on EMD strategy. 6 BioMed Research International Dt Fp1 Af3 F7 F3 Fc5 T7 Fc1 Cp1 P3 Po3 O1 C4 Cp2 Pz Oz P4 Fp1 Af3 0.02 F8 F7 0.01 Fc6 Fc2 Cz Cp5 P7 F4 Fz C3 0.03 Fp2 Af4 Cp6 T7 P7 −0.02 Po4 O2 Cp1 P3 F8 T8 C4 Cp2 Pz Oz 0.01 Fc6 Fc2 Cz Po3 O1 −0.03 F4 Fz C3 0.02 Fp2 Af4 Fc1 Cp5 −0.01 P8 F3 Fc5 0 T8 0.03 Dp 0.04 P4 Po4 O2 0 Cp6 P8 −0.01 −0.02 −0.04 −0.03 (a) (b) EＨＩＬＧ 0.015 Fp1 Af3 F7 F3 Fc5 T7 Fc1 Cp1 P3 Po3 O1 F8 0.005 Fc6 Fc2 Cz Cp5 0.01 F4 Fz C3 P7 Fp2 Af4 C4 Cp2 0 Cp6 −0.005 Pz Oz T8 P4 Po4 O2 P8 −0.01 −0.015 (c) Figure 4: Fisher distance of different channels with subject 1. Features are extracted from component IMF1. For each channel, Fisher distance is calculated among features extracted from 480 labeled emotion samples of subject 1. (a) Fisher distance under feature 𝐷𝑡 . (b) Fisher distance under feature 𝐷𝑝 . (c) Fisher distance under feature 𝐸norm . Fisher distance is an efficient criterion of divisibility between two classes, which is broadly used in pattern recognition. It computes the ratio of between-class scatter degree and within-class scatter degree between two classes. Larger ratio means larger divisibility of the two classes. In our experiment, we used fisher distance to mark important electrodes under condition that IMF1 is used for feature extraction. For each channel, fisher distance is calculated among features extracted from one subject’s total 480 labeled emotion samples. Figure 4 gives fisher distance on valence dimension with subject 1. Figure 4(a) shows that, under feature 𝐷𝑡 , electrodes Fp1, Fp2, FC6, Cp1, O1, and Oz have larger values. Figure 4(b) shows that, under feature 𝐷𝑝 , Fp1, FC6, Cp1, Cp2, O1, Oz, P7, and P8 have larger values. Figure 4(c) shows that, under feature 𝐸norm , F7, F8, T7, T8, P7, P8, O1, O2, and Oz have larger values. Based on the analysis of all the subjects, we selected the following 8 electrodes Fp1, Fp2, F7, F8, T7, T8, P7, and P8 for channel reduction verification. Table 2 gives 𝐹1 score and classification accuracy with 8 channels selected for emotion recognition. We see that 𝐹1 score is 0.7374 for valence and 0.7769 for arousal. The classification accuracy with 8 channels is 69.10% for valence and 71.99% for arousal, slightly lower than accuracy with total 32 channels. We also applied 𝑡-test to examine whether the performance of 8 channels is similar BioMed Research International 7 80 performance between classical method and our method. The null hypothesis is “the performance is similar” and if 𝑝 value is larger than 𝛼, the null hypothesis is accepted. The results of 𝑡-test in Table 3 show that the performance of our method is more splendid than fractal dimension, sample entropy, and differential entropy of Beta band with 𝑝 far less than 0.05. It also shows that the performance of our method is similar and better than the differential entropy of Gamma band. EMD strategy outperforms time domain method, including fractal dimension and sample entropy. This is because compared to methods in time domain, EMD has the advantage of utilizing more oscillation information. Compared to time-frequency method DWT, EMD can decompose EEG signals automatically, getting rid of selecting transform window first. The classification accuracy is also higher than DWT. So the experiment results infer that our method based on EMD strategy is suitable for emotion recognition from EEG signals. 70 Accuracy (%) 60 50 40 30 20 10 0 Valence Arousal 4. Discussion FD SampEn DWT + DE (Beta) DWT + DE (Gamma) Our method Figure 5: Classification accuracies of different methods. “FD,” “SampEn,” and “DE” in the figure are corresponding to fractal dimension, sample entropy, and differential entropy, respectively. The mean accuracy was computed among all the subjects. Error bars show the standard deviation of the mean accuracies across all subjects. to total 32 channels. The null hypothesis is “the performance is similar” and if 𝑝 value is larger than 𝛼, the null hypothesis is accepted. The 𝑡-test result shows that the performance under 8 channels and 32 channels is similar, with 𝑝 = 0.4194 for valence and 𝑝 = 0.9521 for arousal. So in practical use, we just need to extract features from IMF1 with 8 channels. Our offline experiment used every 5 s EEG signals as a labeled emotion sample. This infers that our method may provide a new solution for real-time emotion recognition in BCI systems. 3.4. Results Comparison with Other Methods. In this subsection, we compared our proposed method with some classical methods, including fractal dimension (FD), sample entropy, differential entropy, and time-frequency analysis DWT. We used box counting for fractal dimension calculating. The parameter for sample entropy SampEn(𝑚, 𝑟, 𝑁) was set as 𝑟 = 0.2, 𝑚 = 2, and 𝑁 = 128. We used “db4” decomposition to realize DWT. Then the differential entropy of Beta (16–32 Hz) and Gamma (32–64 Hz) bands is extracted as features. Our method used IMF1 for feature extraction of 𝐷𝑡 , 𝐷𝑝 , and 𝐸norm . For all the methods, 8 selected channels FP1, FP2, F7, F8, T7, T8, P7, and P8 are used for feature extraction. From Figure 5 and Table 3, we see that our method yields the highest accuracy, 69.10% for valence and 71.99% for arousal. We applied 𝑡-test (𝛼 < 0.05) to examine the Emotion recognition from EEG signals has achieved significant progress in recent years. Previous methods are usually conducted in time domain, frequency domain, and timefrequency domain. In this paper, we propose a method of feature extraction for emotion recognition in EMD domain, a new aspect of view. By utilizing EMD, EEG signals can be decomposed into different oscillation components named IMF automatically. The characteristics of IMF are utilized as features for emotion recognition, including the first difference of time series, the first difference of phase, and the normalized energy. Compared to methods in time domain, EMD has the advantage of utilizing more frequency information. The experiment results show that the proposed method outperforms method in time domain, such as fractal dimension in [3, 4] and sample entropy in [5]. Compared to time-frequency methods, such as STFT and DWT, EMD can decompose EEG signals automatically, getting rid of selecting transform window first. The classification accuracy is also higher than DWT in [18]. We investigate the role of each IMF in emotion classification. Features extracted from IMF1 yield the highest accuracy. IMF1 is corresponding to the fastest changing component of EEG signals, so our study confirms the deduction that emotion is more relative to high frequency component. This consists with findings in [26] that Beta (16–32 Hz) and Gamma (32–64 Hz) bands are successfully selected more often than other bands. Finally, we selected 8 informative channels based on EMD strategy, namely, FP1, FP2, F7, F8, T7, T8, P7, and P8. Our proposed method just needs to extract features from IMF1 with 8 channels, which will save time and relieve computation burden. Also in our experiment, every 5 s EEG signals are extracted as a sample, so it may provide a new solution for real-time emotion recognition in BCI systems. Our limitation is that now we just test it on DEAP dataset, so in the future we want to experiment it on more emotional datasets to verify the method comprehensively. Also we will 8 BioMed Research International Table 3: The mean accuracy of different kinds of methods (Fp1, Fp2, F7, F8, T7, T8, P7, and P8) (standard deviation shown in parentheses; statistical analysis shown in column 𝑡-test). Methods Fractal dimension Sample entropy DWT + differential entropy (Beta) DWT + differential entropy (Gamma) Our method Accuracy (%) 53.08 (19.14) 57.44 (11.66) 60.87 (11.74) 67.36 (6.61) 69.10 (6.95) Valence 𝑡-test (our method) 𝑝=0 𝑝=0 𝑝 = 0.0013 𝑝 = 0.3185 𝑝=1 utilize more strategies such as feature smoothing and deep network to improve the classification accuracy. 5. Conclusion In this paper, an emotion recognition method based on EMD using three statistics is proposed. An extensive analysis has been carried out to investigate the effectiveness of the features for emotion classification. The results show that the three features are suitable for emotion recognition. Then the effect of each IMF component is inquired. The results reveal that, among the multilevel IMFs, the first component IMF1 plays the most important role in emotion recognition. Also the informative channels based on EMD strategy are investigated and 8 channels, namely, FP1, FP2, F7, F8, T7, T8, P7, and P8, are selected for feature extraction. Finally, the proposed method is compared with some classical methods and our method yields the highest accuracy. Conflicts of Interest The authors declare that there are no conflicts of interest regarding the publication of this paper. Acknowledgments This work was supported by the grant from the National Natural Science Foundation of China (Grant no. 61701089). References [1] C.-H. Han, J.-H. Lim, J.-H. Lee, K. Kim, and C.-H. Im, “Datadriven user feedback: an improved neurofeedback strategy considering the interindividual variability of EEG features,” BioMed Research International, vol. 2016, Article ID 3939815, 7 pages, 2016. [2] K. 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Decorum. An Ancient Idea for Everyday Aesthetics? Elisabetta Di Stefano Everyday Aesthetics was born in the 21st Century as a sub-discipline of Anglo-American Aesthetics and it has spread in the international debate. However, the contribute of historical perspective has not properly explored yet. Is it possible to trace the history of everyday aesthetics before the official birth of this discipline? I will try and give an affirmative answer by focusing on an exemplary category: that of the decorum. Using the history of ideas, I will analyse the Greek concept of prepon and the similar Latin concepts of decorum which express the idea of ‘convenience’ or ‘fitness to purpose’ in the ethical and rhetorical sphere. Later I will analyse the evolution of the concept of decorum in the theory of Ancient and Renaissance architecture (Vitruvius, Leon Battista Alberti). My goal is to demonstrate that in Ancient and Renaissance culture decorum is a category that refers to the objects and practices of everyday life but also a principle that regulates appropriate behaviour in the sphere of good manners. Consequently, given its pervasiveness in the different areas of everyday life, the concept of decorum can be a paradigmatic example to trace the history of everyday aesthetics. | Keywords: Decorum, Everyday Aesthetics, Prepon, Good Manners, History of Ideas 1. Introduction Originated at the start of the new Millennium as a sub-discipline of AngloAmerican aesthetics focusing on art, Everyday Aesthetics has distinguished itself for its shift towards practices and objects of everyday life. Several fields and varied aesthetic categories have been included in its inquiries, nevertheless one line of investigation has received little attention until now, that is to say, the historical realm. This essay aims to go back to the early origins of Western culture in the attempt to trace the steps of the history of everyday aesthetics before its official birth. To this aim I will rely on the methodology of the history of ideas in the footsteps of the Polish philosopher Władysław Tatarkiewicz (1980). His method turns out to be particularly fitting for my inquiry seeing as Tatarkiewicz developed the history of aesthetics including all ideas affecting aesthetic issues, even when presented under different names, and belonging to other disciplines not only philosophical. Vol. 10/2 2021 25 Tatarkiewicz’s historiographical approach allows us to find a way out of the traditional impasse inherited from the early historians of aesthetics, Robert Zimmerman (1858) and Benedetto Croce (1902). They firmly held onto a general framework excluding all contributions not produced by philosophers, and inevitably got bogged down while attempting to legitimize a history of aesthetics also predating the eighteenth-century foundation of the discipline. By separating the history of words from that of concepts, Tatarkiewicz claimed that aesthetic thinking began in Europe more than two-thousand years before a name was found for it and an autonomous field of research was established. Considering that everyday practices and objects had rarely attracted philosophical interest before, usually being seen as too trivial, this method, that targets on implicit aesthetics, proves to be suitable above all for everyday aesthetics. The reason for this is that it aims at including a variety of concepts touching upon the field of aesthetics by relying not only on philosophical texts but also on artistic and literary contributions, technical textbooks and private documents. Needless to say, a full outline of the history of everyday aesthetics is an ambitious goal far beyond the limits of this essay. I will therefore limit myself by outlining the origins of one single aesthetic idea, that of decorum. This notion has an exemplary value, inasmuch as it cuts across several cultural realms. While changing its names in the course of different periods, it preserves a consistent meaning: ‘aptness’, ‘convenience’, ‘fitness to purpose’. Tatarkiewicz includes this notion among the varieties of beauty and focuses on its terminological transformations: From ancient times regarded as a variety of beauty has been aptness, specifically the aptness of things to the task the things were meant to fulfil, to the purpose that they served. The Greeks called this quality πρέπον; the Romans translated the expression as decorum. […] Later, in Latin, the name aptum was used more frequently, but in the Renaissance decorum returned. Frenchmen of the ‘Great [17th] Century’ most often called this property bienséance, Poles of the age spoke of przystojność. Today one speaks rather of suitability, appropriateness, purposefulness and functionalism as a quality of certain arts and the cause of the pleasure that we find in them. The terminology has varied, but the concept itself has persisted.” (Tatarkiewicz, 1980, p. 159) Among the above listed terminological variations, decorum is in my opinion the most interesting, inasmuch as it qualifies as a norm regulating the beauty of both behaviour and architecture, two realms in which the Italian language still uses the same Latin-derived word decoro. The notion of decorum was first conceptualized by Cicero as a norm of rhetoric and as a principle of everyday life (Orat. 21, 70): “In an oration, as in life, nothing is harder than to determine what is appropriate. The Greeks call it πρέπον; let us call it decorum or ‘propriety’” Cicero, 1952, p. 357). Starting from Cicero’s early definition and in reference to the history of ideas, I will first explore the Greek notion of πρέπον from which the Latin concept stems; then I will focus on decorum as conveying the idea of ‘convenience’ and ELISABETTA DI STEFANO Decorum. An Ancient Idea for Everyday Aesthetics? 26 ‘fitness to purpose’ in the rhetorical and ethical sphere. Finally, I will analyse the evolution of decorum in the Ancient and Renaissance architectural theory (Vitruvius, Leon Battista Alberti) and in the field of good manners. My goal is to demonstrate that decorum – and its analogous terms – is a category that not only refers to the objects and practices of everyday life but is also a principle that regulates appropriate behaviour. Consequently, given its pervasiveness in the different areas of everyday life, the concept of decorum can be a paradigmatic example to trace the history of everyday aesthetical ideas. 2. The concept of πρέπον in Greek culture As emphasised by Pohlenz (1933, p. 53), the substantivized adjective to prepon has many meanings. In archaic Greek it designates a shining quality, a conspicuousness which, like the virtues of Homeric heroes, stands out before the eyes of the beholders (Homer, Iliad, XII, 104). However, the timelessness of the myth fades away when the term to prepon is coupled with the noun kairos, ‘occasion’. The temporal determination provides a new meaning to to prepon and connects it to what Mario Perniola defines as ‘actual beauty’, that is to say, a type of beauty waiving off its absolute and universal value to adapt to given circumstances (Perniola, 1982, p. 45). The connection between ‘convenience’ (prepon) and ‘occasion’ (kairos) is abundantly documented in Greek literature and it is particularly powerful in technical writings about medicine, politics and rhetoric. This bond describes words and actions being effective, since they are placed in a given space-time configuration (i.e. the decisive place, the crucial moment) and therefore they are well adapted to the situation (Trédé, 1992). In the field of rhetoric, the link between prepon and kairos was first established by Gorgias and then reinforced by Isocrates, according to whom discourses cannot be beautiful if they are not attuned to the circumstances and befitting the topic (Soph. 13). Furthermore, Isocrates was the first to confer an educational and political value to the notion of prepon connecting it to the issue of paideia, that is to say, the education of youth in relation to which the rhetor is the life mentor. In the realm of philosophy, Socrates plays a key role in the understanding of the idea of prepon. Since he left no autographic writtings, his legacy can be evinced from the dialogues of Plato and Xenophon, which often feature Socrates in the main role. In the Platonic dialogue, Hippias Major (289d-290e), talking about utensils, Socrates claims that not all materials are well adapted to all shapes, but only to those for which they are ‘appropriate’. For stirring a bean soup, a fig wooden spoon is more appropriate than a golden one because “it makes the soup smell better, and at the same time, […] it won’t break our pot, spill out the soup, put out the fire, and make us do without a truly noble meal, when we were going to have a banquet” (Plato, 1982, p. 13). However, while Plato’s Socrates is mainly concerned with metaphysical ELISABETTA DI STEFANO Decorum. An Ancient Idea for Everyday Aesthetics? 27 questions and the quest for beauty in itself (i.e. the idea of beauty), Xenophon’s Socrates launches the notion of functional beauty. This latter, which is to be found again in the Latin concept of aptus (appropriateness to purpose), will survive through Medieval theoretical contributions – as testified by Agustine’s surviving treatise, De pulchro et apto (Tatarkiewicz, 1980, p. 160) – until modern functionalism many centuries later. Socrates’s key role in the elaboration of an aesthetic category pertaining to everyday life is made clear by his many references to domestic environments and objects of daily use. In a passage from Xenophon’s Memorabilia (III, 8, 4), Socrates claims that a golden shield, although beautiful to look at, is not suited to a battle since gold is too fragile to guarantee safety. The careful choice of materials then translates into care for the people who are going to use them and for their needs. This is clearly stated in a later section of the Memorabilia (III, 10, 9-13). Talking to the armorer Pistias, who is bragging about his breastplates and how they are well-made because they are well-proportioned, Socrates claims that an armour should not be perfectly well-proportioned, but in relation to the wearer. As a matter of fact, unfitting armours, as they hang from one’s shoulders or burden some other part of the body, are oppressing and difficult to wear, while those which are fitting should look like “an accessory rather than an encumbrance” (Xenophon, 1979, pp. 237-239). Furthermore, the notion of convenience is abundantly discussed by Aristotle in his contributions both to rhetoric and to philosophy. He defines convenience as the right correspondence between the language and the character of the rhetor (Reth. III, 7,1408 a-b) and in the Poetics (13, 1454a) he emphasises how each character is supposed to use words and perform actions that are consistent with their character. This line of thought has then been further developed in Theophrastus’ Characters. Finally, among the most important Greek theoretical contributions on the notion of prepon, one should mention Panetius, a member of the middle Stoa. While breaking with the early Stoa and its idea of the wise life as isolated from the world, Panetius addresses all people, who while dealing with everyday affairs have to make choices and perform actions. According to Panetious, people should use their abilities and gifts to the benefit of society by following the prepon which guides everybody to do the right thing at the right time (Cicu, 2000, pp. 136-137). 3. Cicero and decorum The close correspondence between the Greek word prepon and the Latin decorum is clearly stated by Cicero in the texts Orator (21,70) and De Officiis (I, 93). Although the two expressions are similar, the notion of decorum undergoes some evolution in meaning in the transition between the text on rhetoric written in 46 BC to the one on ethics written two years later. It would actually be wrong to believe that the notion of decorum as presented in the Orator is simply a tool of rhetoric. This latter is not only an operational field connected to politics, but also one in which decorum is presented as ELISABETTA DI STEFANO Decorum. An Ancient Idea for Everyday Aesthetics? 28 a guiding principle, whose validity extends beyond the rhetoric to the arts and life in general (Guérin, 2009, p. 125). Nevertheless, an even stronger connection between ethics and aesthetics can be found in the De Officiis, inasmuch as decorum is introduced as the fourth part of the honestum dealing with the appropriateness of discourses and daily actions performed within one’s community. In the rhetorical field, decorum is a virtue of style, harmoniously regulating the relations between speaker, message, audience, and register of communication (Orat. 21, 71). Latin rhetoric distinguishes three styles, genus tenue or plain, genus medium and genus grande or grand. Decorum should see that each genre is matched with the appropriate arguments, styles and audiences. Despite appearing at first as a normative principle with codified rules, decorum is introduced by Cicero when dealing with the difficulties connected to understanding what is appropriate both in discourses and in life in general (Orat. 21, 70: “ut […] in vita sic in oratione nihil est difficilius quam quid deceat videre”; “In an oration, as in life, nothing is harder than to determine what is appropriate” Cicero, 1952, p. 357). It should be added that, in the realm of ethics, grasping the appropriateness of words and actions with respect to circumstances is the result not of rational intelligence but of a form of sensibility which is able to perceive nuances, atmospheres and moods. Jumping ahead to a notion engendered within the milieu of Romanticism, one might say, with André Demouliez (1976, p. 286), that convenience belongs to the realm of taste, provided it is not taste itself. This remark is confirmed by Quintilian (Inst. Or. 11, 1, 91); while connecting decorum to measure and temperance, he states that convenient behaviour cannot be guided by pre-established rules but only by sensitivity. We might refer to it as a certain taste, as suggested by Quintilian’s analogy with food, where, rules are of little value. Inspired by the philosopher Panetius, in De officiis Cicero emphasises the bond between decorum and moral integrity, which is applied to interpersonal relations within the community, in the following terms: We have next to discuss the one remaining division of moral rectitude. That is the one in which we find considerateness and self-control, which give, as it were, a sort of polish to life; it embraces also temperance, complete subjection of all the passions, and moderation in all things. Under this head is further included what, in Latin, may be called decorum (propriety); for in Greek it is called πρέπον. Such is its essential nature, that it is inseparable from moral goodness; for what is proper is morally right, and what is morally right is proper. The nature of the difference between morality and propriety can be more easily felt than expressed. (Cicero, De off., I, 27, 93; 1928, pp. 95-97) Self-control, temperance and moderation make up the essence of decorum and provide aesthetic value to everyday life (quidam ornatus vitae, ‘a sort of polish to life’), making one’s manners gentle and one’s behaviour refined, while respecting not only those who belong to the high society but to all citizen. As it was in Greek culture, also in Latin culture decorum is a form of beauty. ELISABETTA DI STEFANO Decorum. An Ancient Idea for Everyday Aesthetics? 29 This is confirmed by Cicero’s analogy between the harmony of the body and the appropriateness of one’s conduct. However, whereas physical beauty is visible to the eye, decorum is a ‘relational’ aesthetic category. In other words, it is a behavioural beauty which shines through words and daily actions and sparks approval in the community. For, as physical beauty with harmonious symmetry of the limbs engages the attention and delights the eye, for the very reason that all the parts combine in harmony and grace, so this propriety, which shines out in our conduct, engages the approbation of our fellow-men by the order, consistency, and self-control it imposes upon every word and deed. We should, therefore, in our dealings with people show what I may almost call reverence toward all men – not only toward the men who are the best, but toward others as well. […] It is the function of justice not to do wrong to one's fellow-men; of considerateness, not to wound their feelings; and in this the essence of propriety is best seen. (Cicero, De off., I, 28, 98-99; 1928, pp. 101-103) This form of beauty that glows in a correct behaviour is the distinctive feature of the vir bonus (honest man), whose ideal portrait is painted by Cicero in De officiis. This would be someone who is active in public and political life, and who spends their free time (otium) engaging in the arts and philosophical studies. By nurturing a sense for aesthetics and abiding to the principles of honesty and convenience, this person achieves elegance in their being and acting. As he outlines this ethical and aesthetic model of the ideal person, Cicero includes in De officiis (I, 126-134) some sort of ‘manual of good manners’, suggesting hygiene and behavioural norms inspired by the right measure and avoiding all excess. He recommends bodily hygiene without affectation but also without negligence; a way of walking neither too fast, nor too slowly; a clear and fluent way of conversing, which avoids excluding others from the conversation or falling into tittle-tattle (Cicu, 2000, pp. 150-154). As a result, Cicero’s ideal person, in close resemblance to Roman models such as Scipio Aemilianus, is well-read, balanced, never out of place or out of measure, with a strong sense of attachment to their homeland and community. As Guérin (2009, p. 126) points out “the decorum described in Cicero’s De officiis is […] a principle of coherence between the ethical agent and his actions, a means to reach a state of general appropriateness, the rational convenientia by the virtue of which one can make the choices and accomplish the actions which correspond to his own nature”. As a result, Cicero applies to his vir bonus model the same criterion of convenience, based on which the poets choose only those words and actions that are befitting of a character – according to the previously mentioned Aristotelian principle consolidated by Theophrastus. Along the same line one can place Quintilian’s contribution on the topic of convenience in the XI book of his treatise, Institutio oratoria. According to Quintilian as well, decorum is a norm regulating the choice of content, the distribution of words, the style and even the performance of the rhetor. ELISABETTA DI STEFANO Decorum. An Ancient Idea for Everyday Aesthetics? 30 However, as it shares its lexical root with the impersonal verb decet (it is convenient, it is befitting), decorum qualifies also as a moral principle, guiding choices and behaviours even to the detriment of personal interest. Quintilian mentions the example of Socrates, who refrained from simple personal defence in court, which would have saved him from being sentenced to death, since it was contrary to his moral values. Hence, the rhetor has to refrain from persuasion if this latter clashes against a higher value (Inst. Or. 11, 1, 11). As a result, also for Quintilian, decorum is a rhetorical norm regulating the rhetorical performance as much a moral principle, guiding one’s lifestyle and the consistency of thinking and acting. Indeed, in the footsteps of Cicero (De or. 3, 212), he claims that the main virtue of the orator is prudentia (practical sagacity). This is a moral virtue that can be acquired with experience and its effects derive from the ability to grasp what is appropriate in each occasion. In this regard, Latin culture establishes decorum as an ethical and aesthetic category which describes the ideal of an honest person (Cicero) and of a rhetor engaged in the rightful and appropriate administration of public affairs (Quintilian) for the good of the community. 4. The Architectural Theory: Vitruvius and Leon Battista Alberti The migration of the notion of decorum into the area of art – already inaugurated by the numerous architectural metaphors employed by Cicero (De or. III, 152 and Or. 50.) – was further legitimized by Vitruvius, who imported into architecture many terms and concepts from rhetoric. According to Vitruvius, architects have to design a building taking into account beauty (venustas), utility (usus) and appropriateness (decor) (De arch. VI, 10). In De architectura (IV, I, 7-8) decor is the principle that connects the use of the various orders of architecture to the character of the divinity to whom a temple is dedicated: the Doric style, simple and sober, is appropriate to gods who manifest strength and military valor; the Ionian style is appropriate to female deities; the Corinthian style with its delicate ornaments and floral motifs to the youngest and most graceful gods. Whereas Vitruvius is mainly receptive to the normative aspect of the principle of convenience, Leon Battista Alberti also picks up on its moral value. In line with Cicero’s and Quintilian’s teachings, he claims that a good architect must be able to evaluate ‘what is fitting’ to each building since each of them has its own specific ‘character’ (Alberti 1988, IX, 10, p. 315: “The greatest glory in the art of building is to have a good sense of what is appropriate”). Paradigmatic remarks are then provided concerning the differences between the palace of a prince and the fortress of a tyrant: A royal palace should be sited in the city center, should be of easy access, and should be gracefully decorated, elegant, and refined, rather than ostentatious. But that of a tyrant, being a fortress rather than a house, should be positioned where it is neither inside nor outside the city. Further, whereas a royal dwelling might be sited next to a showground, a temple, or the houses of noblemen, that of a tyrant should be set well back on all sides from any buildings. In either case an appropriate and useful guideline, which will lend the building ELISABETTA DI STEFANO Decorum. An Ancient Idea for Everyday Aesthetics? 31 dignity, will be to construct it in such a way that, if a royal palace, it should not be so large that it is impossible to throw out any troublemaker, or, if a fortress, not so constricted that it resembles a prison more than the apartment of a fine prince.” (Alberti 1988, V, 3, pp. 121-122, emphasis mine) Decorum is therefore connected to the idea of dignitas (dignity)1, which conveys the ethical and aesthetic distinctive features of individuals with respect to their character and social status. While developing this line in book VIII of his treatise on architecture, Alberti claims that decorum establishes the amount and type of ornaments for different buildings taking into account hosted functions and the social prestige of their inhabitants.2 Alberti then outlines an ascending scale of aesthetic values from private dwellings to public and religious buildings. These latter are said to require greater decorative richness as no house can be more beautiful than the house of God. As a result, the ornament is no longer just an additional and decorative element, and is perfectly integrated to the structure, characterizing each building according to the principle of decorum, understood as both an ethical and aesthetic measure. Besides the decorum of the rhetorical tradition, Alberti also retrieves – although just in a quick implicit remark – the Socratic notion of prepon as functional beauty. Socrates’ criticism of the golden shield, which despite being aesthetically pleasing is of little use in battle, seems to find an echo in Alberti’s words, as they criticize the “doors like those about which we read in historians and poets, so heavily weighed down with gold, ivory, and reliefs that they could be opened only by a team of men, and would give off a terrifying creak” (Alberti 1988, VII, 12, p. 226). On the contrary, those less elaborate and light, “that are easy to open and close”, should be appreciated more because they are more functional. The idea of beauty as aptness or fitness to purpose, already launched by the Socratic prepon, is reborn along the centuries under many other names. As emphasised by Tatarkiewicz: During the Enlightenment, the concept of beauty became still more strongly bound up with the concept of aptness; in that period, aptness had advocates especially among the philosophers, essayists and aestheticians of Britain: no longer now in the sense of social aptness, but once again in the sense of utility, as formerly in Greece. David Hume wrote (Treatise, 1739, vol. II) that the beauty of many human works derives from their utility and fitness for the purpose which they serve. Likewise Adam Smith (Of the Beauty which the Appearance of Utility Bestows upon all Productions of Art, 1759, part IV, chap. I): The effectiveness of any system or machine in producing the purpose for which they were designed, lends beauty to the entire object. And in the same vein, Archibald Alison (Essays on the Nature and Principles of 1 The connection between the concept of ‘dignity’ (dignitas) and that of ‘convenience’ (decor / decorum) is found in the treatise De pictura II, 38, (Alberti 1980, p. 67: “Dignity must be observed in everything. It would not be suitable to dress Venus or Minerva in a servant's hood nor to dress Mars or Jupiter in female clothes” (my transl.). 2 Alberti (1988 VIII, 3, p. 250) claims: “In these matters I do feel, however, that even when the dignity of the individual is considered, a sense of measure must be maintained, and that even kings may be criticized for overexpenditure.” ELISABETTA DI STEFANO Decorum. An Ancient Idea for Everyday Aesthetics? 32 Taste, 1790) said that there is no shape that does not become beautiful when it is perfectly suited to its purpose. For these writers the field of beauty continued to be split: some objects possess their own beauty, others acquire it thanks to their utility. (Tatarkiewicz, 1980, p. 161) While the Socratic prepon will launch a line of thinking on architecture and objects of use which will reach modern functionalism, the ethical and aesthetic notion of decorum will sustain the debate on good manners and what is befitting of those who wish to be part of ‘high society’. 5. The Good Manners The ethical notion of dignity (in Italian decoro) recurs in a lot of books on good manners. Already in The Book of the Courtier written by Baldassare Castiglione in 1528, one reads that the perfect courtier must display courtesy and most of all ‘discretion’ – that is to say, the ability to act appropriately according to the circumstances (Castiglione 1901, par. 7-8, pp. 82-83). The Courtier enjoyed great literary success in sixteenth-century Europe, and it would be misleading to take it as just a handbook of good manners, like those popping up in the following centuries starting with Giovanni Della Casa’s Galateo. Already bucking the trend of his time and the decline of court values, Castiglione aims to outline the ideal figure of the courtier, who is able to bestow beauty on everyday life through an elegant and graceful behaviour. The distinctive sprezzatura which shapes all of the courtier’s behaviours is a refined art which avoids all artificiality. Virtue is indeed achieved, according to Castiglione, when that affectation is kept at bay, that is to say, when each activity is distinctively natural, simple and modest. However, Castiglione’s ideal courtier belongs to the already faded Renaissance world. In the sixteenth century, the courts of the great Italian lords resemble more a theatre stage, where a role needs to be played, that is to say, one’s behaviour needs to be adapted to what ensures the prince’s or leader’s benevolence; as a result, courtesy turns into sterile formalism and etiquette. The several treatises on good manners of the following centuries bear testimony to the need to establish a code of norms defining the behaviour of high society. Social aesthetics is hence developed on the basis of a shared ceremonial, which is often more a matter of appearance than a real expression of virtue. As Tatarkiewicz points out, over time the notion of aptness appears under other names, particularly in seventeenth-century French classicist theory: ‘convenance’, ‘justesse’, and especially ‘bienséance’.3 The change was not only one of terminology. A fairly significant shift in thinking had taken place: the concern now was less with qualities of things fitting them to their use, and more with qualities of a man fitting him for his social station: a man is pleasing when his appearance and behaviour match his estate and dignity. (Tatarkiewicz, 1980, p. 161) 3 As Tatarkiewicz (1980, p. 161) points out: “According to Dictionnaire de l'Academie Française (1787 edition), ‘bienséance’ signifies ‘convenance de ce qui se dit, de ce qui se fait par rapport a l’age, au sexe, au temps, au lieu etc.” ELISABETTA DI STEFANO Decorum. An Ancient Idea for Everyday Aesthetics? 33 The issue of behavioural appropriateness finds ample development in French and English debates in the seventeenth and eighteenth century, leading to the origins of the person ‘of good taste’, a refined, elegant person, who is tactful and able to select appropriate words and perform appropriate actions in each moment of everyday life. 6. Decorum as a Category for Everyday Aesthetics This short historiographical journey has shown that the notion of convenience has crossed several cultural fields (philosophy, rhetoric, art, good manners) and has received many names (prepon, decorum, decor, biénseance). What remains to be investigated is whether this notion can be rightfully credited with exemplary value with regard to everyday aesthetics. Although Cicero presents prepon/decorum as operating within every aspect of life, the propounders of Everyday Aesthetics do not seem to be familiar with the Greek-Latin notion, despite often speaking of fitness for purpose and convenience in relation to circumstances. Within the Anglo-American contemporary debate, only Glenn Parson and Allen Carlson (2008, pp. 2-4) directly refer to the Socratic prepon in order to provide their theory of functional beauty with a historical foundation. However, their inquiry focuses on the question of whether beauty results from ‘being fit’ or ‘looking fit’ for function. They thus forget that, according to the rhetorical theory of prepon/decorum, there is no dyscrasia between the two options. Both Aristotle and Cicero (De or. III, 45, 179) conceive the perfection of the discourse on the model of the human body, that is beautiful because each organ fits its specific function. Following the rhetorical tradition, the architect Leon Battista Alberti compares buildings to living organisms (“a building is very like an animal”; Alberti 1988, IX, 5, p. 301), and on this ground points to the unity of ‘being’ fitting to purpose and ‘looking’ fitting to purpose: Take the case of a horse: they realized that where the shape of each member looked suitable for a particular use, so the whole animal itself would work well in that use. Thus, they found that grace of form could never be separated or divorced from suitability for use. (Alberti 1988, VI, 3, p. 158) Without mentioning rhetoric but staying in the same line, Jane Forsey (2013, p. 238) connects the idea of function with those of ‘character’ and ‘active use’ that are reminiscent of the Aristotelian ideas of ‘character’ and ‘enèrgheia’4: “The functional beauty […] marks out its everyday character, which can be experienced only through active use as demanding singular and specific attention”. Closer to Everyday Aesthetics, the contributions of Japanese-American scholar Yuriko Saito and Finnish philosopher Ossi Naukkarinen deserve particular attention here. In her famous book Everyday Aesthetics (2007, p. 7), focusing on “the care and 4 According to Aristotles, (Met. IX, 6, 1048b e) the act (enérgheia) is the existence itself of the object. ELISABETTA DI STEFANO Decorum. An Ancient Idea for Everyday Aesthetics? 34 respect for the materials, users, and dwellers”, Saito echoes, unawarely, the notion of appropriateness or adaptedness to purpose already developed in the classical era through the Greek word prepon and the Latin decorum. Furthermore, while repeatedly mentioning Archibald Alison, Saito mainly references his association theory and the emotions evoked by natural environments. She therefore does not seem to grasp the sense of Alison’s contribution to the development of an idea of beauty as aptness or fitness to purpose.5 As already discussed elsewhere (Di Stefano 2020), Alison retrieves, without particular theoretical rigor, the topos of the expressive qualities of architectural orders6, which is first to be found in Vitruvius’s De architectura, but then focuses in particular on everyday objects. Concerning furniture, machines and tools, he claims, “nor is there any form which does not become beautiful, where it is found to be perfectly adapted to its end.” (Alison 1821, p. 281) Although Saito (2017, p. 125) discusses the issue of the appropriateness of clothing to different circumstances and cultures, she fails to acknowledge that Alison had already developed a wide investigation on this topic. According to the Scottish philosopher, no garment is beautiful in absolute terms: the colours of clothes should fit the situation and the person wearing them (Alison 1821, pp. 176-178). Bright colours suit young people, sober ones the elderly, and the colours of a prince’s garments are different from those of a farmer: “the dresses in every particular performance had some relation to the character of that performance, and to the emotion it is destined to excite in our mind.” (Alison, 1821, p. 247) Alison’s remarks influenced the American cultural debate of the late nineteenth century and had an impact on the theoreticians of modern functionalism. One can indeed hear the echo of Alison’s words in American sculptor Horatio Greenough, one of the greatest propounders of American functionalism (Ringe, 1960, pp. 314-321), as he says that: “The most beautiful chairs invite you by a promise of ease.” (Greenough, 1947, p. 122) According to Greenough, beauty is the ‘promise of function’ and it expresses a proportion in relation to action and character.7 Alison also influenced Ralph Waldo Emerson, who established an insoluble connection between beauty and convenience.8 Along the line traced by Greenough and Emerson, also Louis H. Sullivan developed contributions which have earned him the title of father of modern functionalism (Di Stefano 2012). Over the centuries the category of decorum has undergone several 5 See Tatarkiewicz (1980), pp. 159-161, in particular on Alison, p. 161. 6 Alison (1821, p. 256) claims: “The Tuscan is distinguished by its severity; the Doric by its simplicity; the Ionic by its elegance; the Corinthian and Composite by their lightness and gaiety. To these characters, their several ornaments are suited with consummate taste.” 7 Greenough (1947, p. 71) points out: “I define Beauty as the promise of Function; Action as the presence of Function; Character as the record of Function […] but so long as there is yet a promise of function there is beauty, proportioned to its relation with action or with character.” 8 See Hopkins (1951, pp. 78-80). ELISABETTA DI STEFANO Decorum. An Ancient Idea for Everyday Aesthetics? 35 transformations in relation to its different philosophical backgrounds. In Saito’s contributions, for instance, the care for the materials, the users, and the inhabitants is enriched with perceptual and sensorial nuances previously unheard of, especially in the aesthetic debate predating the eighteenth century. As a result, Saito’s concept of convenience acquires connotations which are closer to sensibility than to normativity. The importance given to sensibility is clearly expressed when Saito attempts to reconcile aesthetic and functional criteria in the design of objects and buildings, by emphasizing the need to take into account the physical and psychological effects that objects and environments will produce in their users: “a design process also engages the moral capacity of care and respect for other people”, overall people with special needs, for instance, children, elderly people, patients, people with disabilities and refugees (Saito 2017, p. 227). Along the same line Saito polemicizes against the narcissistic and selfreferential trends in contemporary architecture, which make it arrogant if not alienating, and hopes instead for “an architecture of courtesy and attention.” (Saito 2007, p. 221) Connecting the field of architecture and good manners, Saito is unconsciously in line with Tristan Edward’s teaching (Edward 1944), who wrote a book titled Good and Bad Manners in Architecture, thus reconciling the two evolutionary lines of decorum: the architectural theory and the behavioural precepts. According to the Welsh architectural critic and town planner, there are selfish and presumptuous buildings, like skyscrapers, or even rude buildings. To contrast these, he suggested polite and sociable buildings. As we have seen, courtesy would in origin stand for the respect of given rules of conduct and the being endowed with certain virtues (kindness, generosity) which used to be a prerequisite for the members of the court. In a leap from the Renaissance to the present time, within the line of the notion of appropriateness and convenience applied to everyday behaviour, the remarks on tact presented by another supporter of Everyday Aesthetics, the Finnish scholar Ossi Naukkarinen, find an appropriate collocation. He claims that tact is to be understood as a behavioural mode which is appropriate to given circumstances and respectful of others (Naukkarinen 2014). To behave tactfully is really important in all those sectors where a given behavioural etiquette is in place (e.g. at work; in politics; in social relations). In the globalized and multiethnic world of today, it is easy to crash against customs stemming from different cultural values and behavioural norms. In this context, to use tact means to follow a form of sensibility related to the situation, selecting actions based on circumstances. 7. Conclusion Following Władisław Tatarkiewicz’s example and traced path, I tried to show that, since Antiquity and through the Renaissance, decorum has been a category encompassing both objects and practices of everyday life as well as the principle that regulated appropriate behaviour. ELISABETTA DI STEFANO Decorum. An Ancient Idea for Everyday Aesthetics? 36 Although it might at first seem a normative principle, since Antiquity decorum has had an ancipital meaning: it has both a normative aspect and an aspect connected to natural instinct. The former appears to be prominent in rhetoric and architecture, where the fitting ornaments for each discourse (rhetorical figures) and for each building are established. The notion of aptness regulates also the design of objects and affects also the realm of behaviour, whenever strict rules of conduct need to be followed (e.g. etiquette, diplomacy). However, rules do not always provide the fitting solution in relation to changing circumstances, and a natural instinct need to take over. In the realm of social relations, this kind of sensibility is called tact. By means of this short historical journey, I have tried to demonstrate to what extent the concept of decorum has pervaded all spheres of everyday life and to what extent it not only belongs to the categories of everyday aesthetics, but it also allows us to trace the history of Everyday Aesthetics before its official birth. References Alberti, L.B. (1980) De pictura. ed. by C. Grayson, Roma-Bari: Laterza. Alberti, L.B. (1988) On the Art of Building in Ten Books. Translated by J. Rykwert, N. Leach, R. Tavernor. Cambridge Massachusetts - London England: The MIT Press. Alison, A. (1821) Essays on the Nature and Principles of Taste. Artfor: George Goodwin & Sons. Anastatic reprint Hildescheim: G. Olms, 1968. Castiglione, B. (1901) The Book of the Courtier. Translated by L. Eckstein Opdycke, New York: Charles Scribner’s Sons. Cicero (1928) De Officiis. Translated by W. Miller. London: William Heinemann Ltd- New York: G.P. Putnam’s Son. Cicero (1952) Orator. Translated by H.M. Hubbell. London: William Heinemann Ltd/ Cambridge (Mass.): Harvard University Press. Cicu, L. (2000) ‘Cicerone e il prepon’, Paideia, LV, pp. 123-162. Croce, B. (1994) Aesthetic as Science of Expression and General Linguistic. London: Routledge. Demouliez, A. (1976) Cicéron et son goût. Essai sur une définition d'une esthétique romaine de la fin de la République. Bruxelles: Latomus. Di Stefano, E. (2012) ‘The Aesthetic of Louis H. Sullivan: Between Ornament and Functionality’, in J. Gleiter (ed.) Ornament Today. Bolzano: Bozen University Press, pp. 64-75. Di Stefano, E. (2020) ‘Archibald Alison e l’estetica del quotidiano? Una prospettiva neoestetica’, Aesthetica Edizioni Preprint n. 113, pp. 123-132. DOI: 10.7413/03938522018. Dowling, Ch. (2010) ‘The Aesthetics of Daily Life’, The British Journal of Aesthetics, 50:3, pp. 225-242. Edwards, A. T. (1944) Good and Bad Manners in Architecture: An Essay on the Social Aspects of Civic Design. London: John Tiranti. Forsey, J. (2013) The Aesthetics of Design. Oxford: Oxford University Press. Greenough, H. (1947) Form and Function. Remarks on Art, Design and Architecture. Ed. by H. A. Small Berkeley; Los Angeles; London: University of California Press. Guérin, Ch. (2009) ‘Philosophical Decorum and the Literarization of Rhetoric’, in F. Woerther (ed.) Literary and Philosophical Rhetoric in the Greek, Roman, Syriac and Arabic World. Hildesheim: Georg Olms Verlag, pp. 119-139. Hopkins, V.C. (1951) Spires of Form: A Study of Emerson's Aesthetic Theory. Cambridge (Mass.): Harvard University Press. ELISABETTA DI STEFANO Decorum. An Ancient Idea for Everyday Aesthetics? 37 Naukkarinen, O. (2014) ‘Everyday Aesthetic Practices. Ethics and Tact’, Aisthesis, 1, pp. 23-44. Parsons, G. - Allen, C. (2008) Functional beauty. Oxford: Clarendon Press. Perniola, M. (1982) Dal πρεπόν al decorum. Note sulla bellezza effettiva, Rivista di estetica, 12, XXII, pp. 44-52. Plato (1982) Hippias Major. Translated by P. Wooddruff. Oxford: Basil Blackwell. Pohlenz, M. (1933) ‘To πρεπόν. Ein BeitragzurGeschichte des griechischenGeistes’, Nachrichten der Gesellschaft der Wiss. zu Göttingen, Philolog.-histor. Kl., 16, pp. 53-92. Quintilian (2002) The Orator's Education. Edited and translated by D.A. Russell. Cambridge, MA: Harvard University Press. Ringe, D.A. (1960) ‘Horatio Greenough, Archibald Alison: And the Functionalist Theory of Art’, College Art Journal, 19(4), pp. 314-321. Saito, Y. (2007) Everyday Aesthetics. Oxford: Oxford University Press. Saito, Y. (2017) Aesthetics of the Familiar. Everyday Life and World-Making. Oxford: University Press. Tatarkiewicz, W. (1980) A History of Six Ideas: An Essay in Aesthetics. The Hague; Boston; London: MartinusNijhoff. Warszawa: PWN/Polish Scientific Publishers. Trédé, M. (1992) Kairos. L’à-propos et l’occasion. (Le mot et la notion, d'Homère à la fin du IVe siècle avant J.-C.). Paris: Klincksieck. Vitruvius, (1999) Ten Books on Architecture. Translated by I. D. Rowland and Th. Noble Howe Cambridge: Cambridge University Press. Wusten H. van der (2016) ‘Decor and decorum in diplomacy’, in V. Mamadouh and A. van Wageningen (eds.) Urban Europe. Amsterdam: Amsterdam University Press. Xenophon (1979) Memorabilia, Oeconomicus. Translated by E.C. Marchant. Cambridge (Massachusetts): Harvard University Press-London: William Heinemann. Zimmerman, R. (1858) Geschichte der Aesthetik als Philosophischer Wissenschaft. Wien: Braumüller. Elisabetta Di Stefano University of Palermo, Department of Humanities viale delle Scienze, build. 12 90128 Palermo - Italy elisabetta.distefano@unipa.it ELISABETTA DI STEFANO Decorum. An Ancient Idea for Everyday Aesthetics? 38 

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Hindawi Publishing Corporation International Journal of Antennas and Propagation Volume 2012, Article ID 490327, 9 pages doi:10.1155/2012/490327 Research Article Modal Resonant Frequencies and Radiation Quality Factors of Microstrip Antennas Jan Eichler, Pavel Hazdra, Miloslav Capek, and Milos Mazanek Department of Electromagnetic Field, Faculty of Electrical Engineering, Czech Technical University in Prague, Technická 2, 166 27, Prague, Czech Republic Correspondence should be addressed to Pavel Hazdra, hazdrap@fel.cvut.cz Received 9 August 2011; Revised 10 January 2012; Accepted 13 January 2012 Academic Editor: Charles Bunting Copyright © 2012 Jan Eichler 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 chosen rectangular and fractal microstrip patch antennas above an infinite ground plane are analyzed by the theory of characteristic modes. The resonant frequencies and radiation Q are evaluated. A novel method by Vandenbosch for rigorous evaluation of the radiation Q is employed for modal currents on a Rao-Wilton-Glisson (RWG) mesh. It is found that the resonant frequency of a rectangular patch antenna with a dominant mode presents quite complicated behaviour including having a minimum at a specific height. Similarly, as predicted from the simple wire model, the radiation Q exhibits a minimum too. It is observed that the presence of out-of-phase currents flowing along the patch antenna leads to a significant increase of the Q factor. 1. Introduction Evaluation of the basic properties of microstrip patch antennas (MPA) has been numerously discussed in literature, see, for example, [1–3]. The two main MPA attributes are resonant frequency (or frequencies but we will deal mostly with the dominant mode) and the radiation Q factor. So far, only approximate results and semianalytic equations have been published. To our knowledge, this is the first time that these important characteristics have been studied in a rigorous way. The antennas are treated by using a modal approach (hence we do not a priori consider any feeding to be connected), namely, by the theory of characteristic modes (TCM), [4, 5]. Evaluation of the radiation Q is performed both by the TCM from the eigenvalues slope and by novel rigorous equations derived by Vandenbosch [6] and Vandenbosch and Volski [7]. 2. The Theory of Characteristic Modes For completeness, let us formulate the basics of the characteristic modes for perfectly conducting bodies of area S. The scattered field Es is related to the electric surface currents J by the electric field integral equation (EFIE) [8]  L(J) − Ei  tan = 0. (1) Equation (1) is usually treated within the method of moments (MoMs) [8] framework and, due to the structure discretization, the L operator is known as the “complex impedance matrix” [Z] = [R] + j[X]. Then the associated Euler’s equation to be solved is XJn = λn RJn . (2) Equation (2) is a standard weighted eigenvalue equation leading to a set of real characteristic eigencurrents Jn and associated eigenvalues λn . Properties of eigenvalues are described in [9], at this moment it is important to note that λn reflects the amount of net reactive power (thus λn = 0 means resonance). Instead of eigenvalues, the so-called characteristic angles αn are introduced to show more visible behavior with frequency [9]. Characteristic currents form a complete orthogonal set, and hence the total current on a conducting body may be expressed as a linear combination of these mode currents [10]. 2.1. Implementation of the Characteristic Modes Theory. Implementation of the modal decomposition process has been done in the MATLAB [11] environment using Makarov EFIE codes [12] with the RWG basis functions [13]. This usage is restricted to arbitrary 3D PEC structures with air 2 International Journal of Antennas and Propagation dielectrics. Our developed TCM tool [14] has the following main advantages: T1 (x,y,z) (i) Comsol Multiphysics [15]/MATLAB’s PDE TooLbox mesh import, (ii) Optional Green’s function for infinite ground plane simulations, (iii) Single solver/multicore solver/distributed solver (within a computer network with installed MATLAB). In [6] a novel theory able to rigorously calculate radiated power and stored energies directly from currents flowing along the antenna has been presented. The radiation Q factor is then readily evaluated by the definition [16]:  m , W e max W  Pr .  1 8πωε0  Ω1 Ω2 Ω J2 y r2 (0,0)  Q = QZ = ω0 ∂Z ω0 ∂R ∂X . + = 2 ∂ω 2 ∂ω ∂ω Ω1 1 e = (Ie − IR ), W 16πω2 ε0 (5) 1 (Im − IR ), 16πω2 ε0 (6) Ω2 k2 J(r1 )J(r2 ) − ∇ · J(r1 )∇ · J(r2 ) Ie = Ω1 Im = k 2 Ω1 Ω2 J(r1 )J(r2 ) cos(kr21 ) dΩ1dΩ2, r21 (7)  ω0 ∂X ω0 ∂    e 2ω Wm − W = 2 ∂ω 2 ∂ω ω0 ∂λ = Qeig , = 2 ∂ω (8) (9) (10) (13) (14) providing that  3.1. The Modal Radiation Q Factor. The modal radiation Q factor may be evaluated from the slope of modal eigenvalues [18]: ω0 dλ . 2 dω   1  m − W e Pr + j2ω W 2 |I |  m − W e . λ = 2ω W where k is a free-space wavenumber, J is the surface current density, and r21 is the distance between interacting current elements. The tilde denotes that the radiation contribution IR has been subtracted from the stored energies at every point in space [17]. It is assumed that the currents are flowing in a vacuum. Qeig = (12) valid for lossless antennas [19] and using the fact that Pr = 1, (12) results in Q = QX = × sin(kr21 )dΩ1dΩ2, cos(kr21 ) ∇ · J(r1 )∇ · J(r2 ) dΩ1dΩ2, r21 Ω2 (11) Inserting (4) where k 2  For parallel or series RLC circuit (hence, for one mode), the “impedance QZ ” equals the exact “current Q” [6]: Z = R + jX = IR = T2 (x,y,z) x e , W m . Q = 2ω max W k2 J(r1 )J(r2 ) − ∇ · J(r1 )∇ · J(r2 ) sin(kr21 ) × dΩ1dΩ2, r21 m = W r1 In [18], (10) is supposed to be an approximation of the radiation Q, but in resonance it is actually exact. Since characteristic modes are normalized to radiate unit power Pr = 1 [4], (3) reduces to (3) The equations for radiated power Pr and stored electric and e , W m are magnetic energies W Pr = r21 = |r2 − r1 | Figure 1: Distance between nonoverlapping current elements [23]. 3. The Radiation Q Factor Q = 2ω J1 (15) It is therefore concluded that the modal Qeig equals the QX by definition, and it can be proven (using the reactance theorem [20, 21]) that in resonance it also equals the radiation Q defined from energies by (11). 3.2. Software Implementation. The above equations were implemented in MATLAB for the RWG triangular mesh where two different interaction situations occur: (a) Distant Elements. When the triangular elements are not overlapping, current density on triangles may be simply approximated as point sources located at the centre of triangles [22], see Figure 1. No actual integration is then needed. This centroid approach is very fast with satisfactory accuracy as will be shown later (however it may fail for patches located very close to the ground plane). International Journal of Antennas and Propagation P3 (x3 , y3 ) h13 (0,1) A P1 (x1 , y1 ) h12 P2 (x2 , y2 ) r1 x (0,0) T1 (α ,β,γ) 0.01 P0 h23 r2 y z β T1 (x,y,z) P0 3 (0,0) α 0.005 (1,0) 0 2H −0.005 (a) y (b) Figure 2: Self-term evaluation. (a) Original problem, (b) simplex coordinates transformation [23]. x −0.01 0.015 0.01 (b) Overlapping (Self) Elements. As known from the method of moments, the so-called “self ” contributions are of great importance when dealing with calculations on discrete elements (meshes). Here, the self-interaction occurs when two triangles are overlapping each other. Due to the behavior of integral kernels, only rapidly varying term cos(kr21 )/r21 has to be carefully treated. Since k0 R21 → 0 (R21 being the longest side of the triangle T) is satisfied, one needs only to use the first term in the Taylor series expansion. The dominant singular static part is 1/r21 and the integral to be worked out is 1 T T (x 2 − x )  + y − y   2 dx d y dx dy , −0.01 −0.015  4. Applications: Rectangular Patch Antenna Let us first concentrate on a rectangular patch antenna of dimensions L = 50 mm and W = 30 mm (further noted as R50 × 30) placed in air at a height H above an infinite ground plane. Only the dominant TM01 mode will be studied. The reason for choosing a patch with L/W = / 1 is that we do not have to deal with degenerated modes. Using the image theory, the radiator in the XY plane at height z = H above an infinite electric ground plane is modelled as two patches separated by 2H. The total number of triangular elements is 676. In the TCM analyser, a proper out-of-phase mode is selected (Figure 3). The resonant frequency of the dominant mode is shown as a function of height H, see Figure 4. It has been evaluated from a modal resonant condition for eigenvalues λ = e ) = 0 employing an adaptive frequency m − W 2ω(W sweep for each height. The behaviour is quite peculiar, especially for greater heights. For low heights (H < 10 mm or −0.01 4 Regular behaviour 3 Min. f res 2.5 Min. Q 2 1.5 1 where A is the triangle area, hi j are the edge lengths (see Figure 2), and L is the perimeter of the triangle. −0.02 0.02 4.5 3.5 4 ln(1 − 2h12 /L) ln(1 − 2h13 /L) ln(1 − 2h23 /L) I = − A2 + + , 3 h12 h13 h23 (17) −0.03 0.01 0 Figure 3: Model of MPA above infinite ground plane for H = 10 mm, dominant mode TM01 shown. (16) where T = T  is a triangular area. Using simplex coordinates transformation (Figure 2), the result is [23, 24]  0 −0.005 f res (GHz) I= 0.005 0 5 10 15 20 25 30 35 40 45 50 55 60 H (mm) f r (TCM) f r (analytic equation) Figure 4: R50 × 30 resonant frequency of the dominant TM01 mode. The dashed red curve is a quasianalytical equation from [1]. H/λres < 0.08), the resonant frequency decreases “regularly,” and quasianalytical formulas (see, e.g., [1, 3]) based on the fringing field concept are valid below this range. For H ∼ = 25 mm (H/λres ∼ = 0.188) there is absolute minimum of the TM01 resonant frequency. Further on, the resonant frequency rises to reach its maximum for H ∼ = 40 mm (H/λres ∼ = 0.51). Around this specific height the patch also shows the minimum of the radiation Q. The above described process repeats periodically. It is yet unclear to the authors as what is the physical background to the resonant frequency discontinuity around H/λres ∼ = 0.5. e ) obtained m , 2ωW e , and 2ω(W m − W The terms 2ωW from (5)–(9) and eigenvalues λ are plotted at Figure 5 for 4 International Journal of Antennas and Propagation an approximate analytical solution is available and in [25] we showed that the Q is led by the function 20 Reactive energies 15 fQ (H) ∼ = 10 f res tan(k2H) = k2H, 0  1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 f (GHz) λ 2ω (Wm) 2ω (We) 2ω (Wm-We) Figure 5: Reactive energies and their differences for an R50 × 30 patch at height of 25 mm. H λ  min ∼ 3 − 1 = 0.358. = 2 8 6π (20) For sinusoidal currents on dipoles the minimum (evaluated numerically) occurs for H = 0.36λ. The minimum of the patch under study is obtained at H ∼ = 0.4λ, a value that is remarkably close to the simple dipole model. 4.1. Algorithm Convergence. Since no other methods for calculating modal Q are available, Qeig is taken as a reference, and the relative error percentage is defined as: 50 45 40 relative error = 35 Q (–) (19) and the first nontrivial root of (19) could be approximated as [25] −5 30 25 20 15 10 5 0 (18) After deriving (18), the condition is worked-out 5 −10 2H . k2H − sin(k2H) 0 5 10 15 20 25 30 35 H (mm) 40 45 50 55 60 Q eig QJ Figure 6: The radiation Q for dominant mode of an R50 × 30 patch as a function of height H. H = 25 mm as a function of frequency. There is excellent agreement between the difference in stored energies and the eigenvalues, both obtained in a completely different manner. There is also very good agreement between the exact QJ and Qeig confirming the validity of the proposed algorithm via (14), see Figure 6. Note that Qeig in (10) does not require the currents to be calculated on the structure while QJ is evaluated in a rigorous way from modal currents (11). From Figure 6 it is seen that the radiation Q has a minimum for a specific height. It is deduced that the reason lies in the cancelling of the radiated power between the two out-of-phase currents. Similar behaviour has been observed in the case of two half-wave thin-wire dipoles with opposite sinusoidal currents, separated by d = 2H, see [25] for details. Actually these two out-of-phase dipoles may serve as a very simple model for a patch antenna with a dominant mode. When the dipoles are reduced to elementary (Hertzian) ones, QJ − Qeig Qeig · 100, (21) where QJ is calculated from the currents using (11). Four different heights H were chosen, H = 1 mm (0.01λ), H = 2 mm (0.0185λ), H = 10 mm (0.0803λ), and H = 20 mm (0.151λ), and the relative error was evaluated as a function of total triangular elements (including the mirror), see Figure 7. All quality factors were evaluated at the resonant frequency of the dominant mode for the R50 × 30 patch. As discussed earlier, the centroid approximation became more inaccurate with low heights H. However, even for the lowest analyzed value H = 0.01λ, the relative error is in the order of a few percent for reasonable mesh density (hundreds of elements). Further improvements to the integration routine are considered for the future. 4.2. Fractional Bandwidth of the R50 × 30 Patch Antenna. It is known that the fractional bandwidth (FBW) is related to the unloaded Q factor and the desired matching VSWR level. For VSWR < s we have [26] FBW ∼ = s−1 √ [%]. Q s (22) Using a full-wave simulator CST-MWS [27], an R50 × 30 patch has been simulated and the FBWCST for VSWR < 2 was calculated as: FBWCST = f2 − f1 , f0 (23) where f2 and f1 are margins for VSWR < 2 and f0 is the centre frequency. Only very low heights were studied since we used a simple probe feed which introduces an inductance component to the total input impedance. The comparison in Figure 8 shows good agreement of both fractional bandwidths. Relative error (%) International Journal of Antennas and Propagation 10 9 8 7 6 5 4 3 2 1 0 0 200 400 600 800 1000 Number of triangle elements H = 1 mm (0.01 λ) H = 2 mm (0.0185 λ) 1200 5 J1 J2 σ1 σ2 1400 H = 10 mm (0.0803 λ) H = 20 mm (0.151 λ) Figure 7: Relative error of the Q factor as a function of triangular elements (mesh density). Figure 9: The first two characteristic modes (currents and charges) for the SAU1 structure. 5 4.5 W 4 FBW (%) 3.5 3 2.5 J1 J2 σ1 σ2 L 2 1.5 1 0.5 0 1 1.5 2 2.5 3 3.5 H (mm) 4 4.5 5 FBW (CST) FBW (Q eig ) Figure 8: Fractional bandwidth FBW (VSWR < 2) for a R50 × 30 patch. 0 0 Figure 10: The first two characteristic modes (currents and charges) for the SAU2 structure. 5. Applications: Fractal Antennas In this section, a bit more complex structures will be studied. The first one (the “Self Affine U” fractal, SAU), has been described in [28] and further analyzed in [29]. This kind of radiating motif is employed as a dual-band radiator with mutually orthogonal radiation patterns at both bands. Therefore we are analyzing the first two modes, where the currents are orthogonal. These are depicted in Figures 9 and 10 for first (SAU1) and second (SAU2) fractal iteration, respectively. The current of the first (lower) mode J1 has two out-of-phase components (see Figure 11 for schematic current paths) while the second mode comprises inphase currents only. As we know from previous studies, opposite currents contribute to a rapid increase of the radiation Q, and it is expected that J1 will have a much higher Q than J2 . J1 J2 Original Infinite ground plane Image Figure 11: The main current paths for the first two modes of the SAU1/2 structure. 6 International Journal of Antennas and Propagation 280 260 240 Mode no.3 Mode no.2 200 Mode no.1 180 Q αn 220 160 140 120 100 1 1.25 1.5 1.75 2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 × 109 f (Hz) 100 90 80 70 60 50 40 30 20 10 0 10 Q1 Q2 12 14 16 18 20 22 24 26 28 30 H (mm) (a) (b) Figure 12: Characteristic angles (left) and radiation Q for the SAU2. 0.03 0.03 J1 0.02 0.01 0.01 0 0 −0.03 −0.02 −0.01 0 J2 0.02 0.01 0.02 0.03 −0.03 −0.02 −0.01 (a) 0.01 0.02 0.03 (b) 0.03 0.03 σ1 0.02 0.01 0 0 −0.01 −0.01 −0.02 −0.02 −0.02 −0.01 0 σ2 0.02 0.01 −0.03 −0.03 0 0.01 0.02 0.03 (c) −0.03 −0.03 −0.02 −0.01 0 0.01 0.02 0.03 (d) Figure 13: Degenerated dominant mode J1 , and J2 of the FCL2 antenna (currents and charges). Figure 11 presents a very simple concept showing the main current paths for the J1 and J2 modes discussed above including the mirroring effect of the infinite ground plane. It could be simply stated that more opposing current paths lead to significant increase in Q. We show detailed behaviour only for SAU2 (the situation is similar for SAU1)—see Figure 12 that confirms high Q for the J1 mode. Characteristic angles are calculated for H = 29 mm, the actual height for which the dual-band antenna was designed [29]. 5.1. The FCL-2 Fractal Antenna. The second presented structure is the so-called fractal clover leaf (FCL) of the second iteration, [14]. The antenna is fed by an L-probe [30] that excites its dominant mode and is located at height H = 36 mm. Actually, the dominant mode is composed of two degenerated modes J1 and J2 (Figure 13). The second higher mode J3 is shown at Figure 14 for completeness. Figure 15 shows the main current paths of these modes, and we can again deduce that the dominant mode will exhibit International Journal of Antennas and Propagation 7 0.03 0.03 J3 0.02 σ3 0.02 0.01 0.01 0 0 −0.01 −0.02 −0.03 −0.02 −0.01 0 0.01 0.02 0.03 −0.03 −0.03 −0.02 −0.01 (a) 0 0.01 0.02 0.03 (b) Figure 14: Second higher mode J3 (currents and charges). Mode 1, 2 J1 + J2 = 8.25 6.87 5.5 4.12 2.75 1.37 0 −3 .6 2 −7 .2 4 −1 0 .8 7 −1 4 .4 9 −1 8 .1 2 −2 1 .7 4 Mode 3 J3 = 7.32 6.1 4.88 3.66 2.44 1.22 0 −3 .7 7 −7 .5 5 −1 1 .3 3 −1 5 .1 1 −1 8 .8 9 −2 2 .6 7 Figure 15: Schematic depiction of the dominant current paths for the dominant (J1 + J2 ) and the second higher J3 modes together with their modal radiation patterns. lower Q compared to J3 . This is confirmed by Figure 16—J3 has more than 200x higher radiation Q. 6. Resonant Properties of Studied Antennas The properties of studied antennas are summarized in this section. At first we observed that microstrip antenna could support different kinds of modes regarding their Q factors (see Figure 17): (a) low Q modes with the current flowing in one direction and not changing its phase (dominant modes of simple shapes like rectangular, circular patch, and so forth.) (b) high Q modes with part of the currents flowing in the opposite direction. These modes exist even on simple “U” shaped patch (Figure 9 left) and on complex (fractal) geometries. Secondly, it has been observed that resonant frequency is quite a complicated function of height. Unfortunately we do not yet have any physical explanation as to why some modes present minimum values of fr . Looking at Figure 18, it is clear (and interesting) that the resonant frequency behaves quite differently for lowQ and high-Q modes. The resonant frequency of low-Q modes is much more sensitive to the height, whereas high-Q modes exhibit almost constant fr when the height is varied. The proposed explanation is that the opposite currents (responsible for high Q) keep reactive fields very close to the radiating structure so the effect of a fringing field coupled to the ground plane becomes almost negligible. 7. Conclusions Modal resonant properties of selected microstrip patch antennas have been studied with the help of characteristic 8 International Journal of Antennas and Propagation 3 260 2.8 240 2.6 220 2.4 αn 200 fr (GHz) 280 Qeig3 = 226.3 180 160 Qeig1 = Qeig2 = 10.5 140 2 High-Q modes 1.8 1.4 1.2 100 1 1 1.1 1.2 1.3 1.4 1.5 1.6 f (GHz) 1.7 1.8 1.9 10 High-Q modes 90 15 20 H (mm) 25 30 35 SAU2 mode 2 R50 × 50 R50 × 30 Figure 18: Resonant frequencies for different antennas/modes. Using proper feeding techniques (like the L-probe) allows us to design wideband compact antennas. The theory now puts current distribution and the radiation Q factor into objective context. Whenever the current mode exhibits opposite components, high Q may appear. Future work is needed to connect the presented theory with parameter sweeps or even optimization, so we will be able to design novel wideband/multimode compact antennas. 100 80 70 60 50 Low-Q modes 30 5 FCL2 SAU1 mode 1 SAU1 mode 2 SAU2 mode 1 Figure 16: Characteristic angles for the FCL2 structure at H = 29 mm. 40 0 2 × 109 Mode 1 Mode 2 Mode 3 Q eig 2.2 1.6 120 80 Low-Q modes 20 Acknowledgments 10 0 0 5 10 15 20 25 30 35 H (mm) FCL2 SAU1 mode 1 SAU1 mode 2 SAU2 mode 1 SAU2 mode 2 R50 × 50 R50 × 30 This work was supported by the Grant Agency of the Czech Technical University in Prague, grant no. SGS11/065/ OHK3/1T/13 and by the Project COST 1102. The authors would like to thank professor Vandenbosch for fruitful discussions, Neil Bell for his comments, and two anonymous reviewers who suggested some improvements to the paper. Figure 17: Radiation Qs for different antennas/modes. References modes and the novel theory published by Vandenbosch. It has been found that the resonant frequency of a simple rectangular patch antenna is quite a complicated function of its height above the infinite ground. Moreover, the dependency of resonant frequency is also found to be a function of the radiation Q factor (which is now possible to calculate in a rigorous way). Due to the complexity of the problem, no physical explanation for the resonant frequency behaviour has yet been found. It is observed that the radiation Q factor decreases for “standard” heights (<∼0.1λ), however there exists an absolute minimum value of Q that has already been predicted by simple modeling of two elementary out-of-phase dipoles. [1] P. Bhartia, I. Bahl, R. Garg, and A. Ittipiboon, Microstrip Antenna Design Handbook, Artech House, Norwood, Mass, USA, 2000. [2] K. F. Lee and W. Chen, Advances in Microstrip and Printed Antennas, John Wiley & Sons, New York, NY, USA, 1997. [3] J. R. James, P. S. Hall, and C. Wood, Microstrip Antenna Theory and Design, Peter Peregrinus, London, UK, 1986. [4] R. F. Harrington and J. Mautz, “Theory of characteristic modes for conducting bodies,” IEEE Transactions on Antennas and Propagation, vol. 19, no. 5, pp. 622–628, 1971. [5] E. A. Daviu, Analysis and design of antennas for wireless communications using modal methods, Ph.D. dissertation, Universidad Politécnica de Valencia, Valencia, Spain, 2008. [6] G. A. E. Vandenbosch, “Reactive energies, impedance, and Q factor of radiating structures,” IEEE Transactions on Antennas International Journal of Antennas and Propagation [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] and Propagation, vol. 58, no. 4, Article ID 5398856, pp. 1112– 1127, 2010. G. A. E. Vandenbosch and V. 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Glisson, “Electromagnetic scattering by surfaces of arbitrary shape ,” IEEE Transactions on Antennas and Propagation, vol. 30, no. 3, pp. 409–418, 1982. M. Capek, P. Hazdra, P. Hamouz, and M. Mazanek, “Software tools for efficient generation, modelling and optimisation of fractal radiating structures,” IET Microwaves, Antennas and Propagation, vol. 5, no. 8, pp. 1002–1007, 2011. Comsol Multiphysics, http://www.comsol.com/. J. L. Volakis, Ch. Chen, and K. Fujimoto, Small Antennas: Miniaturization Techniques & Applications, chapter 2, McGraw-Hill, New York, NY, USA, 1st edition, 2010. J. S. McLean, “A re-examination of the fundamental limits on the radiation q of electrically small antennas,” IEEE Transactions on Antennas and Propagation, vol. 44, no. 5, pp. 672–676, 1996. R. F. Harrington and J. R. Mautz, “Control of radar scattering by reactive loading,” IEEE Transactions on Antennas and Propagation, vol. 20, no. 4, pp. 446–454, 1972. R. F. 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Eichler, “Radiation Q-factors of thin-wire dipole arrangements,” IEEE Antennas and Wireless Propagation Letters, vol. 10, pp. 556–560, 2011. A. D. Yaghjian and S. R. Best, “Impedance, bandwidth, and Q of antennas,” IEEE Transactions on Antennas and Propagation, vol. 53, no. 4, pp. 1298–1324, 2005. CST Gmbh, http://www.cst.com/. 9 [28] S. N. Sinha and M. Jain, “A self-affine fractal multiband antenna,” IEEE Antennas and Wireless Propagation Letters, vol. 6, Article ID 891519, pp. 110–112, 2007. [29] J. Eichler, P. Hazdra, M. Čapek, T. Kořı́nek, and P. Hamouz, “Design of a dual-band orthogonally polarized l-probe-fed fractal patch antenna using modal methods,” IEEE Antennas and Wireless Propagation Letters, vol. 10, pp. 1389–1392, 2011. [30] C. L. Mak, K. M. Luk, K. F. Lee, and Y. L. Chow, “Experimental study of a microstrip patch antenna with an L-shaped probe,” IEEE Transactions on Antennas and Propagation, vol. 48, no. 5, pp. 777–783, 2000. International Journal of Rotating Machinery Engineering Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 The Scientific World Journal Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal of Distributed Sensor Networks Journal of Sensors Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Control Science and Engineering Advances in Civil Engineering Hindawi Publishing Corporation http://www.hindawi.com Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Volume 2014 Submit your manuscripts at http://www.hindawi.com Journal of Journal of Electrical and Computer Engineering Robotics Hindawi Publishing Corporation http://www.hindawi.com Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Volume 2014 VLSI Design Advances in OptoElectronics International Journal of Navigation and Observation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Hindawi Publishing Corporation http://www.hindawi.com Chemical Engineering Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Volume 2014 Active and Passive Electronic Components Antennas and Propagation Hindawi Publishing Corporation http://www.hindawi.com Aerospace Engineering Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2010 Volume 2014 International Journal of International Journal of International Journal of Modelling & Simulation in Engineering Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Shock and Vibration Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Advances in Acoustics and Vibration Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 

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Malaria Journal Howes et al. Malar J (2016) 15:502 DOI 10.1186/s12936-016-1556-3 Open Access RESEARCH Contemporary epidemiological overview of malaria in Madagascar: operational utility of reported routine case data for malaria control planning Rosalind E. Howes1,2*, Sedera Aurélien Mioramalala3,4, Brune Ramiranirina3, Thierry Franchard3, Andry Joeliarijaona Rakotorahalahy3, Donal Bisanzio5, Peter W. Gething5, Peter A. Zimmerman1* and Arsène Ratsimbasoa3,4,6* Abstract Background: Malaria remains a major public health problem in Madagascar. Widespread scale-up of intervention coverage has led to substantial reductions in case numbers since 2000. However, political instability since 2009 has disrupted these efforts, and a resurgence of malaria has since followed. This paper re-visits the sub-national stratification of malaria transmission across Madagascar to propose a contemporary update, and evaluates the reported routine case data reported at this sub-national scale. Methods: Two independent malariometrics were evaluated to re-examine the status of malaria across Madagascar. First, modelled maps of Plasmodium falciparum infection prevalence (PfPR) from the Malaria Atlas Project were used to update the sub-national stratification into ‘ecozones’ based on transmission intensity. Second, routine reports of case data from health facilities were synthesized from 2010 to 2015 to compare the sub-national epidemiology across the updated ecozones over time. Proxy indicators of data completeness are investigated. Results: The epidemiology of malaria is highly diverse across the island’s ecological regions, with eight contiguous ecozones emerging from the transmission intensity PfPR map. East and west coastal areas have highest transmission year-round, contrasting with the central highlands and desert south where trends appear more closely associated with epidemic outbreak events. Ecozones have shown steady increases in reported malaria cases since 2010, with a near doubling of raw reported case numbers from 2014 to 2015. Gauges of data completeness suggest that interpretation of raw reported case numbers will underestimate true caseload as only approximately 60–75 % of health facility data are reported to the central level each month. Discussion: A sub-national perspective is essential when monitoring the epidemiology of malaria in Madagascar and assessing local control needs. A robust assessment of the status of malaria at a time when intervention coverage efforts are being scaled up provides a platform from which to guide intervention preparedness and assess change in future periods of transmission. Keywords: Malaria, Madagascar, Routine health information systems data, Surveillance, Outbreaks *Correspondence: rosalind.howes@zoo.ox.ac.uk; paz@case.edu; aratsimbasoa@gmail.com 1 Center for Global Health and Diseases, Case Western Reserve University, Cleveland, OH 44106‑4983, USA 3 National Malaria Control Programme, Ministry of Health, Androhibe, Antananarivo, Madagascar Full list of author information is available at the end of the article © 2016 The Author(s). 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. Howes et al. Malar J (2016) 15:502 Background The Malagasy context is one of protracted political instability, most recently in the aftermath of a political coup in 2009 which continues to bear severe economic and social impacts [1] (Fig. 1). A long-term lack of investment in the country’s infrastructure has had direct repercussions for malaria control efforts as well as the country’s broader developmental situation [2]. Madagascar has the seventh lowest per capita gross domestic product (GDP) globally [3] with an estimated 75.3 % of the population living under the national poverty line in 2010 [3]. This was estimated to have increased by more than 10 % since the 2009 crisis, with the World Bank estimating 92 % of the population to be living under $2 a day in 2013 [1]. The population lives predominantly in rural environments (66 %), and almost half (42.4 %) is younger than 15 years, and the median age is 18.4 years [3]. Malnutrition is rife, affecting over half of children, with 25 % of children reported to be severely malnourished [4], exacerbated by periodic famine outbreaks following severe drought [5] and failed harvests from locust infestation [1]. Childhood underweight is reported to be the primary overall risk factor of disease in the Malagasy population [6], increasing vulnerability to infections and development of severe Page 2 of 18 sequelae. In 2012, top causes of consultations at health centres among under 5 year olds were, in descending order, acute respiratory infections, diarrhoea, digestive disorders, and uncomplicated malaria [7]. In the five to 14 years age bracket, uncomplicated malaria represented the second-most common cause of health centre consultations in 2012, while complicated malaria presented the top cause of district-level hospital mortality, at 10.1 % overall, and 21.6 % among under-fives [7]. All 23.9 million people across Madagascar’s 112 health districts are at risk of malaria exposure [8]. In view of this substantial disease burden, malaria represents a main priority for the Malagasy Ministry of Health (MoH) and associated international public healthfocussed partners [9]. Intervention coverage is high, with some of the highest ownership rates of long-lasting insecticide-treated nets (LLINs) in the African region in 2013 [8, 10]. This was supplemented at the end of 2015 with the distribution of 12 million LLINs aiming at universal coverage of one net per two individuals across 92 of the country’s 112 health districts where seasonal or perennial transmission is considered to occur. In the lower transmission districts of the highlands, focalized indoor residual spraying of insecticide (IRS) has been deployed against the Anopheles vectors [11]. Nevertheless, Fig. 1 Timeline of milestones in malaria policy, funding, planning and implementation in Madagascar. Malaria cases (2000–2014) are plotted from data reported by the NMCP published in the WHO’s World Malaria Report 2015 [8]; numbers of RDT confirmed cases for 2015 are from NMCP directly. Intervals between Global Fund disbursement when no financial resources were available to the NMCP were: Sept 2010–Feb 2011; Sept 2014–March 2015; and July 2016 onwards (NMCP, pers. comm., June 2016) Howes et al. Malar J (2016) 15:502 increasing numbers of cases have been reported since 2010. This paper analyses the routine reported malaria rapid diagnostic test (RDT) data from 2010 to 2015 to evaluate trends of malaria transmission across Madagascar. This analysis provides a benchmark assessment of malaria endemicity ahead of the anticipated impact of the large-scale LLIN distribution campaign prior to the 2016 transmission season. December 2015 also represented the end of the Millennium Development Goals era (2000–2015) and Fig. 1 summarizes the malaria control activities that have contributed to the malaria-specific aspects of this initiative. This overview therefore comes at an opportune time to assess progress against the target of “reversing the incidence of malaria” in Madagascar [12, 13]. Transmission of malaria is heterogeneous across Madagascar [14]. The island, the fourth largest globally at over 1500 km in length and with a surface area of 587,000 sq km, similar to California or Sweden, is highly ecologically and climatically diverse [15, 16]. Reflecting these conditions, the epidemiology of malaria across the island shows distinct seasonal trends and transmission intensities between regions [17, 18] with different vector species and behaviours predominant in different areas [19, 20]. Madagascar has a stated aim to reduce the proportion of malaria-attributable fevers and mortality, and start transitioning towards pre-elimination status by end of 2017, the end of the country’s current National Strategic Plan [21] (Fig. 1). The development of spatially specific interventions informed by local transmission characteristics is therefore required to optimize the efficacy of strategies to combat malaria. Mapping the intensity of malaria transmission across the island has been done since the start of control efforts. Joncour et al. published district-level endemicity estimates according to splenic rates in the 1950s, identifying broad categories of malaria transmission [18]. These were updated by Mouchet et al. in the early 1990s, stratifying the country according to climatic and transmission patterns (Fig. 2a [22, 23]). These mapping sub-divisions have subsequently adapted to changing needs, aligning with contemporary administrative boundaries (Fig. 2b [21, 24, 25]), and recently been further simplified from Mouchet’s five zones to two broad zones representing high transmission (east and west coasts) and low transmission (highlands and arid south) by control programme agencies [11, 21, 25]. Here, the boundaries of these subnational divisions are re-evaluated and used to describe the contemporary epidemiology of malaria in Madagascar more than a decade after the country started receiving substantial external funding to roll-out its control programme (Fig. 1). Page 3 of 18 Methods Two methodological steps were followed to address the paper’s goal of assessing Malagasy routine health facility data and its application to programmatic planning. First, regional sub-divisions were reviewed and updated in accordance with contemporary transmission patterns. Second, routine health reports were examined within the updated sub-national stratifications to identify each area’s key epidemiological characteristics which might impact on intervention planning. The data sources accessed in this study are summarized in the Additional file 1. Sub‑national stratification of malaria transmission Transmission intensity, quantified as the communitylevel prevalence of blood stage infection, has been a traditional marker for regional stratification to guide malaria control [26, 27]. This approach bypasses potential weaknesses associated with the surveillance and health reporting systems. In this present study, mean summary maps of the modelled prevalence of Plasmodium falciparum infection across the 2–10 year old age range (PfPR) from the Malaria Atlas Project (MAP; [14, 28]) were used to assess the regional patterns of malaria infection (Additional file 2). These maps are informed by an extensive range of malariometric (PfPR community surveys from Malaria Indicator Surveys [24, 25] and all other available surveys e.g. [29], as well as annual case incidence estimates), intervention coverage [10] and environmental covariate datasets [30]. For the present paper’s objectives, the mean of the 2010–2015 annual surfaces was calculated (PfPR2010–2015; Additional file 2; Fig. 2c). This timeframe ensured consistency with the health facility case data that were available for investigation in this paper and provided a contemporary picture whilst attenuating inter-annual fluctuations in the PfPR estimates. For operational utility, the spatially continuous mean PfPR2010–2015 map was aggregated to current sub-national political boundaries, and then used to sub-divide the country into ecozones of contiguous districts with similar transmission intensity (Fig. 2d). The aim of the stratification was to generate an output that was pertinent for operational purposes and that allowed an intuitive description of the sub-national epidemiology of malaria. The number of ecozones was not prior constrained though the aim was to keep this under ten to ensure programmatic ease and avoid too much duplication across zones in terms of overlapping epidemiology. Automated clustering was attempted using packages in spatial analysis software but these were not well adapted to this present analysis due to the range of mean district level PfPR2010–2015 values which meant that the clustering analysis mainly identified single isolated outliers without also encompassing the practical Howes et al. Malar J (2016) 15:502 Page 4 of 18 Fig. 2 Defining a contemporary sub-national stratification of malaria transmission intensity in Madagascar. a Mouchet et al.’s stratification of malaria transmission across Madagascar (reproduced with permission [22, 23]); this map was adapted to district-level administrative boundaries and b shows the currently used adaptation of the sub-national stratification (Source: NMCP National Strategic Plan [21]). c Represents the Malaria Atlas Project (MAP) modelled map of P. falciparum prevalence in 2–10 year olds (PfPR mean surface from 2010–2015) [14], and d the updated ecozones based off the PfPR map (regional figures correspond to the mean district-level PfPR value (2010–2015), and the min–max PfPR values from the districts in each ecozone. e–h Illustrate some of the environmental covariates which informed the PfPR mapping model, and which therefore underpin the updated sub-national stratifications. e A model of digital elevation (source: Shuttle Radar Topography Mission (SRTM) [59], plotted at 1 km resolution). f Maps mean total annual rainfall (mm) from 2010 to 2014 (source: NASA Tropical Rainfall Measuring Mission (TRMM) 3B43 algorithm available at 0.25º by 0.25º spatial resolution), while g shows the annual temperature suitability for P. falciparum transmission, reproduced with permission from Weiss et al. [60]. h Summarizes land-cover use across Madagascar at 500-m spatial resolution (IGBP MODIS annual landcover product MCD12Q1 [61, 62]) aspects of operational utility. The final stratification approach was therefore driven by two objective criteria including (i) spatial contiguity for operational convenience and (ii) transmission intensity as determined by the district-level mean PfPR2010–2015 map. Some subjective decision-making was necessary in situations when these two criterion could not both be met and instead one needed to be prioritized over the other. Specific examples are given in the Results. Routine health facility data Routine health management information system (HMIS) data reports from January 2010 to December 2015 were evaluated to assess the characteristics of the evidencebase of case reports available to inform decision-making by the Madagascar National Malaria Control Programme (NMCP). The routine HMIS in Madagascar follows a bottom-up pyramid, multi-step process of data aggregation and transfer between levels according to a fixed monthly schedule. Paper-based summaries of all patient consultations at the community-level health centres, either at health facilities or through community health workers, are transferred to the 112 health district offices across Madagascar where data are entered into the database software GeSIS, which is accessible by the regional and central MoH, including the NMCP [31, 32]. Private clinics are on the periphery of the HMIS but are intended to report to the public health system though many do not, so these are acknowledged as being poorly represented in the routine data system [21]. The Malagasy NMCP does not distinguish between species of malaria in its reporting systems. Combination RDTs are used which can distinguish Plasmodium falciparum from other species [33, 34], but this information is not recorded. The clinical cases described here therefore refer indiscriminately to any species of human Howes et al. Malar J (2016) 15:502 Plasmodium infection, though cross-sectional surveys have indicated that infection is predominantly from P. falciparum (>99 % by PCR when aggregated nationally [24]). Outbreak surveillance and definitions Alongside the HMIS, a parallel data reporting channel, the Integrated Diseases Surveillance and Response System, is intended as an outbreak early-warning system for notifiable diseases of epidemic potential, including malaria. Paper or electronic reports are sent weekly to the central NMCP for analysis. In practice, this reporting channel is weak and poorly developed in most health districts; the US President’s Malaria Initiative in Madagascar identified only eight of 112 districts as having functional reporting [11]. Outbreak surveillance is also conducted directly at the health facility level based on weekly trends in observed RDT-positive case numbers. Other surveillance institutions also act as channels to alert NMCP of outbreak situations, such as the Institut Pasteur’s nationally distributed sentinel site network [35, 36]. The NMCP respond to two categories of outbreaks [21]. In areas considered at very low risk of malaria, notably the Central highland and desert South ecozones, evidence of autochthonous transmission triggers an emergency response. In higher transmission zones outbreaks follow epidemic trends, where escalation of case numbers is commonly associated with mortality. In these latter areas, two operational definitions of outbreaks are applied at the health-facility level which are consistent with approaches followed elsewhere [37, 38]: (i) a weekly total of RDT-confirmed cases exceeding a pre-defined weekly health centre-specific threshold (calculated as two standard deviations from the mean number of confirmed cases from at least the preceding 3 years of data); or, (ii) in health centres lacking a defined outbreak threshold, a weekly doubling of RDT-positive cases over 3 consecutive weeks [21, 39]. Outbreak reports from January 2012 to December 2015 were collated from NMCP surveillance records to assess district-level temporal and spatial trends in outbreak occurrence based on these definitions. Demographic data To ensure consistency with NMCP estimates, official demographic data from the Madagascar MoH [40] were used in incidence calculations. As per the MoH protocols, a fixed annual population growth rate of 2.8 % was applied cumulatively at the district level to population data collected from the last official national census in 1993. In the absence of more specific population age distribution data, the MoH also defines standardized Page 5 of 18 age categories, with children under 5 years representing 18 % of the population total. This age bracket differs from the age categories in the malaria case reporting database which reflects anti-malarial treatment dosing age categories, so includes 5 year olds with under-fives. To account for this disparity, the proportion of 5 years old within the 5–14 years category was estimated (28.6 % of the overall population, thus 2.86 %). This therefore corresponded to an estimated 20.86 % of the total population being under 6 years [40]. Proxy indicators of data reporting completeness Indicators of reporting completeness, including the numbers of health centre reports received at the central level and the proportion of distributed RDTs accounted for in the health centre reports, were obtained from MoH records through the NMCP. The 2014 database of drug and RDT stock-outs, which reported the cumulative total days of consumable stock-outs annually, contained blank entries. Stock-out reporting required active participation by each health centre, thus the lack of reporting could not necessarily be interpreted to indicate ‘zero stockouts events’. To address this characteristic of the dataset, a conservative approach was followed, whereby health centres that reported no specific value for RDT stock-out events but did report on the status of other consumables, was taken to indicate zero stock-out days of RDTs. Health centres that did not report at all were excluded from this analysis. Related variables, including the proportion of the population resident further than 5 km from a health facility and district office accessibility from health centres, were also collated and evaluated. Results An updated map to reflect the contemporary stratification of malaria transmission The MAP’s PfPR map predictions for 2010–2015 (Fig. 2c; [14]) were summarized to the district level and examined for regional trends (Additional file 2). Figure 2e–h illustrate a subset of the main environmental covariates used in the PfPR modelling process. The overall range of district-aggregated mean PfPR values was relatively narrow, with only 17 districts exceeding 10 % (these summary metrics mask sub-district variation). This therefore limited the applicability of the traditional malaria endemicity categorization, which has 10 % PfPR as the upper threshold of its lowest endemicity category: ‘hypoendemic’ [27]. Instead, narrower intervals were necessary to distinguish local transmission intensity patterns. Overall, eight distinct transmission zones emerged from the PfPR2010–2015 output map Howes et al. Malar J (2016) 15:502 (Fig. 2d). Ensuring spatially contiguous zones meant that some overlap in the ranges of mean PfPR2010–2015 values was necessary. In different areas, it was sometimes necessary to prioritise differently the two stratification criteria of (i) spatial contiguity and (ii) local transmission intensity similarities. For example, the east coast districts of Mananjary and Ifanadiana (approximately two-thirds down the east coast) had lower PfPR2010–2015 than the neighbouring east coast districts. However, to ensure spatial contiguity in the ecozones, these two isolated districts were included in the Southeast ecozone. Moreover, these two districts showed greater similarity in terms of transmission intensity to the Southeast districts than to the Highland fringe districts. In contrast, the high altitude central highland areas include small islands of very low/negligible autochthonous transmission. The unique epidemiology in these areas justified their amalgamation as a single ecozone despite lacking spatial continuity as they would require distinct intervention planning and therefore distinct ecozone classification. Similarly, Maroantsetra district was classified into the Highland fringe west ecozone due to its transmission characteristics being more similar to those than to the Northeast ecozone districts with which Maroantsetra shared north and south borders. The spatial continuity of the Northeast ecozone was not considered broken by this as the resulting Northeast ecozone included the full northern section of the east coast. The stratification therefore aimed to be objective but nevertheless included some subjective decision-making to optimise the final product for operational utility. Relative to previous stratifications (Fig. 2a, b), the new PfPR-based stratification (Fig. 2d and tabulated in Additional file 3) uses a larger number of classes (eight versus four to five in previous versions). The eight zones came about directly from the dataset, the number was not predetermined. These additional divisions mean that greater resolution in the epidemiology of each zone can be characterized. Areas at each extreme of the transmission spectrum are more clearly differentiated from neighbouring zones. For example, the previous single ‘east coast’ zone is now split into two areas of quite distinct transmission characteristics across the northern and southern east coast districts, allowing the high transmission areas to be more clearly identified. Page 6 of 18 Trends in routine malaria case reports A total of 175,061 health facility reports were available for analysis which had been reported from Madagascar’s 3924 health facilities to the central NMCP over the 2010– 2015 time frame. During that time, absolute numbers of reported RDT-confirmed malaria cases almost quadrupled to a maximum in 2015 of 738,996 reported cases, a steady increase from 2010 when 201,135 cases were reported to the NMCP (Fig. 3a; Additional file 4A). The greatest year-on-year increases were between 2011–2012 and 2014–2015, when there were 65 and 97 % increases (i.e., near doubling) of reported cases, respectively. All ecozones other than the Central highlands reported increases of reported cases between 2010 and 2015. In the Northeast, there was only a 30 % increase (37,884– 78,716 cases), while in other zones the reported increase ranged from doubling to tenfold increases (Fig. 3a). The recent 2014–2015 increase in reported cases was evident from all zones, with five zones reporting more than a doubling of cases in that 1 year period and the South ecozone reporting a tenfold increase from 4238 reported cases in 2014 to 47,583 in 2015. The Southeast ecozone had a 41 % increase in reported cases from 2014 to 2015 (218,675–308,192 cases), accounting for just under half (42 %) the country’s total reported cases for 2015 despite having only 16 % of the country’s population [40]. The Southeast ecozone was the only area to have also reported a substantial increase in case numbers from 2013 to 2014 (66 % increase), other areas had seen a decrease or only very slight increase ahead of the major increases into 2015 (Fig. 3a). Seasonal trends are largely consistent through the time window evaluated, peaking in April–May except in the two east coast ecozones where an earlier February peak is generally observed. Trends in the lower endemic Central highlands are less consistent. Additional file 4A–D provides a more detailed analysis of the temporal structure of the data through autocorrelation plots of the time series, and linear regression and generalized additive models. Incidence of reported RDT-confirmed cases nationally peaked for 2010–2015 in April–May 2015 at 3.8–4.0 cases/1000 population/month, having increased significantly since 2010, increasing from annual incidence of 9.65/1000 RDT+ cases in 2010 to 30.87/1000 reported in 2015 (linear regression model p < 0.001; Additional (See figure on next page.) Fig. 3 Descriptive plots of the RDT-confirmed case data available in the NMCP database for 2010–2015. a Raw reported confirmed case numbers in the NMCP database, aggregated monthly into the eight ecozones. Additional plots including individual ecozone plots are available in Additional file 2. b Raw reported age-specific monthly incidence of RDT-confirmed cases per 1000 population. Population size is estimated annually based on a fixed growth rate (see “Methods” section). All-age population estimates for 2015 by ecozone are (Ministry of Health, 2015): A Northwest: 1.907 million; B Northeast: 4.470 million; C Southwest: 2.083 million; D Southeast: 3.855 million; E Highland fringe west: 2.907 million; F Highland fringe east: 5.759 million; G South: 1.298 million; H Central highlands: 1.659 million. Additional plots and tables relating to this data set are included in Additional file 2 Howes et al. Malar J (2016) 15:502 Page 7 of 18 Howes et al. Malar J (2016) 15:502 file 4B). As detailed in Additional file 4B, the fastest rates of increase in incidence were reported from the southern ecozones: the Southeast, Southwest and South (in descending order) had the sharpest rates of increase. Negative trends were identified from the Northeast and Central highland ecozones, indicating a decrease in incidence, though these trends were non-significant (linear model p > 0.05). Monthly incidence across two age categories (0–5 and >5 years; Fig. 3b) revealed significantly different age-specific incidence in all ecozones except the Highland fringe east (Wilcoxon Signed-Rank test with 95 % confidence). The Central highland ecozone was the only area where the incidence of malaria in over 5 years olds exceeded that of infants (0–5 years), in all other ecozones where there was a significant difference, the caseload was greater in infants under-five (Fig. 3b). This was particularly marked in the Southeast, where the underfive incidence was on average 3.7 times greater than in the older age category (range: 2.3–5.5). To account for the potential distortion of temporal trends in raw reported numbers resulting from incomplete reporting or diagnostic kit stock-outs, RDT-confirmed case counts were adjusted to the total reported number of consultations and the overall number of reported RDT results (Additional file 4C–D). HMIS reported consultations increased by 51 % from 4.8 million in 2010 to 7.3 million in 2015. During the same time period, total reports of diagnostic tests performed more than doubled from 603,727 to 1,487,745. Trends in reported RDT use closely reflect the temporal trends in confirmed case reports (Additional file 4D). Trends in these adjusted datasets were less apparent than from the raw reported data, but linear regression models still indicated overall increasing burden in both adjusted metrics nationally. Across the country, the annual diagnostic test positivity rate increased steadily from 33 % in 2010 to 50 % in 2015, indicating that despite substantial increases in the use of diagnostic testing, there was nevertheless an increase in the proportion of fever cases seeking treatment that were positive for malaria. The increasing burden of malaria was particularly apparent in the south where the change in RDT positivity rate increased significantly over the 6-year reporting period in the Southwest (35 % Page 8 of 18 in 2010 to 60 % in 2015), Southeast (39 to 60 %), South (16 to 58 %), and both Fringe (west: 28 to 41 %; east: 9 to 24 %) ecozones (p < 0.05 by linear regression; Additional file 4D). Other zones had a much smaller effect size of change [negative for the Northeast (37 to 35 %) and Central highlands (17 to 15 %)], which was non-significant. In the Southwest, positive malaria diagnoses were relatively rare before 2013, with only 3 months reported to have >10 % of consultations attributable to malaria; in contrast, health centre reports from 2013 to 2015 indicated 25 months to have had >10 % of consultations concluding with an RDT+ diagnosis (Additional file 4C). In the higher-endemic Southeast, health facility reports noted only 2 months during 2010 when RDT+ consultations were greater than 12 % of all consultations; in contrast, this occurred during all months of 2015. Seasonal trends from these reporting-adjusted figures are less prominent than from the raw RDT positive counts (Fig. 3a), but nevertheless apparent. An annual lag in autocorrelation across the time series of diagnostic positivity rate was particularly apparent from the east coast ecozones (Northeast, Southeast and Fringe areas), indicative of a seasonality effect, though this was less evident from western areas (Additional file 4D). Completeness of the HMIS data Of the 3924 health facilities intended to contribute monthly reports to the HMIS in 2015 [41], 214 were hospitals or specialist institutions, 2563 were community-level public health facilities staffed by either doctors (63 %) or nurses (37 %), and 1147 were private-sector institutions. Reporting improved across the 6-year period reviewed, from a mean monthly reporting rate of just over half of health facilities (55 %) in 2010, increasing to three-quarters (76 %) in 2015 (Fig. 4a). Some inter-annual fluctuation in total facility count is anticipated due to facility closures and openings, but fluctuations in the numbers of received reports are likely to be principally attributable to underreporting. An additional source of malaria case data comes from the network of community health workers (CHW) active across the country [41, 42]. However, these volunteers report their activities through their health facility, not (See figure on next page.) Fig. 4 Indicators of the completeness of routine HMIS reporting. a Monthly health centre reports received by the NMCP. The solid black line indicates the total number of health facilities in Madagascar (n = 3924 [41]). The right-hand axis refers to the % of overall health centres reporting across all zones. Data represent a monthly report being submitted, but provide no indication of the completeness of the submitted reports. b Proportion of distributed RDTs for which a result (either positive or negative) was reported to the HMIS in 2014. Overall, 1.86 million RDTs were distributed to health districts nationally, and results for 0.94 million (50.5 %) were reported into the HMIS database. c Reported RDT stock-out incidence. The map shows the district-level mean RDT stock-out days in 2014. Only data from health centres that submitted at least one report for drug/diagnostic availability in the year were included in the mean calculation. Plots show stock-out status from 2012 to 2014 per ecozone. Points correspond to the mean number of stock-out days annually (left hand y axis) and bars represent the reporting rate within each ecozone (right hand y axis) Howes et al. Malar J (2016) 15:502 Page 9 of 18 Howes et al. Malar J (2016) 15:502 directly to the HMIS. Increasing numbers of active CHW should not affect the number of monthly reports submitted to the HMIS, though it will impact on the numbers of fever-related consultations performed. While Fig. 4a data indicate that a report was received at the central level from the health facility, it provides no indication of the quality of the report. A proxy indicator of reporting completeness may be represented through evaluation of the proportion of RDTs distributed for which a result was reported into the NMCP database (Fig. 4b). Important limitations exist to assuming this proxy: not necessarily 100 % of RDTs distributed would be needed within the year, RDT distribution may not be constant through time (some health facilities may receive large deliveries some years and carry lower stocks in other years), and the risk that not all distributed RDTs are fit for use (some may be discarded due to void test results or overdue expiry dates). Nevertheless, the broad categories used in Fig. 4b indicate that even making allowance for distributed RDTs remaining unused in the health centres, 79 of 112 health districts (71 %) had more than half of their distributed RDT stock results in 2014 unaccounted for in the HMIS; in 34 districts, reports were received for fewer than 30 % of the distributed RDTs. Overall, 50.5 % of the distributed RDTs across Madagascar in 2014 (n = 1.856 million) were accounted for in the HMIS results. When aggregated to the ecozone scale, reporting from Southeast ecozone was highest, accounting for 78 % of distributed RDTs, while the South ecozone had poorest reporting, accounting for only 24 % of distributed RDTs in 2014. Rates in all other ecozones ranged between 29 and 56 %. Case management policies in Madagascar require RDT confirmation for a case to be treated with anti-malarials and counted as a confirmed case in the reporting system. RDT stock-outs will therefore impact directly on the numbers of cases reported. Reporting of presumed cases is not consistently distinguished from confirmed cases in the monthly reports, introducing uncertainty into the reports (NMCP, pers. comm.). Nationally in 2014, 51 % of health facilities sent no reports of any stock inventories (including of drug stocks), while those that did report indicated a mean number of 30.5 days with RDT stock-outs (range: 0–330 days). Figure 4c summarizes the district stock-out data for 2014 (map) and reporting characteristics by ecozone from 2012 to 2014 (graphs). Bar plots (right hand y axis) show the health centre reporting rates over time, indicating the large numbers of health centres failing to report, and thus the difficulty with interpreting the data on numbers of stock-out days, which otherwise indicate that stock-outs are relatively infrequent events. Health centre isolation was evaluated as a potential factor contributing to reporting incompleteness (Fig. 5). Page 10 of 18 Figure 5a maps population density across the country, while Fig. 5b charts the proportion of each district’s population living within 1 h walk of their nearest health facility (5 km). The colour index in Fig. 5c indicates the relative difficulty for health centre staff to access the health district office to submit their monthly health report. Overall, 56 % of the population are reported to live further than 5 km from their nearest health centre. This was generally consistent across all ecozones (range: 51–64 %), peaking in the Southwest. Nearly half of health facilities were situated more than 50 km from their district office (1125 of 2690 health facilities for which location details were available), with 13 % (n = 147) of those accessible only by foot or river/sea. Health centre isolation data (quantified at the district level as the mean travel time from each health centre to the central district office) were analysed for potential correlations with 2014 data on: (i) RDT result underreporting; and, (ii) the RDT stock-out reporting rate. No correlation emerged between these datasets, which were only available aggregated to the district level. Outbreaks The nature of transmission events classified as ‘outbreaks’ differs substantially between affected geographic areas, as previously defined. Nevertheless, here these are considered together, irrespective of the particular outbreak types. This was partly due to data availability on the classifications of outbreak events, but also because all types of outbreaks require NMCP intervention, irrespective of the circumstances. Overall, therefore, January 2012 to December 2015 yielded a total of 292 outbreaks reported to the NMCP’s surveillance system as shown in Fig. 6 (instances of autochthonous transmission in low transmission ecozones, or an unexpected excess of cases in endemic areas; definitions provided in “Methods” section). Total reported outbreaks per year were 42 in 2012, 107 in 2013, 60 in 2014, and 83 in 2015. Over half (n = 63) of the country’s 112 districts reported an outbreak during this period; 31 of these reported more than one outbreak. Overall, half the total reported outbreaks occurred in just ten districts. At the commune level (administrative sub-division of a district), 193 of 1579 communes were affected by outbreaks between January 2012 and December 2015 (12.2 %), though 73.1 % of these reported only a single outbreak. Only 7.3 % (n = 14) of affected communes reported more than two outbreaks during the 4 year reporting period examined. The data suggest that although districts could be categorized as being at higher or lower risk of epidemics, at the communelevel outbreaks do not appear to have a strong temporal association. Howes et al. Malar J (2016) 15:502 Page 11 of 18 Fig. 5 District-level summaries of health centre accessibility, a population density in 2015; b health centre accessibility to catchment populations and b health centre accessibility to the health district headquarters where monthly data must be reported. a Maps population density in 2015 (WorldPop [63]) together with cities estimated to have populations >100,000). b Shows the proportion of the population resident more than 5 km from their nearest health facility (equivalent to further than approximately one h walk). The underlying colour indicates the total population resident in the district, while the pie charts show the proportion of the population per district resident further or closer than 5 km from their nearest health facility. This is based off a subset of 2690 health facilities (69 % of all health facilities) for which data were available. c Maps pie charts representing different categories of accessibility for each health centre per district. Colours represent the available mode of transport, while colour intensity indicates distance categories between health centres and the health district headquarters. Pie chart sizes indicate the number of health centres needing to report data per district (range: 3–55). Arrows indicate the increasing difficulty with reporting. Data from the NMCP (Fig. 5b) and the Malagasy Health Sector Development Plan for 2014-2019 [64] (Fig. 5c) The occurrence of outbreaks in relation to the overall burden of malaria is considered in Fig. 7. Records of the outbreaks were incomplete, with 133 of the 292 outbreak reports in the database not specifying the affected month. The outbreaks barplots in Fig. 7 are therefore incomplete, but nevertheless suggest a relationship with the intensity of background transmission. Lower endemicity areas, such as the two Highland fringe ecozones, have peaks in incidence corresponding with months when outbreaks were reported; while higher incidence areas, such as along the East coast and the Northwest ecozone, trends in malaria incidence do not correspond to outbreak periods. For example, in the Southeast, the year with fewest reported outbreaks (2015; n = 4) was when incidence was greatest. Discussion An updated sub-national stratification of Madagascar is proposed which reflects contemporary transmission intensity. Within these limits, the epidemiology of malaria is described as evidenced by routinely reported health facility data made available to the NMCP. From this evidence base, the National Programme formulates control policies, oversees epidemiological monitoring, and assesses policy impacts. The eight ecozones may not necessarily each require bespoke control schemes, but these allow greater sub-national epidemiological insight, and therefore flexibility in targeting of interventions based on local needs. These sub-divisions are not static, but instead must be periodically updated to reflect shifts in the current patterns of transmission, though with a frequency that does not prevent temporal trends being assessed in the short term. The narrow time window presented here is the product of decades of malaria control, so the epidemiology described cannot be interpreted as representing the fundamental niche of malaria in Madagascar. Malaria is a disease of increasing public health concern in Madagascar. Since 2010, the number of reported Howes et al. Malar J (2016) 15:502 Fig. 6 Outbreaks documented in the NMCP records for January 2012–December 2015. Outbreak definitions vary according to expected local transmission characteristics. Colours indicate the total number of outbreaks reported per district during the period examined. Overlain symbols indicate the number of years affected between 2012 and 2015. Grey background indicates no reported outbreaks RDT-confirmed cases has quadrupled across the country. When adjusted for numbers of tests performed (thus adjusting for (i) reporting rates, (ii) the unavailability of diagnostics, and (iii) the scale-up of access to diagnostic testing through community health worker programmes [42]), the RDT positivity rate increased by 50 % between 2010 and 2015, reaching 50 % slide positivity in 2015. Despite this evidence of recent resurgences, the incidence of malaria in Madagascar remains among the lowest of the sub-Saharan African region, with high levels of intervention coverage, particularly of LLINs [8, 14]. This juncture therefore represents an important opportunity to re-evaluate strategies and reverse the recent negative trends. For instance, while LLIN coverage may be high, treatment-seeking and adherence to recommended artemisinin combination therapy (ACT) policies appears weak [8, 43]. Page 12 of 18 Although the majority of the malaria burden remains in the Southeast (42 % in 2015), malaria has increased across all areas in year-round significance, increasing in intensity during both high (December–April) and low transmission periods. The Central highlands seem to be the exception, but the epidemiology differed there, consisting primarily of imported cases (NMCP, pers. comm.), and with very low incidence compared to other ecozones. Outbreaks, defined in this context as unexpectedly high or rapid increases in transmission or evidence of autochthonous transmission in areas considered at low risk, appear to associate closely with overall burden trends in lower-transmission ecozones. A more detailed view of changes in case numbers at health facilities affected by outbreaks would allow more robust insights into the relative contribution of outbreak-driven transmission to overall caseloads across the country. An important missing piece from this study’s narrative is the relative contribution of each Plasmodium species to the regional case burden. This detail is not recorded in the data. Prevalence surveys indicate a predominance of P. falciparum across most areas, but regional pockets of Plasmodium vivax and sporadic cases of Plasmodium ovale and Plasmodium malariae are also detected [24, 25, 29, 44, 45]. A longitudinal dataset on the contribution of each species to morbidity from routine reporting would improve the evidence-base for determining the value of introducing non-P. falciparum-specific intervention policies. The recent trends described in this study stand in stark contrast to the country’s stated ambitions to start the transition towards malaria elimination. The country’s current National Strategic Plan (2013–2017) aims for pre-elimination status by end 2017, with zero malaria deaths and the reduction of test positivity rates to <5 % in 15 % of districts, and the halving of the positivity rate in the remaining districts relative to 2013 baseline data [21, 39]. Overall, however, 2013–2015 saw a 34 % increase in the positivity rate. Acknowledgement of these trends by the NMCP led to a revision of the National Strategic Plan in 2015, with adjustment of their initial goals now more aligned with achieving burden control than elimination [21]. The drive to control and eventually eliminate malaria from Madagascar receives strong financial backing from the international community, with disbursements in excess of $400 million since 2004 when collaborative efforts were galvanized and intensified [8, 11, 46]. In 2006–2010, resources for malaria control in Madagascar corresponded to higher than average percapita funding across the African region [8, 47]. Since this time though, the country’s political crisis has severely impacted on the country’s socio-economic status and Howes et al. Malar J (2016) 15:502 Page 13 of 18 Fig. 7 Temporal correspondence between reported malaria case incidence (left-hand y axis; plotted as points) and outbreak reports (right hand y axis; plotted as barplots) aggregated by ecozone for 2012–2015. Of the 292 outbreaks reports, nearly half (n = 133) provided no supporting information about the affected month so these could not be included in the monthly outbreak plots. The total annual outbreaks reported per ecozone is specified in the plots (fraction denominator), with the numerator summarizing the number of outbreaks missing from the bar plots due to incomplete information associated health system infrastructure and staffing, impeded external funding to the country thus preventing intervention roll-out (see the Global Fund disbursement delays in Fig. 1), and complicated the control programme management by preventing direct bilateral relationships between the MoH and donors [21]. The situation in Madagascar therefore reflects what has been widely reported from other areas, that withdrawal Howes et al. Malar J (2016) 15:502 of core funding is closely associated with rapid and predictable resurgence of malaria [48, 49] (Fig. 1). Despite high coverage rates of interventions around 2013, interrupted funding led to delays in sustaining these levels. Periods of limited funding are when reliable epidemiological data become all the more important to inform efficient planning and optimize resource allocation. Such data require robust processing at all stages of the chain from the probability of patients seeking treatment at a health facility, through diagnosis, through data reporting to data management and evaluation at the central level. More than half the Malagasy population live further than 5 km (roughly corresponding to an hour’s walk) from their nearest health facility. This likely contributes substantially to the treatment-seeking rate that ranged in 2013 from 21.7 to 49.1 % across different parts of the country [24, 43, 50, 51], with many patients suffering fevers resorting to self-medication instead (including both pharmaceutical and traditional herbal remedies) [52]. Therefore, most cases of fever will never interact with the country’s formal health system. The routine HMIS had a largely consistent reporting rate from health facilities across 2010–2014 at approximately 60 %, though rising to 75 % in 2015 (Fig. 4a), meaning that almost half of health centre reports were not recorded in the NMCP’s databases for most of the time period reviewed. Further reinforcement of the incompleteness of the datasets is the disparity between distributed diagnostic kits and the test results reported (Fig. 4b). The received outbreak surveillance data are also incomplete, evidenced by nearly half of reports not being month-specific (Fig. 7). Madagascar has a well-documented history of devastating epidemics, with the earliest reported outbreak around 1887 coinciding with the establishment of rice farming [21]. Outbreaks both in Madagascar and globally have primarily occurred in the context of socio-economic and political upheavals [48] when there has been a breakdown of the control programme structure, including surveillance and reporting channels. Implicit in the programmatic concept of an outbreak is that the event triggers an emergency response from the responsible public health institution to curtail the exacerbation of the situation. An efficient surveillance system will allow outbreaks to be anticipated and intervention resources to be mobilized ahead of transmission developing out of control. The reporting system in Madagascar was described in 2015 by the US President’s Malaria Initiative as “extremely weak”, hindered in part by a critical shortage of health staff at all levels, with human resources unevenly distributed across the country [11]. To further investigate this, a nationally representative review of the health reporting system was commissioned in 2015 to qualitatively and quantitatively assess the system’s Page 14 of 18 functionality (thus supplanting the need for proxy indictors of completeness). This included detailed interviews with staff at all levels of the reporting pyramid, alongside comparisons of raw data to assess the completeness and accuracy of reporting from doctors’ records up to the central database. Preliminary results suggest that both the quantity and quality of reported health facility data were weak. Reporting timeliness was particularly affected, with only 8 of 45 districts reviewed in January 2015 reporting within the MoH’s defined schedule [31]. Reporting completeness varied across the country, being highest in the central highlands, and lowest in the tropical coastal areas and desert south. A further concern identified by the surveys related to the lack of health professionals to make use of the data and identify trends specific to their local catchment population: interpretation of the data was principally limited to the central level, and not performed in situ. The investigators’ perspectives of the NMCP’s data in particular, was that the existence of a database was a strength of their epidemiological monitoring unit. Weaknesses of the data included the complexity of the reporting system, its fragmented nature and the difficulty of assimilating the data for analysis, logic checks are not routinely performed on the data, and confusion existed regarding some of the calculations used to derive the metrics reported [31]. Surveys actively assessing stocks of diagnostic kits in health centres suggest that the passively reported data on stock-out events that were available for review here (Fig. 4c) underestimate the true incidence of stock-out events [49]. This indicates that the true rate of under-diagnosis due to diagnostic stock-outs and knock-on impact on reported case numbers may be even greater than suggested. Stakeholders are aware of these limitations to the reporting system, and a concerted effort to address them is ongoing. Given the need for annual case estimates to assess both national- and global-level trends in malaria incidence and to monitor progress towards pre-defined milestones, the World Health Organization’s (WHO) Global Malaria Programme and collaborators have developed methods to account for weaknesses in routine HMIS data [8, 14, 53, 54]. Two main approaches exist to estimate malaria incidence [53], each best suited to different countries according to their programme control phase and the strength of their disease surveillance system. Method 1 is favoured by the WHO for countries outside Africa, where routine reporting is considered more reliable. This method uses parameters to adjust MoH routine HMIS data to account for (i) incomplete case detection by health facilities; (ii) the likelihood of over-diagnosis of malaria among patients with fevers; and, (iii) public/private treatmentseeking preferences [53, 55]. Method 2 uses a cartographic approach based on population prevalence surveys to Howes et al. Malar J (2016) 15:502 develop spatial and temporal models of the incidence of clinical disease; this has been the main approach followed by MAP to derive global endemicity maps of P. falciparum and P. vivax transmission [14, 56, 57], and is used by the WHO to estimate case numbers from most sub-Saharan African countries where routine reporting is considered too weak for Method 1 estimation. A subset of low-transmission African countries is also considered to have sufficiently strong routine reporting data to allow support for Method 1-based estimates. The implication being that these countries have a sufficiently functional health system to have robust surveillance and reporting systems in place within the context of a functional and effective malaria control programme. The sub-Saharan African countries included in this category are Botswana, Cabo Verde, Eritrea, Namibia, South Africa, Swaziland, and Zimbabwe (all of which are classified as being “on track for >75 % decrease in incidence 2000–2015”), and Madagascar (“less than 50 % change in incidence projected 2000–2015”) [8]. Nevertheless, the WHO lists Madagascar among the eight sub-Saharan African low-transmission countries with sufficiently accurate HMIS data to permit convincing estimates to be made from simple adjustment of the numbers of reported cases. This study explores the routine HMIS data available to the Malagasy NMCP which underpins this perhaps contradictory classification. Of the subset of African Method 1 countries, Madagascar had the highest endemicity in 2015 [8, 14] and among the lowest access to the formal health system [43]. The magnitude of the uncertainty in the reported case numbers is evident by the WHO’s case estimate for Madagascar in 2013. Although 387,045 cases were reported by Madagascar to the World Malaria Report for 2013, once adjusted using Method 1, the estimated case number for 2013 was 1.2 million (0.75– 2.10 million) [8], therefore corresponding to more than three times the reported RDT-confirmed numbers. Given these acknowledged uncertainties and the recent resurgence of transmission with variable sub-national patterns, a re-consideration of the method applied to case estimation in Madagascar would be worthwhile. Regular nationally representative Malaria Indicator Surveys and other prevalence surveys take place, which provide a rich source of malariometric data complimentary to the HMIS data. If Madagascar is to remain within the Method 1 countries and have its case estimates derived from adjustment of the routine reporting data, the spatial heterogeneity of transmission characteristics and of reporting robustness discussed in this paper would be a valuable addition to the methodology instead of relying on a single set of parameters. The data adjustment parameters show widespread variation between ecozones, with treatmentseeking rates in the Fringe areas less than half that of the South (21.7 and 49.1 %, respectively) [24]. Similarly, Page 15 of 18 health facility reporting rates differ between areas, and, although no correlation could be identified from the aggregated district-level data reviewed here, it is likely that some association between health centre isolation and reporting quality and timeliness exists. No single set of national-level summary adjustment parameters, as per the current WHO Method 1 protocol, could capture this heterogeneity. The sub-national variation in transmission intensity is reflected by factors such as age-specific incidence patterns (Fig. 3b). The Central highlands ecozone, where transmission is lowest, has no significant age-specific patterns with the whole population at similar risk of infection. In higher endemic areas, younger children suffer the highest burden of disease, likely due to the development of immunity in older age groups from repeated exposure in childhood. To adequately adjust health facility data from the HMIS, this spatial variability ought to be taken into account. In parallel to this, large numbers of prevalence surveys have been performed across the country as part of three completed MIS surveys from 2011, 2013, and April 2016 (results expected end 2016), among others [29]. These community-based surveys have shown the same increase in prevalence as the HMIS health-facility based case data. While these prevalence surveys are generally better standardized than HMIS data, they are much less comprehensive in space and time. Comparison of the estimates from these two independent methods (routine data-based and cartographic) and datasets would be valuable. Conclusion Madagascar is highly diverse, not least in terms of its malarial epidemiology, and accordingly requires spatially specific control planning. This paper presents an overview of the last 5 years of HMIS data, the period immediately following a political crisis and the nation’s subsequent exclusion from the international community until May 2014. Associated with these events, malaria control efforts have been hindered, resulting in rapid resurgences of transmission [49], with ecozones previously considered to be low transmission zones, such as the Southwest and South, now becoming stable endemic areas, reflected by differential patterns in age-specific incidence, for example. No single approach for malaria control can be applied across the island, or even binary high/low transmission approach given the peculiarities of transmission in different ecozones. Regular epidemiological assessments are therefore recommended at the ecozone levels. The NMCP is frequently restricted to being reactive rather than proactive and unable to anticipate demands (e.g., in terms of anticipating commodity needs or anticipating outbreaks). Closer analysis of data trends in real-time, Howes et al. Malar J (2016) 15:502 would be valuable to support the NMCP. For instance, it will be important to closely evaluate the 2016 data to assess the impact of the 2015–2016 LLIN distribution programme, and determine how rapidly the sharp increase of cases in 2015 can be reversed to shift the country in the direction of its elimination goals. This study comes at the end of the Millennium Development Goal era (2000–2015), which aimed to have “halted by 2015 and begun to reverse the incidence of malaria” (Target 6.C) [12]. In Madagascar this was clearly being achieved from 2000 to 2009, though the reverse has occurred since 2010 (Figs. 1, 2, 3). Using the year 2000 as a baseline descriptor may not be a very pertinent approach for evaluating the current status of progress in malaria control. The situation in 2000 was one of essentially no financial or political commitment to large-scale intervention roll-out. Most reported cases would have been presumptively diagnosed based on symptoms (likely over-estimating case numbers), given that rapid field-based diagnostics were only introduced in 2007. More pertinent than noting a decrease in caseload since 2000 is the concerning increase in burden since 2010 when funding sources were substantially increased and malaria control was widespread across Madagascar. While it is important to interpret the imperfect health facility data with caution [42, 58], the available data do provide detailed insight into the relative differences in epidemiology sub-nationally. However, given that between a quarter and half of health facility reports are missing from the NMCP’s database, the absolute number of cases reported is evidently a major underestimate. Approaches to quantify estimates of the absolute annual burden would be useful in attempting to deduce a more robust estimate of overall burden rather than relying on an incomplete database as is currently the case by the NMCP. Nevertheless, the HMIS data present a convincing case for the growing challenge of malaria across all areas of Madagascar. Additional files Additional file 1. Summary of data sources consulted. The table provides details of the data sources used for each figure in the paper. Additional file 2. Malaria Atlas Project (MAP) modelled maps of Plasmodium falciparum parasite prevalence (PfPR) in 2010–2015. This document provides additional information about the modelled prevalence map that was used to stratify Madagascar into contemporary ecozones. Additional file 3. Tabulation of regions and districts by ecozone. Administrative regions and districts of Madagascar classified according to the newly designed ecozones. Additional file 4. Additional plots of the trends of reported malaria case data in Madagascar (2010–2015). This file provides additional analysis of the temporal trends in the reported malaria data. Data are adjusted for reporting and diagnostic shortages, and trends are examined using autocorrelation plots, generalized additive models and linear regression models. Page 16 of 18 Authors’ contributions REH wrote the first draft and conceived the study with PAZ and AR. SAM, BR, TF, and AJR contributed the data. DB and PWG advised on the analysis. RH, PAZ and AR were responsible for data interpretation. All authors edited the final version of the manuscript and approved its submission for publication. All authors read and approved the final manuscript. Author details 1 Center for Global Health and Diseases, Case Western Reserve University, Cleveland, OH 44106‑4983, USA. 2 Department of Zoology, University of Oxford, Oxford OX1 3PS, UK. 3 National Malaria Control Programme, Ministry of Health, Androhibe, Antananarivo, Madagascar. 4 Department of Public Health, Faculty of Medicine, University of Antananarivo, Antananarivo, Madagascar. 5 Oxford Big Data Institute, Li Ka Shing Centre for Health Information and Discovery, University of Oxford, Oxford OX3 7BN, UK. 6 Faculty of Sciences, University of Antananarivo, Antananarivo, Madagascar. Acknowledgements The authors thank two anonymous reviewers for their helpful suggestions towards improving this work. The authors are also grateful to Harry Gibson, Zhi Huang, Mike Thorn and Dan Weiss of the Malaria Atlas Project for help with the temperature suitability index figure, elevation and rainfall raster surfaces. Competing interests SAM, AJR, BR, TF and AR are employees of the National Malaria Control Programme of Madagascar. The authors declare that they have no competing interests. Availability of data and materials Data are available from the National Control Programme of Madagascar. Consent for publication All authors approved the manuscript’s submission for publication. Funding The authors, except DB and PWG, acknowledge funding from the NIH to PAZ (R01 AI097366). REH is also funded by a Wellcome Trust Senior Research Fellowship to Simon I Hay (095066). AJR acknowledges funding from the Global Fund to Fight AIDS, Tuberculosis and Malaria. PWG acknowledges support from the Bill and Melinda Gates Foundation (#OPP1068048, #OPP1106023), which also supports DB. Received: 5 August 2016 Accepted: 5 October 2016 References 1. The World Bank. Madagascar: Measuring the impact of the political crisis. Washington; 2013. http://www.worldbank.org/en/news/feature/2013/06/05/madagascar-measuring-the-impact-of-the-politicalcrisis. Accessed 31 Jan 2016. 2. Barmania S. Madagascar’s health challenges. Lancet. 2015;386:729–30. 3. World Bank Country Profile. http://www.data.worldbank.org/country/ madagascar. 4. World Food Programme-UNICEF. Rural Madagascar comprehensive food and nutrition security and vulnerability analysis. 2011. 5. Famine Early Warning Systems Network (FEWS NET). http://www.fews. net/southern-africa/madagascar. Accessed 31 Jan 2016. 6. Global Burden of diseases, injuries, and risk factors study 2010. GBD Profile; 2010. http://www.healthdata.org/results/country-profiles. 7. Service des Statistiques Sanitaires—Ministère de la Santé Publique. Annuaire des Statistiques du Secteur Santé de Madagascar. Antananarivo: Ministry of Health; 2012. 8. 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Predicting plant diversity patterns in Madagascar: understanding the effects of climate and land cover change in a biodiversity hotspot. PLoS One. 2015;10:e0122721. 16. Battistini R, Richard-Vindard G, editors. Biogeography and ecology in Madagascar. Berlin: Spring-Science + Business Media; 1972. 17. Lepers JP, Ramanamirija JA, Andriamangatiana-Rason MD, Coulanges P. Recent findings on the epidemiology of malaria and on the distribution of plasmodial species in Madagascar in 1987 (in French). Arch Inst Pasteur Madag. 1988;54:151–67. 18. Joncour G. The fight against malaria in Madagascar (in French). Bull World Health Organ. 1956;15:711–23. 19. Laventure S, Rabarison P, Mouchet J, Andrianaivolambo L, Rakotoarivony I, Rajaonarivelo E, et al. Malaria: research perspectives in medical entomology in Madagascar (in French). Sante. 1995;5:406–10. 20. Pock Tsy JM, Duchemin JB, Marrama L, Rabarison P, Le Goff G, Rajaonarivelo V, et al. Distribution of the species of the Anopheles gambiae complex and first evidence of Anopheles merus as a malaria vector in Madagascar. Malar J. 2003;2:33. 21. National Malaria Control Programme of Madagascar. National strategic plan for malaria control in Madagascar 2013–2017: consolidating the gains with a view to elimination of malaria from Madagascar, 2015–2017 revision. 2015. 22. Mouchet J, Blanchy S, Rakotonjanabelo A, Ranaivoson G, Rajaonarivelo E, Laventure S, et al. Epidemiological stratification of malaria in Madagascar (in French). Arch Inst Pasteur Madag. 1993;60:50–9. 23. Mouchet J, Blanchy S. Particularités et stratification du paludisme à Madagascar. Cahiers Santé. 1995;5:386–8. 24. Institut National de la Statistique (INSTAT), Programme National de Lutte contre le Paludisme (PNLP), Institut Pasteur de Madagascar (IPM), and ICF International. Madagascar Malaria Indicator Survey. Enquête sur les indicateurs du paludisme (EIPM). Calverton: INSTAT, PNLP, IPM and ICF International; 2013. 25. Institut National de la Statistique (INSTAT), Programme National de Lutte contre le Paludisme (PNLP), and ICF International. Madagascar Malaria Indicator Survey. Enquête sur les indicateurs du paludisme (EIPM). Calverton: INSTAT, PNLP, and ICF International; 2011. 26. Mendis K, Rietveld A, Warsame M, Bosman A, Greenwood B, Wernsdorfer WH. From malaria control to eradication: the WHO perspective. Trop Med Int Health. 2009;14:802–9. 27. Hay SI, Smith DL, Snow RW. Measuring malaria endemicity from intense to interrupted transmission. Lancet Infect Dis. 2008;8:369–78. 28. Malaria Atlas Project (MAP). http://www.map.ox.ac.uk. 29. Kesteman T, Randrianarivelojosia M, Mattern C, Raboanary E, Pourette D, Girond F, et al. Nationwide evaluation of malaria infections, morbidity, mortality, and coverage of malaria control interventions in Madagascar. Malar J. 2014;13:465. 30. Weiss DJ, Mappin B, Dalrymple U, Bhatt S, Cameron E, Hay SI, et al. Reexamining environmental correlates of Plasmodium falciparum malaria endemicity: a data-intensive variable selection approach. Malar J. 2015;14:68. 31. Ly M, N’Gbichi J-M, Lippeveld T, Yazoume Y. Report on the evaluation of the performance of the Health Management Information System (HMIS) and Integrated Diseases Surveillance and Response System (IDSRS). MEASURE Evaluation, funded by the President’s Malaria Initiative (PMI) via USAID; 2016. 32. Department of Health Statistics of the Ministry of Health of Madagascar. User guide to the GeSIS software for Madagascar (Version 2011). Antananarivo: Ministry of Health; 2012. Page 17 of 18 33. Ratsimbasoa A, Ravony H, Vonimpaisomihanta JA, Raherinjafy R, Jahevitra M, Rapelanoro R, et al. Management of uncomplicated malaria in febrile under 5-year-old children by community health workers in Madagascar: reliability of malaria rapid diagnostic tests. Malar J. 2012;11:85. 34. Ratsimbasoa A, Randriamanantena A, Raherinjafy R, Rasoarilalao N, Menard D. Which malaria rapid test for Madagascar? Field and laboratory evaluation of three tests and expert microscopy of samples from suspected malaria patients in Madagascar. Am J Trop Med Hyg. 2007;76:481–5. 35. Rajatonirina S, Rakotomanana F, Randrianasolo L, Razanajatovo NH, Andriamandimby SF, Ravolomanana L, et al. Early-warning health and process indicators for sentinel surveillance in Madagascar 2007–2011. Online J Publ Health Inform. 2014;6:e197. 36. Randrianasolo L, Raoelina Y, Ratsitorahina M, Ravolomanana L, Andriamandimby S, Heraud JM, et al. Sentinel surveillance system for early outbreak detection in Madagascar. BMC Publ Health. 2010;10:31. 37. WHO. Systems for the early detection of malaria epidemics in Africa. An analysis of current practices and future priorities. Geneva: World Health Organization; 2006. 38. Hay SI, Simba M, Busolo M, Noor AM, Guyatt HL, Ochola SA, et al. Defining and detecting malaria epidemics in the highlands of western Kenya. Emerg Infect Dis. 2002;8:555–62. 39. National Malaria Control Programme of Madagascar. National strategic plan for malaria control in Madagascar 2013–2017. Reinforcing the gains of malaria control with a perspective towards malaria elimination in Madagascar. 2012. 40. Ministry of Health of Madagascar. Reference manual of principal health system indicators (in French). Antananarivo: Ministry of Health of Madagascar; 2014. 41. National Malaria Control Programme of Madagascar. Monitoring and evaluation plan revised version for 2015–2017. 2015. 42. WHO. The utility of estimates for health monitoring and decision-making: global, regional and country perspectives. Report of a technical meeting. Geneva: World Health Organization; 2015. 43. Battle KE, Bisanzio D, Gibson HS, Bhatt S, Cameron E, Weiss DJ, et al. Treatment-seeking rates in malaria endemic countries. Malar J. 2016;15:20. 44. Barnadas C, Ratsimbasoa A, Tichit M, Bouchier C, Jahevitra M, Picot S, et al. Plasmodium vivax resistance to chloroquine in Madagascar: clinical efficacy and polymorphisms in pvmdr1 and pvcrt-o genes. Antimicrob Agents Chemother. 2008;52:4233–40. 45. Menard D, Barnadas C, Bouchier C, Henry-Halldin C, Gray LR, Ratsimbasoa A, et al. Plasmodium vivax clinical malaria is commonly observed in Duffynegative Malagasy people. Proc Natl Acad Sci USA. 2010;107:5967–71. 46. The Global Fund to Fight AIDS Tuberculosis and Malaria. Grant Portfolio for Madagascar. http://www.theglobalfund.org/en/portfolio/ country/?loc=MDG. Accessed 31 Jan 2016. 47. Pigott DM, Atun R, Moyes CL, Hay SI, Gething PW. Funding for malaria control 2006–2010: a comprehensive global assessment. Malar J. 2012;11:246. 48. Cohen JM, Smith DL, Cotter C, Ward A, Yamey G, Sabot OJ, et al. Malaria resurgence: a systematic review and assessment of its causes. Malar J. 2012;11:122. 49. Kesteman T, Rafalimanantsoa SA, Razafimandimby H, Rasamimanana HH, Raharimanga V, Ramarosandratana B, et al. Multiple causes of an unexpected malaria outbreak in a high-transmission area in Madagascar. Malar J. 2016;15:57. 50. Marks F, Rabehanta N, Baker S, Panzner U, Park SE, Fobil JN, et al. A way forward for healthcare in Madagascar? Clin Infect Dis. 2016;62(Suppl 1):S76–9. 51. Pach A, Warren M, Chang I, Im J, Nichols C, Meyer CG, et al. A qualitative study investigating experiences, perceptions, and healthcare system performance in relation to the surveillance of typhoid fever in Madagascar. Clin Infect Dis. 2016;62(Suppl 1):S69–75. 52. Mattern C, Pourette D, Raboanary E, Kesteman T, Piola P, Randrianarivelojosia M, et al. “Tazomoka is not a problem”. Local perspectives on malaria, fever case management and bed net use in Madagascar. PLoS One. 2016;11:e0151068. 53. Cibulskis RE, Aregawi M, Williams R, Otten M, Dye C. Worldwide incidence of malaria in 2009: estimates, time trends, and a critique of methods. PLoS Med. 2011;8:e1001142. Howes et al. Malar J (2016) 15:502 54. Hay SI, Okiro EA, Gething PW, Patil AP, Tatem AJ, Guerra CA, et al. Estimating the global clinical burden of Plasmodium falciparum malaria in 2007. PLoS Med. 2010;7:e1000290. 55. WHO. World Malaria Report 2014. Geneva: World Health Organization; 2014. 56. Gething PW, Elyazar IR, Moyes CL, Smith DL, Battle KE, Guerra CA, et al. A long neglected world malaria map: Plasmodium vivax endemicity in 2010. PLoS Negl Trop Dis. 2012;6:e1814. 57. Gething PW, Patil AP, Smith DL, Guerra CA, Elyazar IR, Johnston GL, et al. A new world malaria map: Plasmodium falciparum endemicity in 2010. Malar J. 2011;10:378. 58. Rowe AK, Kachur SP, Yoon SS, Lynch M, Slutsker L, Steketee RW. Caution is required when using health facility-based data to evaluate the health impact of malaria control efforts in Africa. Malar J. 2009;8:209. 59. Farr TG, Rosen PA, Caro E, Crippen R, Duren R, Hensley S, et al. The shuttle radar topography mission. Rev Geophys. 2007:45. Page 18 of 18 60. Weiss DJ, Bhatt S, Mappin B, Van Boeckel TP, Smith DL, Hay SI, et al. Air temperature suitability for Plasmodium falciparum malaria transmission in Africa 2000–2012: a high-resolution spatiotemporal prediction. Malar J. 2014;13:171. 61. Friedl MA, Sulla-Menashe D, Tan B, Schneider A, Ramankutty N, Sibley A, et al. MODIS collection 5 global land cover: algorithm refinements and characterization of new datasets, 2001–2012, collection 5.1 IGBP land cover. Boston: Boston University; 2010. 62. Channan S, Collins K, Emanuel WR. Global mosaics of the standard MODIS land cover type data. College Park: University of Maryland and the Pacific Northwest National Laboratory; 2014. 63. WorldPop project. http://www.worldpop.org. 64. Chandler R, Lang E. Budgeting the Malagasy Health Sector Development Plan: supplementary analyses, using the OneHealth tool. Washington: Futures Group, Health Policy Project; 2015. 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 

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UC Irvine UC Irvine Previously Published Works Title Evaluation of daily home spirometry for school children with asthma: new insights. Permalink https://escholarship.org/uc/item/3db1m0h1 Journal Pediatric pulmonology, 41(9) ISSN 8755-6863 Authors Thompson, Rohan Delfino, Ralph J Tjoa, Thomas et al. Publication Date 2006-09-01 DOI 10.1002/ppul.20449 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed eScholarship.org Powered by the California Digital Library University of California Pediatric Pulmonology 41:819–828 (2006) Evaluation of Daily Home Spirometry for School Children with Asthma: New Insights Rohan Thompson, MD,1,2 Ralph J. Delfino, MD, PhD,3* Thomas Tjoa, MPH, Eliezer Nussbaum, MD,1,2 and Dan Cooper, MD2 3 MS, Summary. Home spirometers are useful for monitoring asthma therapy and for research, but the validity of maneuvers in children is in question. We evaluated the quality of PEF, FEV1, and FVC data obtained from 67 children with persistent asthma who self-administered spirometry at home using the hand-held ndd EasyOne Frontline Spirometer with full expiratory curve data, electronic measurements of maneuver quality, and on-screen incentives. Half were studied in 2003 in one region, and half in 2004 in another region of Southern California. Subjects were followed at home weekly over 2 months and daily over 10 consecutive days. We retained completed spirometry sessions (9,916) consisting of three of six best maneuvers in the morning, afternoon, and evening. Percent compliance, software assessed repeatability and acceptability modified from American Thoracic Society criteria, and visually assessed quality of maneuvers, were compared across daily and weekly follow-up, study regions, and subject characteristics. Compliance was higher for daily (>90%) than for weekly follow-up (>84%), but not significantly different, and was consistent across subject characteristics. The number with two reproducible and acceptable maneuvers was significantly lower in the first than second region for daily (70 vs. 90%) and weekly follow-up (66 vs. 87%). Of 22,926 software accepted maneuvers, 1,944 (8.5%) were visually rejected (variable effort, cough, glottic closure). Maneuver quality was significantly lower for subjects age 9–12 versus 13–18 years, for subjects not taking anti-inflammatory medications, and for subjects with <80% predicted FEV1. Longitudinal data collection is possible in children with asthma by employing repeated home training and follow-up, and using spirometers with built in quality assurance and incentive software. Region, age, and multiple indicators of persistent asthma, predict ability to perform reliable and accurate lung function maneuvers. Pediatr Pulmonol. 2006; 41:819–828. ß 2006 Wiley-Liss, Inc. Key words: asthma; spirometry; reproducibility of results; patient compliance; data collection; evaluation research. INTRODUCTION Spirometry and its variable peak expiratory flow (PEF) have been widely used as an index of measurement for asthma both in the clinical setting and in research trials. The use of PEF can be relatively useful when a skilled technician or medical professional directly guides technique and interpretation. However, the validity of selfadministered PEF is questionable. Some previous studies have even shown that it can be less sensitive than a simple symptom diary for revealing acute exacerbations in asthma.1,2 In addition, subject compliance and ability to perform maneuvers accurately at home, especially among children is also questionable.3,4 Typical home monitoring is done with non-electronic devices. Evidence from three studies proved that around a fourth to a third of nonelectronic PEF data was falsified.3–5 Despite these and other limitations, daily PEF is still being widely used in clinical asthma trials due to its simplicity and low cost. The best standard for measurement of pulmonary function has always been the laboratory spirometer and body Plethysmograph. These devices are not used frequently in clinical trials due to expense of use and lack ß 2006 Wiley-Liss, Inc. of portability. Recently more portable hand held devices that have some similar capabilities in measuring lung 1 Pediatric Pulmonology Division, Miller Children’s Hospital, Long Beach, California. 2 Department of Pediatrics, School of Medicine, University of California at Irvine, Irvine, California. 3 Department of Medicine, Epidemiology Division, School of Medicine, University of California at Irvine, Irvine, California. Grant sponsor: National Institute of Environmental Health Sciences (NIEHS); Grant number: ES011615. *Correspondence to: Ralph J. Delfino, M.D., Ph.D., Department of Medicine, Epidemiology Division, School of Medicine, University of California, Irvine, 100 Theory Dr., Suite 100, Irvine, CA 92697-7555. E-mail: rdelfino@uci.edu Received 10 February 2006; Revised 23 March 2006; Accepted 7 April 2006. DOI 10.1002/ppul.20449 Published online 17 July 2006 in Wiley InterScience (www.interscience.wiley.com). 820 Thompson et al. function have become available. These devices have the capability of measuring forced vital capacity (FVC), forced expiratory volume in one second (FEV1), forced expiratory flow at 25%–75% of FVC (FEF25–75), and PEF. They are superior to peak flow meters by their very ability to measure all of the aforementioned variables as well as compliance because they store their data until retrieved. For the investigator, this mode of data collection in the field could lead to measurements that are more objective and accurate, and include detailed parameters of lung function and maneuver quality checks. This study aims to evaluate the compliance and technical quality of home spirometry data from school children with asthma, for use in research studies. We obtained home spirometry data from subjects who selfadministered spirometry at home using the hand-held ndd EasyOne Frontline Spirometer (ndd Medical Technologies, Chelmsford, MA). Mortimer et al.6 previously validated this portable spirometer against an office spirometer (12 L dry rolling seal). They showed a 1.1% difference in FEV1 and
0.9% difference in FVC. The data and subjects used in the present study are part of a much larger study to evaluate the effects of air pollutants on school children with asthma in Southern California. We report results from 67 subjects who completed 9,916 spirometry sessions, with each session consisting of at least three maneuvers. Each curve was evaluated and measured independently for acceptability by selected criteria. These curves were then further evaluated for visual acceptability by pediatric pulmonology physicians. MATERIALS AND METHODS Population and Design The design of this study involved repeated measures of health outcomes and environmental exposures in children with asthma, and it is referred to as a panel study. We followed 67 school children with asthma in two regions of ABBREVIATIONS ATS BEV dFEV1 dFVC dPEF EOTV FEF25–75 FET FEV1 FVC GCRC IBD PEF PEFT American Thoracic Society Back-extrapolated volume Difference between highest and 2nd highest FEV1 within in a test session Difference between highest and 2nd highest FVC from an acceptable effort Difference between highest and 2nd highest PEF from an acceptable effort End-of-test volume Forced expiratory flow between 25% and 75% of FVC Forced expiratory time Forced expiratory volume in one second Forced vital capacity General Clinical Research Center Inhaled bronchodilators Peak expiratory flow Time to peak expiratory flow Southern California, Riverside City and Whittier. These two regions were specifically chosen due to their high levels of air pollution. Although key to the aims of the overall study, the health impacts of air pollutants measured is beyond the scope of this article. The Institutional Review Boards of the University of California, Irvine approved the study protocol. Informed written consent was obtained from all subjects and one of their legal guardians. Subjects were recruited if they were ages 9– 18 years and had an established diagnosis of asthma based on self-reported physician-diagnosed asthma, including at least a 1 year history of episodic symptoms involving wheezing, cough, or dyspnea. The study targeted recruitment of children with mild to moderate persistent asthma based upon a modified severity scale established by the National Heart Lung and Blood Institute.7 This included a history in the last 12 months of exacerbations of asthma symptoms requiring the use of prescribed as-needed, oral or inhaled, bronchodilator(s) on 2 or more days per week, regardless of anti-inflammatory medication use. Subjects were also recruited if they were taking oral or inhaled antiinflammatory medications, regardless of recent symptom frequency. Finally, subjects were recruited with <80% predicted FEV1 from office spirometry at their baseline visit to the General Clinical Research Center (GCRC), University of California, Irvine. Subjects were ineligible if they smoked or if someone smoked in the subject’s home. Out of 97 eligible subjects evaluated at the GCRC, 13 were unable or unwilling to participate, and did not enter the panel study. Of the 84 who entered, shortly after entry 10 dropped out and 7 were dropped due to noncompliance. Two subjects were dropped later due to non-compliance, but had a sufficient amount of spirometry data for evaluation (66 and 55 sessions) and they were retained for the present analysis of spirometry data quality. Another subject withdrew later in the study and data was retained for the present analysis (59 sessions), leaving 67 subjects. Prior to entry in the panel study, subjects were administered a background questionnaire. For 67 subjects in the panel study, responses showed 5 subjects (7.5%) reported having been admitted to the hospital in the past 12 months, and 17 subjects (25%) had been seen in the emergency room at least once in the past 12 months for their asthma. In addition, 22 subjects (33%) reported the need for inhaled bronchodilators (IBD) multiple times per week, 9 subjects (13%) required IBD at least weekly, and 35 subjects (52%) required IBD less frequently or could not recall usage frequency. Only 11 subjects (17%) reported not having used some form of inhaled corticosteroid as a controller medication for their asthma in the last year. Seven of these subjects did not frequently use IBD either, but five of them had percent predicted FEV1 <80%, and the other two had several recent asthma exacerbations requiring physician evaluations. Evaluation of Daily Home Spirometry for School Children with Asthma The first site of study was Riverside where two groups of approximately 16 asthmatics each were followed in alternating 1-month blocks over a 4-month period in the late summer through late fall of 2003 with daily measures of health outcomes from each subject. This was repeated at a second site in the late summer through late fall 2004 in Whittier. This time of the year was chosen because of historically high levels of particulate air pollution in the Los Angeles basin. Subjects performed forced expiratory flow maneuvers first on an office spirometer during their baseline visit to the GCRC. Then, subjects were trained in home on how to perform expiratory flow maneuvers with the ndd portable spirometer during a 5-day run-in period (around an hour on the first day, then around 15–30 min per day for the next 4 days as needed). That data is not included in the present analysis. Subjects were instructed to perform spirometry in the morning, afternoon, and evening, and to complete an electronic diary every two waking hours reporting health outcomes, asthma medications, and time place activity information. Subjects were followed in two blocks of 1 month each for a total of 56 days when they performed thrice-daily spirometry and completed the diary. Subjects further participated in a more intensive phase of study over a continuous 10-day period within one of each subject’s 2 months of follow-up. This additionally involved wearing a personal air sampler to measure exposure to air pollutants, and providing a daily measurement of exhaled nitric oxide. Generally, four subjects were followed in each 10-day period and on-site validity checks were conducted daily in their homes. During days when subjects were not wearing the personal air sampler (approximately 46 days) they were followed once each week at their home for validity checks to ensure compliance. Four of the 67 total subjects did not participate in the 10-day exposure assessment and their data are only included in the analysis of spirometry data from the weekly follow-up. Spirometry Methods and Assessment of Repeatability and Acceptability The ndd portable spirometer is a small hand held device that uses an ultrasonic flow sensor to measure air flow, thus eliminating many problems with other techniques because measurements are independent of temperature, humidity, pressure, and gas composition. It further uses ndd’s disposable mouthpiece, spiretteTM, with no sensor elements and thus does not contribute or affect air flow measurement. It has a large memory capacity (700 sessions). A graphic display of 64  160 pixels along with a 14-button rubber keypad allow for subject visualization of maneuver performance messages and input of data (see below). 821 The ndd portable spirometer has software that complies with American Thoracic Society (ATS) criteria for acceptability and repeatability.8,9 Acceptability parameters are standardized measurements of the technical quality of a maneuver. Repeatability is a measure of the repeatability of expiratory parameters from one maneuver to the next, and is measured by the differences between the highest and second-highest FVC, FEV1, and PEF (dFVC, dFEV1, dPEF). Because studies have shown that the ATS criteria,9 designed primarily for adults, can be difficult for children to achieve,8,10 we modified ATS criteria for children with asthma (Table 2).6,8,10 We lowered the ATS9 recommended forced expiratory time (FET) from 6 to 4 sec given the findings of Arets et al.,8 who found only 15% of children with obstructive lung disease who were experienced in spirometry were able to achieve FET >6 sec for otherwise acceptable maneuvers, similar to the findings of Desmond et al. in children with obstructive lung disease (19%).10 Enright et al.11 found a majority of a general population sample of children achieved FET >6 sec (65%). We also focused evaluation on premature termination of effort or abrupt ending based on the end-oftest volume (EOTV) (Table 2). To assess accurately EOTV and thus abrupt ending, we calculated FET and EOTV to end of the expiratory plateau and did not include negative (inspiratory) flow. The end of test was marked by NDD software where inspiration >150 ml occurred or a plateau was detected (volume change <45 ml over 2 s for FET <4 s or 60 ml for FET 4 s). We also considered the fact that children with asthma show considerable variability in pulmonary function due in part to bronchial reactivity. Further, ATS criteria are intended for in-clinic spirometry guided by technicians using a laboratory spirometer. Therefore, we used a more relaxed goal for repeatability than that recommended by the ATS,9 by setting FEV1 and FVC repeatability at 10% instead of 5%, and PEF at 20% instead of 10%. Because the curves were also visually evaluated, we were able to reject unacceptable curves that met less stringent repeatability criteria. When almost the entire second best curve was ‘‘smooth’’ and perfectly acceptable, but a small portion of the curve showed ‘‘a departure,’’ it was reasonable to accept rather than reject the test session. We visually determined variable effort and other quality problems where there was more than around a 10% deviation from the expected smoothed curve. Maneuver sessions that met acceptability and repeatability criteria by the software were independently reviewed by physicians trained in pediatric pulmonology. Physicians classified each maneuver rejected based on the reasons for rejection (cough, glottic closure, variable effort, or a combination of errors). Figure 1 gives examples. Quality problems were defined by reference to available standardized flowvolume curves showing established abnormal or disrupted curves. 822 Thompson et al. Acceptability and repeatability criteria were custom programmed into the software for this study by ndd technicians. The ndd EasyOne Frontline model stores raw data for all three best maneuvers in memory. We developed custom SAS (Cary, NC) programs (available upon request) to output performance variables and plot three best curves for analysis and improved visual review over the ndd printout of the single best curve. Specific protocols and instructions were given to all technicians doing field training and follow-up. Each spirometer was checked for calibration (3%) with a 3 L syringe according to the manufacturer’s specifications before the start of each 1-month follow-up and before each 10-day follow-up. Subjects were instructed to use nose clips. The software programming on the ndd portable spirometer automatically stopped after three Fig. 1. Examples of curves with visual scoring errors. A: Variable effort: intermittent deceleration and acceleration in flow, which may also be intermittent breaths during expiratory flow maneuver; (B) Glottic Closure: abrupt interruption in expiratory flow causing a sharp spike downward indicating an abrupt stop and start in flow; (C) Cough: erratic interruption in expiratory flow, along with repetitive glottic closures. Evaluation of Daily Home Spirometry for School Children with Asthma 823 Fig. 1. (Continued ) good maneuvers were obtained, but would give each participant up to six chances to meet acceptability and repeatability criteria (Table 2). If the participant were unable to meet repeatability and acceptability criteria after six attempts, the spirometer would automatically store the best three attempts based on calculation of the largest FEV1 þ FVC. The spirometer also gave intermittent instructions to subjects to aid them in performing acceptable and reproducible maneuvers based on the error of each attempt (Table 2). To unmask the effects of air pollutants, it was emphasized to all subjects the importance of doing sessions prior to the use of an IBD unless necessary and to wait at least 4 hr after the use of an IBD before performing a session. Furthermore, subjects were required to answer yes/no questions generated by the spirometer after each spirometry session [(1) Did you have any cough during this test? (2) Did you need to use your rescue medication in the last hour?]. Answers specific to each spirometry session were stored in memory. Subjects received a handbook that contained detailed instructions on use of their spirometer and in the appropriate technique for doing spirometry sessions. In addition, a large and laminated one-sheet guide with stepwise illustrations was given to subjects and placed in a prominent place in the home where maneuvers were done. They were encouraged to refer to the handbook and onesheet guide if needed. During follow-up, research technicians downloaded data into laptops during home visits and checked compliance with the three daily sessions, and the acceptability of maneuvers as generated by the ndd software (v 2.6). Subjects were retrained as needed. The data manager using our custom SAS programs checked data again the next morning. Problems were reported to field staff and subjects were retrained if needed that daily follow-up or before the next weekly follow-up. Compliance was further encouraged by audio reminder alarms on the ndd portable spirometer, and by a monetary sliding scale based on the number of maneuvers performed with each session. Subjects performing most of the maneuvers (17–21 per week) received a bonus payment as well. The ndd software also awarded 500 points for sessions with three acceptable and two reproducible maneuvers, but only 200 points if all six attempts failed acceptability and repeatability criteria. This was used as an additional behavioral incentive. Analysis We compared the percentage compliance, and repeatability and acceptability of maneuvers between daily and weekly follow-up, between study regions (Riverside vs. Whittier), and across key subject characteristics to better understand the underlying determinants of data quality. Key subject characteristics included gender, use of controller medications, age group (9–12 vs. 13–18 years old) and percent predicted FEV1 <80 versus 80% based on repeated measurements of acceptable and reproducible maneuvers. Percent-predicted normal FEV1 were calculated for a given height, age, gender, and race-ethnicity using normal lung function equations from the National Health and Nutrition Examination Survey III.12 Tests for significant differences in data quality were performed across these binary group characteristics using a two-sided 824 Thompson et al. TABLE 1— Study Group Characteristics Subject variables Data Subject number Riverside Whittier Age, mean (range) Gender N (%) Female Male Race N (%) White Black Hispanic Asian Mean percent predicted FEV1 (SD) Riverside Whittier 33 34 13.5 (9–18 years) 25 (37.3) 42 (62.7) 25 (37.3) 15 (22.4) 24 (35.8) 3 (4.5) 80.8 (16.3) 89.5 (12.7)* *P < 0.05 for difference between Riverside and Whittier in percent predicted FEV1. t-test with Satterthwaite’s method in SAS v 9.1 Proc TTEST.13 RESULTS The 67 subjects in this repeated measures study were of diverse racial and ethnic background, and ranged in age from 9 to 18 years (mean 13.5 years) (Table 1). There were more males than females. Mean percent-predicted FEV1 from all valid maneuvers during daily home follow-up was lower for subjects in Riverside compared with Whittier (80.8% vs. 89.5%, respectively). Despite the prolonged period of study, all home spirometers assigned to subjects for the duration of this study were returned undamaged. One spirometer was found to have minimal loss of calibration (4%) after 4 weeks of use and was sent back to the manufacturer for calibration. The distribution of performance variables is shown in Table 3. Evaluation of acceptability and repeatability of maneuvers is shown in Table 4. Daily versus weekly follow-up values of acceptability and repeatability did not differ significantly, although there was a consistent trend of higher values during daily follow-up. There was no statistically significant difference between the more strict criteria of three acceptable, two reproducible maneuvers versus two acceptable and reproducible maneuvers, hereafter referred to as criteria A and B, respectively. As expected, the lowest percentage of acceptability and repeatability occurred when all three flow-volume criteria were evaluated simultaneously, but values remained >75% across all parameters. Table 5 shows the frequency of subject errors detected by ndd EasyOne software, which labeled the spirometry curves as unacceptable. The most common error made by subjects was abrupt ending and invalid time to peak expiratory flow (PEFT). Table 6 shows causes of physician visual rejection of software accepted maneuvers. Variable effort was the most common cause of rejection, often in combination with cough and glottic closure. Glottic closure followed by cough was the next most common. Overall frequency of causes for visual rejection was small being 10.4% and 8% of overall acceptable and reproducible maneuvers for daily and weekly follow-up, respectively. Evaluation of compliance showed excellent overall compliance of >90% for daily follow-up and >84% for weekly follow-up data (Table 7). This finding remained consistent across study region and subject characteristics. However, the quality of maneuvers differed across a number of factors. Subjects in Riverside performed worse than subjects in Whittier for all comparisons. There were also significant differences across subject characteristics for both daily and weekly follow-up data for criteria A and B, and for visually acceptable criteria test sessions. These TABLE 2— Acceptability and Reproducibility Criteria Acceptability criteria Instruction to subjects if criterion not met Back-extrapolated volume (BEV) must be <150 ml or 5% of the FVC Time to peak expiratory flow (PEFT) must be >40 msec and 160 mseca a. We later added an additional instruction in the second half of study for maneuvers where PEFT was 40 msec: ‘‘wait until buzz before blowing out.’’ No abrupt ending, which is when EOTV >100 ml is accumulated in the last 0.5 sec before the end of test or for age <12 year, 50 ml in the last 0.25 sec before the end of test. EOTV is calculated from the forced expiratory time (FET) (0.5 or 0.25 sec to end of expiration). Abrupt ending also occurs if FET is <2 sec [end-of-test volume (EOTV)] Repeatability Criteria Difference between the highest PEF and 2nd highest PEF (dPEF) within in a test session must be within 20% Difference between the highest FEV1 and 2nd highest FEV1 (dFEV1) within in a test session must be within 10% Difference between the highest FVC and 2nd highest FVC (dFVC) within in a test session must be within 10% Do not hesitate at the beginning of the maneuver Blast out faster when starting the maneuver Blow out longer Blast out harder when starting the maneuver Take a deeper breath Take a deeper breath Evaluation of Daily Home Spirometry for School Children with Asthma TABLE 3— Distribution of Variables for the Quality of the Best Maneuver 825 1 Percentiles BEV (ml) BEV (% FVC) PEFT (msec) EOTV (ml) FET (sec) dFVC (%) dFEV1 (%) dPEF (%) 5th 10th 25th 50th 75th 90th 95th 18.1 0.7 50.0 2.5 2.5 0.3 0.3 0.5 26.9 1.0 60.0 3.8 2.8 0.6 0.6 1.1 42.5 1.5 60.0 6.9 3.4 1.4 1.6 2.7 62.5 2.1 80.0 12.5 4.6 3.4 3.7 6.2 86.3 2.8 100.0 24.4 6.2 7.0 7.5 12.4 114.4 3.6 120.0 42.5 8.4 14.0 15.5 25.2 133.7 4.1 140.0 55.6 10.0 24.8 26.9 38.9 1 The maneuver with the highest FEV1 þ FVC for each of 9,916 spirometry sessions. characteristics included age group 9–12 years performing worse than 13–18 years, and subjects with FEV1 80% predicted performing significantly better than their counterparts. Subjects not on controller medications performed worse than those taking them, but this difference was statistically significant only for daily follow-up. Performance between daily and weekly follow-up was not significantly different. Weekly follow-up performance between months one and two of subject follow-up was not significantly different. DISCUSSION Overview of Findings and Comparison with the Literature Mortimer et al.,6 evaluated data from asthmatic children using the ndd portable spirometer, which had quality control software with modified ATS acceptability and repeatability criteria similar to the present study, including the relaxed goal for repeatability and end-of-test criteria tailored for asthmatic children. They conducted an asthma panel study of 92 children with asthma and found overall compliance was 87% for 2 weeks (2,151 sessions), similar to our compliance for weekly follow-up over 2 months (85%–90%). They also found the proportion of sessions with at least two acceptable and reproducible maneuvers by software criteria was 62% overall, which is lower than the present study (76%–80%). However, their study differs from the present in that subjects were younger (ages 6–11), and no exclusion of training period data was reported. We found a non-significant small increase in the quality of the maneuvers from month 1 to 2, and no change in compliance. Wensley and Silverman14 analyzed the quality of data and compliance in 90 asthmatic school children ages 7–14 years who used a Vitalograph home spirometer and were followed at home monthly over 4 months. They found some deterioration of collected data over time primarily due to decreasing compliance (from 81% to 70%) rather than any deterioration in maneuver quality (from 82% to 80%). However, quality could be only assessed by percent variability of FVC þ FEV1 for the two best maneuvers. We previously used the Vitalograph 2110 (Vitalograph, Inc., Lenexa, KS) in a panel study of 19 children with asthma each followed daily for 14 days, and found 80% compliance and 81% of sessions had acceptable and reproducible FEV1 data.15 Although these results are roughly comparable to the present study, quality assurance TABLE 4— Acceptability and Repeatability of Self-Administered Spirometry for Daily Compared with Weekly Staff Follow-Up* At least three acceptable, two reproducible maneuvers Parameters FEV1, FVC, PEF FEV1 only FVC only PEF only Total no. of spirometry sessions Daily follow-up 1 78.7 85.6 84.6 89.2 1814 Weekly follow-up 75.2 82.0 81.4 85.4 8102 At least two acceptable, two reproducible maneuvers Daily follow-up 79.9 87.1 86.3 91.2 1814 Weekly follow-up 76.4 84.0 83.6 87.8 8102 *There were no P < 0.05 differences between frequencies for daily versus weekly follow-up or between three acceptable, two reproducible maneuvers versus two acceptable, two reproducible maneuvers. 1 Percent of total spirometry sessions collected (three maneuvers per session). 826 Thompson et al. TABLE 5— Frequency of Maneuver Errors Error type1 Abrupt ending PEFT PEFT þabrupt ending BEV BEV þabrupt ending BEV þ PEFT BEV þ PEFT þabrupt ending Total maneuvers Best maneuver2 no. (% total) 2nd best maneuver no. (% total) 3rd best maneuver no. (% total) 32 (0.32) 18 (0.18) 3 (0.03) 10 (0.10) 2 (0.02) 14 (0.14) 1 (0.01) 9916 76 (0.77) 79 (0.80) 20 (0.20) 26 (0.26) 6 (0.06) 42 (0.42) 8 (0.08) 9916 170 (1.71) 209 (2.11) 32 (0.32) 73 (0.74) 9 (0.09) 111 (1.12) 15 (0.15) 9916 1 See Table 2 for definitions. Maneuvers are ranked in order of best effort (FEV1 þ FVC). 2 software was limited to PEFT (acceptable at 40– 290 msec) and prompting of subject maneuvers until PEF readings were within 10%. Given that subjects in the present study were usually not coached by technicians, the distribution of performance variables shows the quality of the data is reasonably good (Table 3). Enright et al.11 assessed the quality of technician-administered spirometry done annually for 3 years in a large cohort study of over 4,000 Southern California school children ages 9–18. The distribution of our PEFT and Back-extrapolated volume (BEV) variables in Table 3 are similar to theirs. We show no negative EOTV in the lower distribution as they did (see Materials and Methods), but our 95th percentile was only 56 ml. The FET in the general population sample of Enright et al.11 was longer (median 6.6 s) compared with a median of 4.6 sec for the persistent asthmatics of our study. Our repeatability results were worse. The median dFEV1 in Enright et al.11 was 1.2% whereas ours was 3.7%. Further evaluation of the quality of maneuvers in our study was made by evaluating the visual acceptability of curves that met software criteria for at least two acceptable and two TABLE 6— Causes of Visual Rejection of Maneuvers That Met Acceptability Criteria Causes of visual rejection Cough Glottic closure2 Variable effort Cough/variable effort Cough/glottic closure Cough/glottic closure/ variable effort Glottic closure/ variable effort Total rejected Total acceptable 1 Daily follow-up 1 Weekly follow-up 45 (1.0) 33 (0.7) 262 (6.0) 65 (1.5) 9 (0.2) 9 (0.2) 152 (0.8) 223 (1.2) 724 (3.93) 71 (0.4) 71 (0.4) 14 (0.1) 31 (0.7) 240 (1.3) 454 (10.4) 4350 (100) 1496 (8.0) 18576 (100) Number rejected (percent of total acceptable maneuvers for the follow-up frequency). 2 Includes problems at the end of the curve, which may not have had an effect on FEV1 or FVC. reproducible curves. Variable effort was the most frequent cause of visual rejection (Table 6). In the present study, we found that both compliance and quality of curves were higher for subjects followed up daily as compared with weekly (Tables 4 and 7), but differences were not statistically significant. This consistency in technique and compliance could be attributed to the spirometer itself, which as mentioned above, contains inherent quality assurance measures that do not require the presence of a field team member to correct subjects on common errors during maneuver attempts (Table 2). Subjects were also given a manual and a small poster size colored pictograph describing and depicting a subject doing appropriate spirometry technique. In addition, compliance was regularly encouraged by monetary incentives, an on-screen point system, and audio alarms in the ndd software to remind subjects during selected periods (see Materials and Methods). We found quality of maneuvers was significantly worse in younger subjects. This age difference in the quality of home spirometry was also found by Mortimer et al.,6 Enright et al.,11 and Pelkonen et al.16 Multiple indicators of persistent asthma also predicted ability to perform reliable and accurate lung function maneuvers as shown by significantly better maneuver quality among subjects with 80% predicted FEV1 and subjects on controller medication. We speculate that the improved performance with better lung function is due to the intrinsic nature of the subjects’ lung disease (asthma). There were no significant differences in the distribution of controller medication use by percent predicted FEV1. The quality of maneuvers was also significantly worse in Riverside as compared with Whittier. Underlying group characteristics might explain this. Riverside and Whittier were significantly different in the number with <80% predicted FEV1 (42% vs. 18%, respectively, Fisher’s exact P < 0.05). However, the regional groups were similar in use of maintenance asthma controller medications (64 vs. 68%). The groups did differ by age sub-group with the Riverside group having more subjects 9–12 years old than the Whittier group (48.5% vs. 17.7%). However, there was Evaluation of Daily Home Spirometry for School Children with Asthma 827 TABLE 7— Compliance, Acceptability and Repeatability of Self-Administered Spirometry: Differences by Panel, Age, Controller Medication Use and Lung Function Daily Follow-up Characteristic Riverside Whittier Age group 9–12 13–18 Controller medication On Not on Mean percent predicted FEV1 80 <80 Riverside Whittier Age Group 9–12 13–18 Controller Medication On Not on Mean percent predicted FEV1 80 <80 Follow-up Month 1 Month 2 No. of Subjects Compliance: No. test sessions collected (% expected) Percent1 with 3 acceptable, 2 reproducible Percent with 2 acceptable and 2 reproducible Percent visually acceptable with 2 acceptable and 2 reproducible 31 32 881 (92.8) 933 (95.2) 68.82 88.1 69.72 89.6 54.92 81.7 21 42 607 (95.0) 1207 (93.6) 66.72 84.8 67.92 86.0 56.52 74.8 42 21 1232 (94.6) 582 (92.8) 83.82 68.0 84.82 69.6 72.3 61.0 45 18 84.62 68.6 73.72 56.4 33 34 82.92 68.4 Weekly follow-up 3918 (84.8) 64.72 4184 (89.7) 85.0 65.52 86.6 52.02 78.4 21 42 2801 (87.4) 5301 (87.2) 64.62 80.8 65.72 82.1 54.42 71.5 42 21 5288 (87.4) 2814 (87.0) 78.0 69.9 79.5 70.6 66.9 63.1 45 18 5576 (87.2) 2526 (87.5) 81.52 61.2 82.72 62.6 74.32 46.4 67 62 4210 (87.8) 3892 (86.7) 72.7 77.8 73.8 79.3 64.0 67.3 1289 (93.6) 525 (95.1) 1 Percentages are the number with the stated characteristic divided by the number of spirometry sessions collected. P < 0.05 t-test with Satterthwaite’s method for differences in performance between region, age, controller medication group, percent predicted FEV1, and month of follow-up. 2 no difference in maneuver quality by age sub-group in Whittier, and performance was a significantly better in Whittier for both 9–12 and 13–18 year old subjects (not shown). We speculate that unmeasured characteristics led to this regional difference in quality of maneuvers. Application of Findings to Research In field studies of chronic lung diseases that require frequent measurements of pulmonary function, the use of peak flows has been one of the most common measurements available to investigators.2 This is largely due to the ease of use of peak flow meters, portability and low cost. Unfortunately, measurement of peak flows has been shown to have many disadvantages for use in research or clinical management of patients with asthma or other chronic lung diseases.1–5,17–20 Previously, full measurement of pulmonary function that allows for evaluation of other critical variables of air flow (FEV1, FVC, FEF25–75) has been limited only to the office or laboratory, thus precluding repeated daily measurements. Portable spirometers such as the one used in this study demonstrate several advantages over peak flow meters. First, its ability to generate true flow-volume loops not only allows for evaluation of various parameters of expiratory flows (FEV1, FVC, FEF25–75, PEF), but also allows for evaluation of technique and effort based on professional standards programmed into the software (Table 2). The measurement of these parameters allows for evaluation of both central and peripheral airway function, thus the potential for better and more expansive evaluation of clinical status. Finally, quality assurance is enhanced by the machine’s ability to: (1) give subjects correcting instructions on the next maneuver attempt based on errors evaluated on the current maneuver attempt; and (2) store large volumes of time-date-stamped data. CONCLUSIONS Although there were subgroup differences in compliance and maneuver quality in this study, overall performance with the use of the ndd portable spirometer 828 Thompson et al. was good, with acceptability and repeatability criteria being met at its most stringent (three acceptable and two reproducible maneuvers) more than 61% of the time for all subgroups. The lack of significant differences in quality of maneuvers and compliance for daily versus weekly follow-up supports the effectiveness of quality assurance measures built into the ndd and the ease of performing maneuvers for well-trained subjects even as young as age 9 years. The use of portable spirometers with the specifications described is feasible and results in a high yield of valid data for field research. Handheld spirometers with capabilities such as the ndd EasyOne are superior to simple peak flow meters in allowing for comprehensive measures of lung function, quality assurance measures, compliance markers, and ease of use. This allows researchers to evaluate the impact of clinical interventions or environmental exposures on chronic lung diseases such as asthma. We found data quality was affected by a variety of influences, including clinical severity, treatment regimen, and population differences, all of which should be taken into account when interpreting data. Despite these impacts on data quality, results of this large panel study of pediatric asthma show that durable and portable spirometers offer quality assurance and longitudinal data collection capabilities. Further studies evaluating portable electronic spirometry in research and clinical settings are strongly encouraged. ACKNOWLEDGMENTS The authors declare they have no competing financial interests. The authors thank Dr. Ira B Tager and Dr. Kathleen M Mortimer for their assistance in custom software development for the ndd portable spirometer, our staff Lynn Chuang and Vanessa Phan for their skilled effort in training subjects and collecting lung function data for this study, and Victor Gastanaga for data management and quality control. Most of all we thank our study participants for their diligent efforts. The project described was supported by grant number ES011615 from the National Institute of Environmental Health Sciences (NIEHS), U.S. National Institutes of Health (NIH), and its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS, NIH. REFERENCES 1. Chan-Yeung M, Chang JH, Manfreda J, Ferguson A., Becker A. Changes in peak flow, symptom score, and the use of medications during acute exacerbations of asthma. Am J Respir Crit Care Med 1996;154:889–893. 2. Mortimer KM, Redline S, Kattan M, Wright EC, Keresmar CM. Are peak flow and symptom measures good predictors of asthma hospitalizations and unscheduled visits? Pediatr Pulmonol 2001;31:190–197. 3. Redline S, Wright EC, Kattan M, Keresmar C, Weiss K. Shortterm compliance with peak flow monitoring: results from a study of inner city children with asthma. Pediatr Pulmonol 1996;21: 203–210. 4. Verschelden P, Cartier A, L’Archeveque J, Trudeau C, Malo JL. Compliance with and accuracy of daily self-assessment of peak expiratory flows (PEF) in asthmatic subjects over a three month period. Eur Respir J 1996;9:880–885. 5. Kamps AW, Roorda RJ, Brand PL. Peak flow diaries in childhood asthma are unreliable. Thorax 2001;56:180–182. 6. Mortimer KM, Fallot A, Balmes JR, Tager IB. Evaluating the use of a portable spirometer in a study of pediatric asthma. Chest 2003;123:1899–1907. 7. National Heart, Lung, and Blood Institute (NHLBI). The expert Panel Report 2: Guidelines for the Diagnosis and Management of Asthma. Bethesda MD: NHLBI, National Institutes of Health. Pub no 97-4051, 1997. 8. Arets HG, Brackel HJ, van der Ent CK. Forced expiratory maneuvers in children: do they meet ATS and ERS criteria for spirometry? Eur Respir J 2001;18:655–660. 9. American Thoracic Society. Standardization of spirometry 1994 update. Am J Respir Crit Care Med 1995;152:1107–1136. 10. Desmond KJ, Allen PD, Demizio DL, Kovesi T, Coates AL. Redefining end of test (EOT) criteria for pulmonary function testing in children. Am J Respir Crit Care Med 1997;156:542– 545. 11. Enright PL, Linn WS, Avol EL, Margolis HG, Gong H, Jr., Peters JM. Quality of spirometry test performance in children and adolescents: experience in a large field study. Chest 2000;118: 665–671. 12. Hankinson JL, Odencrantz JR, Fedan KB. Spirometric reference values from a sample of the general U.S. population. Am J Respir Crit Care Med 1999;159:179–187. 13. SAS Institute Inc. SAS/STAT1 9.1 User’s Guide. Cary, NC: SAS Institute Inc., 2004; pp 4775–4794. 14. Wensley DC, Silverman M. The quality of home spirometry in school children with asthma. Thorax 2001;56:183–185. 15. Delfino RJ, Quintana PJE, Floro J, Gastañaga VM, Samimi BS, Kleinman MT, Liu L-JS, Bufalino C, Wu C-F, McLaren CE. Association of FEV1 in asthmatic children with personal and microenvironmental exposure to airborne particulate matter. Environ Health Perspect 2004;112:932–941. 16. Pelkonen AS, Nikander K, Turpeinen M. Reproducibility of home spirometry in children with newly diagnosed asthma. Pediatr Pulmonol 2000;29:34–38. 17. Thiadens HA, De Bock GH, Van Houwelingen JC, Dekker FW, De Waal MW, Springer MP, Postma DS. Can peak expiratory flow measurements reliably identify the presence of airway obstruction and bronchodilator response as assessed by FEV(1) in primary care patients presenting with a persistent cough? Thorax 1999;54: 1055–1060. 18. Giannini D, Paggiaro PL, Moscato G, Gherson G, Bacci E, Bancalari L, Dente FL, Di Franco A, Vagaggini B, Giuntini C. Comparison between peak expiratory flow and forced expiratory volume in one second (FEV1) during bronchoconstriction induced by different stimuli. J Asthma 1997;34:105–111. 19. Hegewald MJ, Crapo RO, Jensen RL. Intraindividual peak flow variability. Chest 1995;107:156–161. 20. Vaughan TR, Weber RW, Tipton WR, Nelson HS. Comparison of PEFR and FEV1 in patients with varying degrees of airway obstruction. 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LENTERA Jurnal Ilmiah Kependidikan Vol. 14 No. 2 (Edisi Dies Natalis XXXIV 2019) 41 - 47 ISSN : 0216-7433 TANTANGAN PENDIDIK DI ERA DIGITAL Diplan1 1. Universitas Muhammadiyah Palangkaraya diplan161181@gmail.com ABSTRAK Di era globalisasi dan informasi ini, guru dan dosen mendapatkan tantangan berupa perilaku siswa yang senang berselancar di dunia maya melalui android, gadget atau handphone canggih yang mereka miliki. Perilaku siswa ini ditandai oleh adanya kecendrungan lebih senang membaca melalui handphone ketimbang buku, senang mencari berbagai informasi sendiri tanpa melakukan recheck lebih dahulu, senang mencari teman melalui akun media sosial, dan terkesan individualis. Untuk itu, guru dan dosen dalam pembelajaran harus mampu memanfaatkan Teknologi Informasi, menggunakan metode yang menyenangkan, memperkaya keilmuan dengan berbagai sumber bahan bacaan, dan mampu melakukan penelitian. Demikian uraian yang berkaitan dengan tantangan pendidik di era Millennial dan upaya yang dilakukan untuk mencari solusi terhadap tantangan yang dihadapi guru. Kata Kunci: Era Digital, Pendidik, Milennial PENDAHULUAN Sejarah revolusi industri dimulai dari industri 1.0, 2.0, 3.0, hingga industri 4.0. Industri 1.0 ditandai dengan mekanisasi produksi untuk menunjang efektifitas dan efisiensi aktivitas manusia, industri 2.0 dicirikan oleh produksi massal dan standarisasi mutu, industri 3.0 ditandai dengan penyesuaian massal dan fleksibilitas berbasis otomasi dan robot. Industri 4.0 selanjutnya hadir menggantikan industri 3.0 yang ditandai dengan penggabungan mesin, alur kerja, dan sistem, dengan menerapkan jaringan cerdas. Istilah industri 4.0 berasal dari sebuah proyek yang diprakarsai oleh pemerintah Jerman untuk mempromosikan komputerisasi diberbagai bidang. Lee et al (2013) menjelaskan, industri 4.0 ditandai dengan peningkatan digitalisasi yang didorong oleh empat faktor: 1) peningkatan volume data, kekuatan komputasi, dan konektivitas; 2) munculnya analisis, kemampuan, dan kecerdasan bisnis; 3) terjadinya bentuk interaksi baru antara manusia dengan mesin; dan 4) perbaikan instruksi transfer digital ke dunia fisik, seperti robotika dan 3D printing. Lifter dan Tschiener (2013) menambahkan, prinsip dasar industri 4.0 adalah penggabungan mesin, alur kerja, dan sistem, dengan menerapkan jaringan cerdas di sepanjang rantai dan proses produksi untuk mengendalikan satu sama lain secara mandiri. Hermann et al (2016) menambahkan, ada empat desain prinsip industri 4.0. Pertama, interkoneksi (sambungan) yaitu kemampuan mesin, perangkat, sensor, dan orang untuk terhubung dan berkomunikasi satu sama lain melalui Internet of Things (IoT) atau Internet of People (IoP). Prinsip ini membutuhkan kolaborasi, keamanan, dan standar. Kedua, transparansi informasi merupakan kemampuan sistem informasi untuk menciptakan salinan virtual dunia fisik dengan 41 Diplan / LENTERA Jurnal Ilmiah Kependidikan Vol. 14 No.2 (Juli – Desember 2019 Edisi Dies Natalis XXXIV) 41-47 memperkaya model digital dengan data sensor termasuk analisis data dan penyediaan informasi. Ketiga, bantuan teknis yang meliputi; (a) kemampuan sistem bantuan untuk mendukung manusia dengan menggabungkan dan mengevaluasi informasi secara sadar untuk membuat keputusan yang tepat dan memecahkan masalah mendesak dalam waktu singkat; (b) kemampuan sistem untuk mendukung manusia dengan melakukan berbagai tugas yang tidak menyenangkan, terlalu melelahkan, atau tidak aman; (c) meliputi bantuan visual dan fisik. Keempat, keputusan terdesentralisasi yang merupakan kemampuan sistem fisik maya untuk membuat keputusan sendiri dan menjalankan tugas seefektif mungkin. Industri 4.0 telah memperkenalkan teknologi produksi massal yang fleksibel (Kagermann et al, 2013). Mesin akan beroperasi secara independen atau berkoordinasi dengan manusia (Sung, 2017). Industri 4.0 merupakan sebuah pendekatan untuk mengontrol proses produksi dengan melakukan sinkronisasi waktu dengan melakukan penyatuan dan penyesuaian produksi (Kohler & Weisz, 2016). Selanjutnya, Zesulka et al (2016) menambahkan, industri 4.0 digunakan pada tiga faktor yang saling terkait yaitu; 1) digitalisasi dan interaksi ekonomi dengan teknik sederhana menuju jaringan ekonomi dengan teknik kompleks; 2) digitalisasi produk dan layanan; dan 3) model pasar baru. Baur dan Wee (2015) memetakan industri 4.0 dengan istilah “kompas digital” Kompas Digital merupakan instrumen bagi perusahaan dalam mengimplementasikan industri 4.0 agar sesuai dengan kebutuhan mereka. Pada komponen tenaga kerja (labor), harus memenuhi; 1) kolaborasi manusia dengan robot; 2) kontrol dan kendali jarak jauh; 3) manajemen kinerja digital; dan 4) otomasi pengetahuan kerja. Demikian pula pada komponen lainnya digunakan sebagai instrumen implementasi industri 4.0. Revolusi digital dan era disrupsi teknologi adalah istilah lain dari industri 4.0. Disebut revolusi digital karena terjadinya proliferasi komputer dan otomatisasi pencatatan di semua bidang. Industri 4.0 dikatakan era disrupsi teknologi karena otomatisasi dan konektivitas di sebuah bidang akan membuat pergerakan dunia industri dan persaingan kerja menjadi tidak linear. Salah satu karakteristik unik dari industri 4.0 adalah pengaplikasian kecerdasan buatan atau artificial intelligence (Tjandrawinata, 2016). Salah satu bentuk pengaplikasian tersebut adalah penggunaan robot untuk menggantikan tenaga manusia sehingga lebih murah, efektif, dan efisien. Unsur-unsur pendidikan dalam definisi di atas terdiri dari usaha (kegiatan) yang bersifat bimbingan dan dilakukan secara sadar, pendidik (pembimbing), anak didik, dasar dan tujuan, dan alat-alat yang digunakan dalama usaha itu. Dengan demikian dapatlah dikatakan bahwa pendidikan sebagai sebuah sistem terdiri dari tujuan, metode, materi (kurikulum), pendidik, anak didik, alat pendidikan, dan lingkungan. Semua unsur ini saling berkaitan satu sama lain. Pendidikan saat ini menghadapi berbagai tantangan, antar lain bisa dilihat dari karakter anak. Karakter anak zaman now semakin nyeleneh dan semaunya sendiri. Anak-anak cenderung egois, tidak suka bekerja sama. Hal ini disebabkan seringnya mereka lebih suka bermain game lewat ponsel android daripada permainan tradisional yang mengajarkan perilaku untuk bekerjasama. Fenomena ini tidak bisa dipungkiri, baik itu di kota maupun di pelosok desa sekalipun. Karakter anak pada sebagian generasi millenial memprihatinkan. Mereka kadang tidak menghargai 42 Tantangan Pendidik di Era Digital orangtua maupun gurunya. Bahkan dari mereka juga terkadang terjebak pada dunia criminal dan narkoba. Generasi millenial dalam minat belajar juga sebagian besar mengalami kemunduran. Di era millennial, kecenderungan dunia pendidikan antara lain: berkembangnya model belajar jarak jauh (Distance Learning), mudahnya menyelenggarakan pendidikan terbuka, sharing resource bersama antar lembaga pendidikan, perpustakaan dan instrument pendidikan lainnya (guru, dosen, laboratorium) berubah fungsi menjadi sumber informasi daripada sekedar rak buku. Lembaga pendidikan akan menghadapi sebuah perubahan yang signifikan akibat proses digital ini. Ini menjadi sebuah peluang dan cara untuk meningkatkan kualitas pendidikan sekaligus tantangan bagi dunia pendidikan di Indonesia. Pada era millenial, manusia mulai meninggalkan cara-cara konvensional dalam menjalani kehidupan, digantikan dengan trend dan gaya hidup yang lebih fresh and youth, atau yang biasa dikenal dengan istilah “kekinian”. Seorang aktivisi HMI, Muhammad Ridal, dalam bukunya yang berjudul “HMI Millenial” mengungkapkan bahwa 33% masyarakat Indonesia saat ini merupakan generasi millenial. Menurut Ridal, era millenial umumnya didominasi oleh orang-orang kelahiran tahun 1980 sampai tahun 2000an, dan berusia 15-34 tahun. Usia ini, tentu saja, merupakan usia dimana individu masih berstatus sebagai pelajar di sekolah. PEMBAHASAN A. Karakteristik Generasi Milennial Generasi millennial memiliki beberapa karakteristik, yaitu: 1. Millennial tidak percaya lagi kepada distribusi informasi yang bersifat satu arah. Mereka lebih percaya kepada konten dan informasi yang dibuat oleh perorangan. Dalam hal pola konsumsi, banyak dari mereka memutuskan untuk membeli produk setelah melihat review atau testimoni yang dilakukan oleh orang lain di internet. Mereka juga tak segan-segan membagikan pengalaman buruk mereka terhadap suatu merek. 2. Millennial lebih memilih ponsel dibanding TV. Televisi bukanlah prioritas generasi millennial untuk mendapatkan informasi atau melihat iklan. Generasi millennial lebih suka mendapat informasi dari ponselnya, dengan mencarinya ke Google atau perbincangan pada forum-forum yang mereka ikuti. 3. Millennial wajib punya media sosial. Komunikasi di antara generasi millennial sangatlah lancar. Komunikasi itu tidak selalu terjadi dengan tatap muka. Banyak dari mereka melakukan semua komunikasinya melalui text messaging atau juga chatting di dunia maya, dengan membuat akun yang berisikan profil dirinya, seperti Twitter, Facebook, hingga Line. Akun media sosial juga dapat dijadikan tempat untuk aktualisasi diri dan ekspresi. Hampir semua generasi millennial dipastikan memiliki akun media sosial sebagai tempat berkomunikasi dan berekspresi. 4. Millennial kurang suka membaca secara konvensional. Bagi generasi ini, tulisan dinilai memusingkan dan membosankan. Generasi millennial bisa dibilang lebih menyukai melihat gambar, apalagi jika menarik dan berwarna. Hobi membaca buku masih tetap ada. Mereka lebih memilih membaca buku online (e-book) untuk tidak perlu repot membawa buku. Sekarang ini, sudah banyak penerbit yang menyediakan format e-book untuk dijual, agar pembaca dapat membaca dalam ponsel pintarnya. 5. Millennial lebih tahu teknologi 43 Diplan / LENTERA Jurnal Ilmiah Kependidikan Vol. 14 No.2 (Juli – Desember 2019 Edisi Dies Natalis XXXIV) 41-47 dibanding orangtua mereka. Kini semua serba digital dan online. Generasi ini melihat dunia tidak secara langsung, yaitu dengan berselancar di dunia maya. Generasi millennial adalah generasi yang sangat modern, lebih daripada orang tua mereka. Mereka sering mengajarkan teknologi pada kalangan orangtua. 6. Millennial cenderung tidak loyal namun bekerja efektif. Mereka juga tidak loyal terhadap suatu pekerjaan atau perusahaan, namun lebih loyal terhadap merek. Millennial hidup di era informasi yang menjadikan mereka tumbuh cerdas. Banyak perusahaan yang mengalami kenaikan pendapatan karena memperkerjakan millennial. 7. Millennial mulai banyak melakukan transaksi secara cashless. Dengan kecanggihan teknologi yang semakin maju ini, generasi millennial pun mulai melakukan transaksi pembelian yang sudah tidak menggunakan uang tunai lagi alias cashless. Generasi ini lebih suka membawa kartu, karena semua pembelian bisa dibayar menggunakan kartu, sehingga lebih praktis, hanya perlu gesek atau tapping. Tantangan Guru Citra dan konsep tentang guru dalam masyarakat kontemporer sangat jauh berbeda dengan konsep masa lampau. Guru masa dahulu berarti orang yang berilmu, yang arif dan bijaksana. Kini guru dilihat sebagai fungsionaris pendidikan yang bertugas mengajar atas dasar kualifikasi keilmuan dan akademis tertentu. Dengan tugas tersebut, guru memperoleh imbalan materi dari negara atau pihak pengelola pendidikan. Dengan demikian, faktor terpenting dalam profesi guru adalah kualifikasi keilmuan dan akademis. Sementara kearifan dan kebijaksanaan yang merupakan sikap dan tingkah laku moral tidak lagi signifikan. Dalam konsep klasik, faktor moral berada di atas kualifikasi keguruan. Berdasarkan penelitian bahwa mayoritas millennial mendapatkan berita bersumber dari media sosial seperti facebook dan twitter, dimana kredibilitas sumber berita sangat sulit untuk diukur. B. Tantangan Pendidik di Era Millennial Kualifikasi akademik seorang guru adalah S1 atau diploma IV. Kompetensi yang harus dimiliki seorang guru adalah kompetensi pedagogik, kompetensi kepribadian, kompetensi social dan kompetensi professional yang diperoleh melalui pendidikan profesi. Masing-masing kompetensi tersebut bila dirinci sebagai berikut: 1. Memiliki kepribadian sebagai pendidik dengan sub kompetensi a. Memiliki kepribadian mantap dan stabil b. Memiliki kepribadian dewasa c. Memiliki kepribadian arif d. Memiliki kepribadian yang berwibawa e. Memiliki akhlak mulia dan dapat menjadi teladan 2. Memiliki kompetensi pedagogik dengan sub kompetensi a. Memahami peserta didik b. Merancang pembelajaran c. Melaksanakan pembelajaran d. Evaluasi hasil belajar e. Pengembangan peserta didik 3. Memiliki kompetensi profesional sebagai pendidik dengan sub kompetensi: a. Menguasai bidang studi secara luas & mendalam b. Memahami materi ajar yang ada dalam kurikulum sekolah c. Memahami struktur, konsep, dan metode keilmuan yang menaungi/koheren materi ajar d. Memahami hubungan konsep antar mata-pelajaran terkait e. Menerapkan konsep-konsep keilmuan dalam kehidupan sehari-hari f. Berkomunikasi secara efektif dengan masyarakat 4. Memiliki kompetensi sosial sebagai pendidik. Tantangan guru di era milenial sangat berat dibanding guru–guru di era terdahulu. Selain menguasai aspek materi keilmuan yang di ajarkan. Guru dituntut memahami teknologi dan selalu menjadi pribadi yang kreatif dan inovatif. Guru harus menjadi role model bagi siswa di generasi millennial, agar siswa memahami 44 Tantangan Pendidik di Era Digital batasan– batasan teknologi, sehingga terhindar dari pemamfaatan yang salah dalam menggunakan teknologi. Tantangan bagi guru tidak berhenti disini, generasi millennial bukan generasi yang bisa dipaksa–paksa, contoh dengan melarang siswa membawa handphone. Guru di era sekarang harus lebih terbuka dengan pemikiran–pemikiran baru. Guru dituntut mendidik siswa sesuai dengan zamanya. Selama tidak bertentangan dengan norma– norma yang ada tentu hadirnya teknologi tidak perlu dipermasalahkan. Pendekatan persuasif baiknya lebih prioritas dilakukan ketimbang melakukan kebijakan–kebijakan yang terkesan otoriter maupun memaksakan kehendak. Guru bisa memberikan pengertian kepada siswa melalui kebijakan–kebijakan konkret dengan memanfaatkan teknologi dalam pembelajaran, seperti e-learning, atau menggunakan sosial media dll. Siswa justru mendapatkan edukasi bahwa teknologi ketika dimanfaatkan dengan baik justru memiliki dampak yang positif. Secara umum, guru hendaknya memahami perubahan sosial yang ada di era sekarang. Dia tidak berhenti belajar mengenai hal–hal baru. Tantangan global di era sekarang juga beda dengan tantangan global di era dahulu. Apapun langkah dan metode yang dilakukan di sekolah pastinya bertujuan membentuk karakter dan menyiapkan SDM yang berkualitas di Indonesia. Masa depan Indonesia ada didalam ruang kelas yang kita ajar. Tantangan lain pendidikan di era millennial sekarang ini adalah informasi datang lebih cepat, massif dan meluas, sehingga tidak bisa diatasi dengan sekedar pergantian kurikulum. Berapa kali pergantian kurikulum tidak akan pernah mampu mengejar percepatan informasi yang ada. Karena itu, pendekatan guru dan murid dalam berinteraksi akan lebih penting. Perlu keteladanan, membangun kehendak, dan menguatkan – dengan bekal ilmu pengetahuan tentunya – sehingga anak didiknya mampu mengatasi tantangan hidupnya. Tantangan terakhir bagi seorang guru adalah adanya empat keterampilan yang diberikan kepada siswa sejak dini, yaitu melahirkan pemikir, melahirkan komunikator, melahirkan kolaborator, dan melahirkan penemu atau pencipta. C. Upaya yang Dilakukan Menghadapi Era Milennial Ada beberapa upaya yang harus dilakukan oleh seorang guru menghadapi tantang dalam mendidik generasi millennial ini. 1. Guru-guru yang lahir pada era revolusi industri ketiga harus mengajar mereka yang lahir pada era berikutnya. Tidak bisa tidak, setiap guru wajib mengikuti perkembangan teknologi. Guru tidak boleh lagi gagap teknologi. Komputer dan gawai harus sudah menjadi keseharian para guru. Media sosial dan berbagai sumber informasi maupun sosialisasi juga harus dipahami para guru, sehingga dalam menjalankan profesinya guru akan kaya dengan materi maupun metode pembelajaran. Siswa pun tidak akan menganggap remeh guru. 2. Selain menguasai perkembangan teknologi, guru dituntut juga memahami kecenderungan yang terjadi terkait perubahan teknologi. Dengan mengikuti perkembangan hasil kemajuan teknologi, guru bakal mampu memberikan sudut Untuk melahirkan pemikir, siswa dilatih menyampaikan gagasan aktual, seperti kemacetan, banjir, tawuran. Untuk melahirkan komunikator, siswa dilatih menyampai ide secara lisan dengan baik. Untuk melahirkan kolaborator, siswa dikelompokan berdasarkan ragam kecerdasannya. Untuk melahirkan penemu, siswa dilatih menjadi inovator. Jejen Musfah, Analisis Kebijakan Pendidikan, Prenadamedia, Jakarta, 2016, hal. 84-86. 45 Diplan / LENTERA Jurnal Ilmiah Kependidikan Vol. 14 No.2 (Juli – Desember 2019 Edisi Dies Natalis XXXIV) 41-47 Revolusi industri pertama ditandai kemunculan mesin menggantikan tenaga manusia dan hewan. Generasi kedua ditandai dengan kemunculan pembangkit tenaga listrik dan motor pembakaran, hingga muncullah pesawat telepon, mobil, pesawat terbang dan sebagainya. Generasi ketiga ditandai dengan kemunculan teknologi digital dan internet. Pada revolusi industri generasi keempat ditandai dengan kemunculan superkomputer, robot pintar, rekayasa genetika dan perkembangan neuroteknologi yang memungkinkan manusia untuk lebih mengoptimalkan fungsi otak. Muncul pola-pola baru ketika disruptif teknologi hadir begitu cepat dan mengancam keberadaan pola lama pandang, alternatif, bahkan solusi kepada para peserta didik. Di sinilah peran guru yang tidak tergantikan oleh teknologi. Dalam pembelajaran, guru harus dapat menguatkan kemampuan olah pikir, olah rasa, olah hati, dan olah ragawi setiap anak. Hal itu penting agar mereka mampu memaknai, mengolah, menginterpretasi, menyaring berbagai informasi yang ada di sekitar. Guru harus memberikan pembelajaran yang relevan dengan siswa. Belajar menjadi lebih berarti ketika mereka memahami aplikasi praktis informasi yang mereka terima. Konten harus spesifik, ringkas, dan cepat. Generasi millenal harus informasi dan akan mencarinya sendiri jika guru tidak menyajikan apa yang mereka anggap relevan. Karena begitu banyak informasi yang selalu tersedia, mereka tidak merasa perlu belajar setiap hal segera. Sebaliknya, mereka ingin diajari bagaimana dan di mana mereka dapat menemukan apa yang mereka butuhkan. Dalam konteks era digital tentu pendidikan karakter sangat penting. Mengingat, internet adalah belantara yang liar, di mana konten-konten informasi yang positif dan negatif bercampur jadi satu. Hanya fondasi moral yang kuat di dalam diri, dalam arti bisa membedakan mana yang baik dan buruklah yang bisa menjamin masa depan generasi muda Indonesia tetep cerah. Oleh karena itu, seorang guru harus menjaga karakter kebangsaan yang potensial terkikis oleh berbagai ideologi radikalisme yang tidak sesuai dengan Pancasila dan Negara Kesatuan Republik Indonesia (NKRI). Di era globalisasi ini, pendidikan harus melakukan reformasi dan inovasi dalam proses belajar mengajar secara terus menerus. Untuk itu, diperlukan dukungan empirik yang dihasilkan oleh kegiatan penelitian. Oleh karena itu, guru harus paham penelitian. PENUTUP Di era globalisasi dan informasi ini, guru dan dosen mendapatkan tantangan berupa perilaku siswa yang senang berselancar di dunia maya melalui android, gadget atau handphone canggih yang mereka miliki. Perilaku siswa ini ditandai oleh adanya kecendrungan lebih senang membaca melalui handphone ketimbang buku, senang mencari berbagai informasi sendiri tanpa melakukan recheck lebih dahulu, senang mencari teman melalui akun media sosial, dan terkesan individualis. Untuk itu, guru dan dosen dalam pembelajaran harus mampu memanfaatkan Teknologi Informasi, menggunakan metode yang menyenangkan, memperkaya keilmuan dengan berbagai sumber bahan bacaan, dan mampu melakukan penelitian. Demikian uraian yang berkaitan dengan tantangan pendidik di era Millennial dan upaya yang dilakukan untuk mencari solusi terhadap tantangan yang dihadapi guru. 46 Tantangan Pendidik di Era Digital DAFTAR PUSTAKA Azra, A. (2000). Pendidikan Islam, Tradisi dan Modernisasi Menuju Millenium Baru, Cet II. Jakarta: Logos. Danim, S. (2002). Inovasi Pendidikan: Dalam Upaya Peningkatan Profesionalisme Tenaga Kependidikan. Bandung: Pustaka Setia. Depdikbud. (1990). Kamus Besar Bahasa Indonesia, Cet. III. In N. Purwanto, Ilmu Pendidikan-Teoritis dan Praktis (p. 1). Jakarta: Balai Pustaka. Fokusmedia. (2003). Undang-undang RI No. 20 Tahun 2003 tentang Sisdiknas beserta Penjelesannya. Bandung: Fokusmedia. Indonesia, C. (2018, Oct 18). Pendidikan dan Digitalisasi di Era Milenial. Retrieved from CNN Indonesia Lestari, R. (2018, Apr 3). Guru BK Kekinian: Solusi Masalah Siswa di Era Millenial. Retrieved from Selasar Mahardika, D. (2018, Oct 18). Tantangan Menjadi Guru di Era Milenial. Retrieved from Dhinar For Indonesia Mucharomah, M. (2017). Kisah sebagai Metode Pembentukan dan Pembinaan AKhlak dalam Perspektif al-Qur'an. Jurnal Edukasia Islamika, 204-207. Mursidin. (2011). Profesionalisme Guru Menurut al-Qur'an, Hadits dan Ahli Pendidikan. Jakarta: Sedaun. Musfah, J. (2016). Analis Kebijakan Pendidikan. Jakarta: Prenadamedia. Nata, A. (2005). Filsafat Pendidikan Islam. Jakarta: Gaya Media Pratama. Purwanto, M. N. (1985). Ilmu Pendidikan Teoritis dan Praktis. Bandung: Remaja Rosdakarya. Purwekerto, U. M. (2018, Aug 27). Tantangan Guru di Era Milenial. Retrieved from Universitas Muhammadiyah Purwekerto Puwanto, Y. (2007). Etika Profesi: Psikologi Profetik Perspektif Psikologi Islami, Cet. I. Bandung: Refika Aditama. Salam, B. (1997). Pengantar Pedagogik (Dasar-dasar Ilmu Mendidik). Jakarta: Rineka Cipta. Semarang, R. (2018, Oct 18). Tantangan Era Milenial dalam Dunia Pendidikan. Retrieved from Radar Semarang. Sukmadinata, N. S. (1997). Pengembangan Kurikulum Teori dan Praktek. 1997: Remaja Rosdakarya. Suwito. (2004). Filsafat Pendidikan Akhlak Ibnu Miskawaih. Yogyakarta: Belukar. Suyanto, & Hisyam, D. (2000). Refleksi dan Reformasi Pendidikan di Indoensia Memasuki Milenium III. Yogyakarta: Adicita Karya Nusa. Tafsir, A. (1982). Ilmu Pendidikan dalam Perspektif Islam. Bandung: Remaja Rosdakarya. Umbara, C. (2005). Undang-undang Republik Indonesia Nomor 14 Tahun 2005 tentang Guru dan Dosen Bab IV Pasal 10. Umbara, C. (2006). Undang-undang Republik Indonesia Nomor 14 Tahun 2005 Tentang Guru dan Dosen Bab IV, Pasal *. Bandung. Yamin, M. (2007). Profesionalisasi Guru dan Implementasi KTSP. Jakarta: Gaung Persada Press. 47 

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Multimodal Technologies and Interaction Article Exploring User Perception Challenges in Vibrotactile Haptic Display Using Resonant Microbeams under Contact with Skin Daehan Wi * and Angela A. Sodemann The Engineering Department, Arizona State University, Mesa, AZ 85212, USA; angela.sodemann@asu.edu * Correspondence: Daehan.Wi@asu.edu Received: 1 April 2019; Accepted: 24 May 2019; Published: 28 May 2019



 Abstract: Resonant vibrotactile microbeams use the concept of resonance to excite the vibration of cantilever beams, which correspond to pixels of an image. The primary benefit of this type of tactile display is its potential for high resolution. This paper presents the concept of the proposed system and human skin contact experiments to explore user perception challenges related to beam vibration during skin contact. The human skin contact experiments can be described in five phases: dried skin contact to metal beam tips, wet and soaped skin contact to metal beam tips, skin contact with a constraint, normal force measurement, and skin contact to the tips of silicone rubber beams attached to metal beam tips. Experimental results are analyzed to determine in what cases of skin contact the beams stop vibrating. It is found that the addition of silicone rubber beams allows the primary metal beams to continue vibrating while in contact with skin. Thus, the vibration response of a metal beam with silicone rubber beams is investigated for the better understanding of the effect of silicone rubber beams on the metal beam vibration. Keywords: resonant microbeams; vibrotactile haptic display; microbeam array; tactile substitution system; skin contact experiment; silicone rubber beams; vibration response; forced response 1. Introduction Haptic displays have been extensively explored in assistive technology solutions for individuals with visual impairments. Tactile vision sensory substitution devices use haptic displays to present visual images to the sense of touch. This ‘haptic display’ area of research can be divided into two possible approaches: (1) conversion of information about pictures to touch, or (2) conversion of complete pictures to touch. In the first approach, an image is first processed by a computer to determine information about the image, such as distance to the nearest object, then vibration or other tactile signals are used to convey this information to the user [1]. Haptic face recognition [2–4] and the human-computer interface system that is studied by D. Hong et al. [5] are examples of this approach. The second approach has the significant benefit of potentially giving the user an effective replacement for vision. This area of research relies upon an assumption that the brain is capable of a type of plasticity known as ‘sensory substitution’, in which information provided via one sense can be interpreted by the brain as information from a different sense [6–8]. In the second approach, there are two main types of haptic display devices. The first type of display is the electrotactile display, commonly placed on the tongue. The second main type of display is the vibration display. Brainport is the popular example of using the electro tactile displays, a device which consists of a video camera mounted on a sunglass and an electrode array [9–11]. The image captured by the camera is converted to a black and white image in software, and pixels from the camera are felt on the tongue via an electrode array [12]. Besides the electro-tactile display, the vibration displays use vibration motors. Multimodal Technol. Interact. 2019, 3, 38; doi:10.3390/mti3020038 www.mdpi.com/journal/mti Multimodal Technol. Interact. 2019, 3, 38 Multimodal Technol. Interact. 2019, 3, x FOR PEER REVIEW 2 of 13 2 of 13 Whilehigh the electrode approach is typically limited can to placement tongue due to its wetness and, thus, conductivity, vibration motor devices be placedon onthe any area of skin. This approach thus, high conductivity, vibration motor devices can be placed on any area of skin. This approach commonly uses vibration motors, camera, and a processing unit [13,14]. The image captured by the commonly uses vibration motors, camera, a processing unitmotors [13,14].corresponding The image captured by the camera is processed into a vibration level forand an array of vibration to the image. camera is processed into a vibration level for an array of vibration motors corresponding to the image. However, all of the haptic display solutions found in the literature have two primary However, all of the haptic display solutions found in the literature have two primary complications: complications: The low resolution and the one-pin-per-element problem. Resolution is the number The low resolution one-pin-per-element Resolution is the number of pixels contained of pixels contained and in anthe image. A visual digitalproblem. image commonly has a resolution such as 640 × 480 in an image. A visual digital image commonly has a resolution such as 640 × 480 or 720 1080 or 720 × 1080 pixels, but the current highest- resolution tactile display has much lower resolution×such pixels, but the highestresolution tactile display has much lower resolution such less than as less than 20 ×current 20. Since a visual image becomes more difficult to interpret, the lower theas resolution 20 × 20. Since a visual image becomes more difficult to interpret, the lower the resolution is, to it ais is, it is expected that the low resolution such as 20 × 20 would not be enough to deliver an image expected that the low resolution such as 20 × 20 would not be enough to deliver an image to a viewer. viewer. Another problem, the one pin per element problem, stems from the need to individually Anothereach problem, one independently pin per elementofproblem, stems from the need to individually each control tactilethe pixel all the others. In order to accomplish thiscontrol individual tactile pixel independently of all the others. In order to accomplish this individual control, a single control, a single pin of a microcontroller is needed for each tactile element. Thus, in order to achieve of a microcontroller is needed for each pins tactile element. Thus, inas order a resolution of apin resolution of even 20 × 20, 400 individual would be required, wellto asachieve 400 individual pulseeven 20 × 20, 400 individual pins would be required,clocks. as well as 400 individual pulse-width modulation width modulation signals and their corresponding signals and their corresponding clocks. To solve these problems and achieve high resolution, we have proposed the resonant microbeam To solve thesedisplay problems and achieve high resolution, weof have the resonantvibrotactile microbeam vibrotactile haptic [15,16]. In this paper, the concept the proposed resonant microbeam vibrotactile haptic display [15,16]. In this paper, the concept of the resonant microbeam vibrotactile haptic display is briefly reviewed. The following human touch experiments are presented: dried skin haptic display is briefly The skin following human touch experiments arebeam presented: dried skin contact, wet and soapedreviewed. skin contact, contact to a constraint and metal tips, maximum contact,force wet measurement, and soaped skin skin contact a constraint metaltobeam tips, beam maximum normal andcontact, skin contact to silicontorubber beamsand attached the metal tips. normal force measurement, and skin contact to silicon rubber beams attached to the metal beam tips. The forced response of a metal beam depending on the silicone rubber beam length is also presented. The forced response of a metal beam depending on the silicone rubber beam length is also presented. 2. The Concept of the Resonant Microbeam Vibrotactile Haptic Display 2. The Concept of the Resonant Microbeam Vibrotactile Haptic Display A resonant microbeam vibrotactile array is a mechatronic system that consists of two A resonant microbeam vibrotactile arraycontroller. is a mechatronic system that consists two subsystems: subsystems: a beam array and a beam array The beam array consists ofofcantilever beams a beam array and a beam array controller. The beam array consists of cantilever beams made of a made of a metal material, which has low damping ratio to reduce the crosstalk between each beam metal material, which has low damping ratio to reduce the crosstalk between each beam vibration. vibration. Each beam in the array is designed to have a unique length and/or cross-sectional area, so Eacheach beam in the is designed to have a unique length and/or cross-sectional that that beam canarray be controlled individually by having a unique natural frequency.area, Theso base ofeach the beam can be controlled individually by having a unique natural frequency. The base of the beam array beam array is attached to a surface transducer or a speaker that is actuated to vibrate, as shown in is attached Figure 1. to a surface transducer or a speaker that is actuated to vibrate, as shown in Figure 1. Figure1.1.AAsurface surfacetransducer transducerproduces producesaawave wavew(t) w(t)equal equalto tothe thesum sumof ofindividual individualamplitudes amplitudesand and Figure frequencies for each beam by using Fourier transform. In this example, five beams are being actuated: frequencies for each beam by using Fourier transform. In this example, five beams are being actuated: threebeams beamsat atan anamplitude amplitudeof of200 200and andtwo twobeams beamsat at an an amplitude amplitude of of 100. 100. three A surface transducer is a device that turns almost any surface into a speaker. In a surface transducer, there is no cone, and a coil is attached to a metal pad so that the vibration transmits the pad into any subject it is attached to. In the proposed device, the forcing vibration from a surface Multimodal Technol. Interact. 2019, 3, 38 3 of 13 A surface transducer is a device that turns almost any surface into a speaker. In a surface transducer, there is no cone, and a coil is attached to a metal pad so that the vibration transmits the pad into any subject it is attached to. In the proposed device, the forcing vibration from a surface transducer excites the beam base. This approach uses the concept of ‘resonance’. When the frequency of the vibration of the surface transducer matches the natural frequency of one of the beams, that beam ‘resonates’—vibrates with large amplitude. Since the natural frequency of each beam is unique, each beam can represent a different pixel of an image. For this approach, there are three important criteria: (1) minimizing natural frequency (2) maximizing vibrational amplitude (3) using material having low damping ratio. Considering that the general frequency area for people to feel static touch is less than 500 Hz [17], it is generally assumed that lower frequencies are more perceivable than higher frequencies even though the perceivable frequency range with dynamic touch is currently unknown. To improve human perception, maximizing vibrational amplitude is important. Since the natural frequency of a beam is inversely proportional to the amplitude of the beam in a lumped mass model of a beam, low frequency is required to maximize human perception [15]. Therefore, the 1st vibration mode of a metal beam is used in this approach to minimize the natural frequency and maximize the amplitude of a beam. Using material having low damping ratio can reduce the bandwidth of beam vibration, which can make it theoretically possible to make more beams in a given frequency range. To achieve the high resolution of the proposed approach, as many beams as possible will be needed without the crosstalk of vibration between each beam. In our previous research [15,16], a beam design simulation algorithm was developed with the consideration of some important criteria mentioned above. It was found that nearly 13,000 beams (96 × 128 resolution) can be made in a single display manufactured by the wire Electric Discharge Machining (EDM) method. Before moving forward for further development of this device, an important problem was found that beam vibration stops when skin contacts beam tips to perceive beam vibration and no beam vibration is perceived. This paper investigates the methods to reduce the friction occurred at the beam tips where skin directly contacts to solve the user perception problem. Diverse skin contact experiments with resonant tactile microbeams were conducted to identify cases where the metal beams do not cease to vibrate. 3. Finger Contact Experiment 3.1. Dried Finger Contact Experiment 3.1.1. The Setup for Dry Skin Contact Experiment In order for the proposed device to be usable for image perception, individual beam vibration must be perceivable by a user contacting the beams with the skin. The first requirement for perceivability is that the beams must continue to vibrate, while in contact with the skin. To investigate human perceivability and determine whether beams continue to vibrate under skin contact, human skin contact experiments are conducted on a small prototype device. For this experiment, a beam array prototype having a total of 12 cantilever metal beams is selected, as shown in Figure 2. The beam array is designed to have less than 1200 Hz as the resonant frequency range for all metal beams. Every beam has the same length, 29.6 mm, so that the skin of a finger can easily contact all beam tips simultaneously. The beam array is manufactured by wire EDM at Arizona State University. Beam number 1 represents the leftmost beam, and beam number 12 represents the rightmost beam. Multimodal Technol. Interact. 2019, 3, 38 Multimodal Technol. Interact. 2019, 3, x FOR PEER REVIEW Multimodal Technol. Interact. 2019, 3, x FOR PEER REVIEW 4 of 13 4 of 13 4 of 13 Figure EDM. Figure 2. 2. The The image image of of 12 12 metal metal beams beams in in aa row row manufactured manufactured by by wire wire EDM. Figure 2. The image of 12 metal beams in a row manufactured by wire EDM. The input of the surface transducer transducer is is connected connected to to the the output output of of an an amplifier amplifier by by USB USB cable, cable, The input its of the surface is connected to the output of an amplifier by USB cable, which receives input from transducer a function generator. The amplifier is provided with a constant The amplifier is provided with constant 5 V which receives its input from a function generator. The amplifier isaaprovided with a constant 5V power source. The function wave specified frequency’, function generator is set set to to generate generate aa sine sine wave of of specified‘starting ‘starting frequency’, powerissource. The function generator isthe setnatural to generate a sine of wave a specified ‘starting frequency’, determined by approximating frequency the of beam from a lumped-mass model which lumped-mass which is determined by approximating the natural frequency of the beam from a lumped-mass model based on onthe theactual actualbeam beam dimensions. amplitude the amplifier is adjusted to aenough small dimensions. TheThe amplitude dial dial of the amplifier is adjusted to a small based on thethe actual beam dimensions. The2nd amplitude dial of the is adjusted to A a highsmall enough amount that the 1stdoes beam does not hit the 2ndwhen beam when the 1st resonates. beam resonates. amount that 1st beam not hit the beam the 1st amplifier beam A high-speed enough amount that the 1stsobeam does not 2nd beam 1st beam resonates. highspeed camera is positioned that the field ofhit view is parallel the axis of the metal beams toArecord camera is positioned so that the field of view isthe parallel to thetowhen axis ofthe the metal beams to record the speed camera is positioned so that the field of view is parallel to the axis of the metal beams to record the beam vibration at the moment a finger touches beam tips. The high-speed camera witha beam vibration at the moment a finger touches thethe beam tips. The high-speed camera is is fitfit with the beam vibration atpositioned the moment a finger touches the beams beam Theinhigh-speed camera is camera fit view with amicroscope microscope lens and positioned so that or three are focused inhigh-speed the high-speed lens and so that twotwo or three beams are tips. focused the camera a microscope lens and positioned so that two or three beams are focused in the high-speed camera view simultaneously. A frame rate of 8819 frames per second is used for video recording to capture simultaneously. A frame rate of 8819 frames per second is used for video recording to capture changes view simultaneously. A frame rate of 8819touches frames per second is used for video to capture changes in beam vibrations when atouches finger the metal beams. Figure 3 shows the experimental in beam vibrations when a finger the metal beams. Figure 3 shows therecording experimental setup changes in abeam vibrations when a finger touches thepresses metal beams. Figure 3 shows the to experimental setup high-speed camera. afinger dried finger thetips beam tips with various forces see with awith high-speed camera. First, aFirst, dried presses the beam with various forces see ifto there setup with a high-speed camera. First, a dried finger presses the beam tips with various forces to see if is a moment when thedoes beam is there a moment when the beam notdoes stop.not stop. if there is a moment when the beam does not stop. Figure 3. The experimental setup with a high-speed camera. Figure Figure 3. 3. The experimental setup with a high-speed camera. 3.1.2. The Result of Dry Skin Contact Experiment 3.1.2. The Result of Dry Skin Contact Experiment The recorded video files are checked with eye, and it is found that when the finger touches the videooffiles checked withdecreases, eye, and and it is the found thatstops when the finger touches the beamThe tip, recorded the amplitude the are vibrating beam beam vibrating. After the skin beam tip,the thebeam amplitude of the vibrating beam decreases, and the beam stops vibrating. Aftervibration the skin contacts tips, the beam stops vibrating within about 6–10 milliseconds. No beam contacts the beam tips, the beam stops vibrating within about 6–10 milliseconds. No beam vibration Multimodal Technol. Interact. 2019, 3, 38 5 of 13 3.1.2. The Result of Dry Skin Contact Experiment The recorded video files are checked with eye, and it is found that when the finger touches the beam tip, the amplitude of the vibrating beam decreases, and the beam stops vibrating. After the skin Multimodal Technol. Interact. 3, x FOR PEERvibrating REVIEW within about 6–10 milliseconds. No beam vibration 5 of 13 contacts the beam tips, 2019, the beam stops is perceived under dry skin contact conditions. Figure 4 illustrates the moment when a finger touches is perceived under skin contact conditions. Figure are 4 illustrates moment when a finger touches the beam tips. Thedry three rectangular parts vibrating the first the three thinnest metal beams, and a the beam tips. The three rectangular parts vibrating are the first three thinnest metal beams, and a white object above the three beams is the finger. white object above the three beams is the finger. Figure 4. 4. The The image image of of aa dried dried finger finger pressing pressing the the beam beam tips. tips. Figure The reason for the beam vibration stopping may be the friction that occurs at the contact surface The reason for the beam vibration stopping may be the friction that occurs at the contact surface of beam tips and the skin. Here, the friction is the only force introduced by the presence of the skin of beam tips and the skin. Here, the friction is the only force introduced by the presence of the skin that is in the direction of the beam vibration (perpendicular to the beam axis), provided that the skin that is in the direction of the beam vibration (perpendicular to the beam axis), provided that the skin does not protrude below the surface of the beam tips. If the skin does protrude below the surface of does not protrude below the surface of the beam tips. If the skin does protrude below the surface of the beam tips, then that skin would exert a normal force in the direction perpendicular to the beam the beam tips, then that skin would exert a normal force in the direction perpendicular to the beam axis. However, from the video captured in this experiment, it is shown that the skin actually does axis. However, from the video captured in this experiment, it is shown that the skin actually does not not protrude down. This means that the friction is the only force that could stop the beam vibration. protrude down. This means that the friction is the only force that could stop the beam vibration. The The two ways to reduce friction are to reduce the coefficient of friction or to reduce the normal force two ways to reduce friction are to reduce the coefficient of friction or to reduce the normal force between the skin and the tops of the metal beams. between the skin and the tops of the metal beams. 3.2. Wet and Soaped Finger Contact Experiment 3.2. Wet and Soaped Finger Contact Experiment In the previous experiment with dry skin, it is revealed that the friction generated at the contact of In the previous experiment with dry skin, it is revealed that the friction generated at the contact beam tips and the skin is the probable reason for the beam vibration damping when the finger touches of beam tips and the skin is the probable reason for the beam vibration damping when the finger it. In this experiment, water and soap are used to reduce the coefficient of the friction. touches it. In this experiment, water and soap are used to reduce the coefficient of the friction. 3.2.1. The Setup for Wet and Soaped Finger Contact Experiment 3.2.1. The Setup for Wet and Soaped Finger Contact Experiment A previous study by O’Meara and Smith [18] reveals that the simple presence of soap and water is A previous study bydecrease O’Mearathe and Smith [18] the simple presence of soap water sufficient to dramatically coefficient ofreveals frictionthat between skin and steel. The sameand setup, as is sufficient to dramatically decrease thesoaped coefficient oftouch friction between skin and steel. The sametouches setup, shown in Figure 3, is used for wet and finger experiment. First, a soaped finger as in Figure 3, is used wet soaped finger touch experiment. First, a soaped theshown beam tips in a vibrating state,for and theand vibration response is recorded by a high-speed camera.finger Then, touches the beam tips in a vibrating state, and the vibration response is recorded by high-speed a wet and soaped finger touches the beam tips, and it is also recorded by the high-speeda camera. camera. Then, a wet and soaped finger touches the beam tips, and it is also recorded by the high3.2.2. The result of Wet and Soaped Finger Contact Experiment speed camera. In the observation of the recorded files with eye, it is still found that when the finger that is wet 3.2.2. The result of Wet and Soaped Finger Contact Experiment or wet and soaped touches the beam tip, the amplitude of the vibrating beam decreases, and the the observation of thethe recorded files with it istips, stillthe found that when the finger that is wet beamInstops vibrating. After skin contacts theeye, beam beam stops vibrating within about or wetmilliseconds. and soaped touches beam tip, the amplitude of the vibrating beamfinger decreases, andbeam the beam 6–10 Figure 5the illustrates the moment when a wet and soaped touches tips. stops vibrating. Afterpart the isskin beammetal tips, the beam vibrating within aboutabove 6–10 The three rectangular the contacts first threethe thinnest beams, andstops a white object with bubbles milliseconds. Figure illustrates the moment the three beams is the5 wet and soaped finger. when a wet and soaped finger touches beam tips. The three rectangular part is the first three thinnest metal beams, and a white object with bubbles above the three beams is the wet and soaped finger. Multimodal Technol. Interact. 2019, 3, 38 Multimodal Technol. Interact. 2019, 3, x FOR PEER REVIEW 6 of 13 6 of 13 Figure 5. The image of a wet and soaped finger pressing the beam tips. 3.3. Finger Contact Experiment with Constraints In this experiment, two constraints of acrylic panels, which have almost the same height as the metal beam tips, are manufactured by a laser cutter to reduce the normal force contributing to the friction. One of the U-shaped acrylic constraints has a height of 29.6 mm, and the other has a height of 29.7 mm, as shown in Figure 6. The image image of of a wet and soaped finger pressing the beam tips. Figure 5. The 3.3.1. The Setup forExperiment Finger Contact Experiment with Constraints Setup 3.3. Finger Contact with Constraints 3.3. Finger Contact Experiment with Constraints The metal beams oftwo a beam array are in the channel of thealmost U-shaped acrylic constraint, In this experiment, constraints of located acrylic panels, which have the same height as the In this experiment, two constraints of acrylic panels, which have almost the same height as the as shown in Figure 7. The same high-speed camera setup, as shown in Figure 3, is used. Since metal beam tips, are manufactured by a laser cutter to reduce the normal force contributing to the metal beam tips, are manufactured by a laser cutter to reduce the normal force contributing to the metal beams dothe notU-shaped vibrate when the U-shapedhas constraint them, a the clearance is maintained friction. One of acrylic constraints a heightcontacts of 29.6 mm, and other has a height of friction. One of the U-shaped acrylic constraints has a height of 29.6 mm, and the other has a height by tape to6.fix the constraint to the beam base, so that the constraint does not touch 29.7using mm,double-sided as shown in Figure of 29.7 mm, as shown in Figure 6. the beams. 3.3.1. The Setup for Finger Contact Experiment with Constraints Setup The metal beams of a beam array are located in the channel of the U-shaped acrylic constraint, as shown in Figure 7. The same high-speed camera setup, as shown in Figure 3, is used. Since the metal beams do not vibrate when the U-shaped constraint contacts them, a clearance is maintained by using double-sided tape to fix the constraint to the beam base, so that the constraint does not touch the beams. The image of U-shaped constraint of Acrylic panels. Figure 6. The 3.3.1. The The Result Setup for Finger Contact Contact Experiment Experiment with with Constraints Constraints Setup 3.3.2. of Finger The beam metal vibration beams of is a beam arraybyare located intip theofchannel of the U-shaped constraint, The perceived tapping the the metal beams, but it isacrylic hard to perceive as shown in beam Figurevibration 7. The same high-speed setup, shown in Figure 3, is used. while Since the any distinct by just touchingcamera the beam tips.asBeam vibration is perceived, the metal still beams do notthrough vibrate when the U-shaped constraint contacts clearance is maintained by beam vibrates the finger pressing (touching) the beamthem, very aweakly. Even though beam using double-sided tape to fix the the constraint to and the beam base, very so that the constraint not touch vibration is perceived by touching beam tip constraints weakly, this maydoes not be suitable Figure 6. The image of U-shaped constraint of Acrylic panels. the beams. for use of the device, since it is impossible to maintain the very weak touch with the beams. Another problem that there is always some vibration with transmitted through the U-shaped constraint, and it 3.3.2. TheisResult of Finger Contact Experiment Constraints The beam vibration is perceived by tapping the tip of the metal beams, but it is hard to perceive any distinct beam vibration by just touching the beam tips. Beam vibration is perceived, while the beam still vibrates through the finger pressing (touching) the beam very weakly. Even though beam vibration is perceived by touching the beam tip and constraints very weakly, this may not be suitable for use of the device, since it is impossible to maintain the very weak touch with the beams. Another problem is that there is always some vibration transmitted through the U-shaped constraint, and it 3.4.1. The Setup for Finger Force Measurement A scale is used to measure the finger force. First, the surface transducer and the beam array with the Acrylic constraint are placed on the scale to measure their weight without a finger press. Then, the vibrating beam tip and constraint are pressed by a finger while the targeted beam vibrates, and Multimodal Technol. Interact. 2019, 3, 38 7 of 13 the weight caused by the finger force including the whole weight of experimental device is measured, as shown in Figure 7. (a) (b) Figure 7. The The image image of of finger finger force force measurement measurement using a scale: (a) (a) Total Total weight of whole experimental device without a finger press; press; (b) (b) Total Total weight weightof ofwhole wholeexperimental experimentaldevice devicewith withaafinger fingerpress. press. 3.3.2. The Result Result of of Finger Finger Force Contact Experiment with Constraints 3.4.2. The Measurement The beam vibrationitisisperceived bythe tapping the tip of by thethe metal beams, but it isforce, hard which to perceive In the experiment, found that weight caused maximum finger does any distinct beam vibration by just touching the beam tips. Beam vibration is perceived, while the =beam not stop beam vibration, is less than 1 gram. 1 gram is equal to 0.001 kg, which is 0.001 × 9.81 0.01 still vibrates the finger (touching)continuously the beam very weakly. Even though N. This forcethrough is too small force pressing to be maintained when people touch the beam beam vibration tips. It is is perceived by touching beam be tipconsidered and constraints this maybased not beon suitable for use of found that other methodsthe should to aidvery the weakly, user perception this experiment the device, since it is impossible to maintain the very weak touch with the beams. Another problem result. is that there is always some vibration transmitted through the U-shaped constraint, and it interferes with recognizing the beam vibration. Moreover, no beam vibration is perceived after the 4th beam, 3.5. Finger Touch Experiment with Silicone Rubber Beams which the resonant frequency is 612 Hz, even though the finger either presses the beam tip very weakly It is found that a new method is needed to reduce the normal force generating from the direct or taps the tip. There might be two possible reasons for the lack of user perception. One is that the finger touch to the beam tips. For this, a material known as ‘dragon skin 10 very fast’, a type of silicone amplitude of the 4th beam may be not enough for the user to perceive the vibration. Another reason is rubber, is selected to make a silicone rubber beam, which is attached to a metal beam tip so that a that the perceivable frequency range with dynamic touch may be similar to the one with static touch, finger could only touch the tips of silicone rubber beams and the normal force of the finger would which is known up to around 500 Hz. To figure out the exact reason, further research will be needed. not be exerted on the metal beam tips directly. 3.4. Finger Force Measurement 3.5.1. The Setup for Finger Touch Experiment with Silicone Rubber Beams It is found that with very weak finger touch to the metal beam tips and constraints, the beam vibration can be perceived. In this experiment, the goal is to measure the maximum normal force that does not stop beam vibration. 3.4.1. The Setup for Finger Force Measurement A scale is used to measure the finger force. First, the surface transducer and the beam array with the Acrylic constraint are placed on the scale to measure their weight without a finger press. Then, the vibrating beam tip and constraint are pressed by a finger while the targeted beam vibrates, and the weight caused by the finger force including the whole weight of experimental device is measured, as shown in Figure 7. 3.4.2. The Result of Finger Force Measurement In the experiment, it is found that the weight caused by the maximum finger force, which does not stop beam vibration, is less than 1 gram. 1 gram is equal to 0.001 kg, which is 0.001 × 9.81 = 0.01 N. This force is too small force to be maintained continuously when people touch the beam tips. It is found that other methods should be considered to aid the user perception based on this experiment result. Multimodal Technol. Interact. 2019, 3, 38 8 of 13 3.5. Finger Touch Experiment with Silicone Rubber Beams It is found that a new method is needed to reduce the normal force generating from the direct finger touch to the beam tips. For this, a material known as ‘dragon skin 10 very fast’, a type of silicone rubber, is selected to make a silicone rubber beam, which is attached to a metal beam tip so that a finger could only touch the tips of silicone rubber beams and the normal force of the finger would not be exerted on the metal beam tips directly. 3.5.1. The Setup for Finger Touch Experiment with Silicone Rubber Beams Multimodal Technol. Interact. 2019, 3, x FOR PEER REVIEW 8 of 13 A and B of ‘Dragon Skin 10 very fast’ are mixed and solidified. The solidified dragon skin is cut to and B of ‘Dragon 10 very fast’ are mixed solidified. solidified dragon is cut makeArectangular shapedSkin silicone rubber beams. Two and silicone rubber The beams are attached to skin the tips of to make rectangular shaped silicone rubber beams. Two silicone rubber beams are attached to the two beams of the metal prototype with an ethyl cyanoacrylate adhesive. The length of silicone rubber tips of two beams thetheir metalthicknesses, prototype with an ethyl cyanoacrylate Thethe length of silicone beams is 11.7 mm,ofand around 1.1mm, are slightlyadhesive. thicker than thickness of a rubber beams is 11.7 mm, and their thicknesses, around 1.1mm, are slightly thicker than the thickness metal beam. Since the silicone rubber beams are cut manually, their thickness is not uniform, as shown of Figure a metal8.beam. Sinceexperimental the silicone rubber beams areincut manually, their thicknessthe is not uniform, in The same setup as shown Figure 3 is used to resonate metal beams as to shownsilicone in Figure 8. The sameare experimental setup as shown in Figure 3 is used to resonate metal which rubber beams attached, and a finger touches the silicone rubber beam tip the when the beamsvibrates to whichinsilicone rubber beams attached, and aOnly finger touches silicone beam tip beam order to perceive theare beam vibration. two metal the beams withrubber silicone rubber when the beam are vibrates in order to perceive the beam vibration. Onlyvibration two metalwith beams beams attached resonated. After investigating the 2nd metal beam the with long silicone silicone nd metal beam vibration with the long rubber beams, beams attached resonated. investigating rubber the long are silicone rubberAfter beams are cut into the the 2half to investigate the difference of the silicone rubber beams, the long silicone rubber beams are cut into the half to investigate the difference 2nd beam vibration. nd of the 2 beam vibration. Figure 8. The and and two two silicone rubber beams for high-speed camera investigation. Figure 8. prototype The prototype silicone rubber beams for high-speed camera investigation. 3.5.2. The Result Result of of Finger Finger Touch Touch Experiment 3.5.2. The Experiment with with Silicone Silicone Rubber Rubber Beams Beams The metalbeam beamvibration vibrationcan can be be perceived perceived by by skin skin contact contact to to the the silicone The 2nd 2nd metal silicone rubber rubber beam beam attached to the 2nd metal beam after cutting the long silicone rubber beam into the half. However, nd attached to the 2 metal beam after cutting the long silicone rubber beam into the half. However, when beam vibration vibration is is observed observed at at around around their their original original resonant resonant when the the vibration vibration test test is is conducted, conducted, no no beam frequencies, which were After the the silicone frequencies, which were used used in in the the previous previous skin skin contact contact experiments. experiments. After silicone rubber rubber beam attached to the 2nd metal beam (the metal beam being observed) is removed from the beam, nd beam attached to the 2 metal beam (the metal beam being observed) is removed from the beam, the the distinct beam vibration is observed at around the resonant frequency. This shows that the distinct beam vibration is observed at around the resonant frequency. This shows that the long long silicone thethe beam vibration, and and the amplitude of theof beam longthe silicone silicone rubber rubberbeams beamsabsorb absorb beam vibration, the amplitude the with beamthe with long rubber beams becomes smaller. It is also found that the resonant frequency of the metal beams can silicone rubber beams becomes smaller. It is also found that the resonant frequency of the metal shift bycan attaching siliconethe rubber beams to the beamtotips. beams shift bythe attaching silicone rubber beams the beam tips. Even though the mechanical properties like damping Even though the mechanical properties like damping ratio ratio between between the the silicone silicone rubber rubber beam beam and metal beam are different, since the total length of the beams increases due to adding and metal beam are different, since the total length of the beams increases due to adding the the silicone silicone rubber beams to to the the top top of of metal metal beams; beams; the the resonant resonant frequency frequency range range of of the the metal metal beams beams are are shifted. shifted. rubber beams For example, the frequency of the 1st metal beam is 511 Hz, and it is shifted to 490 Hz after attaching st For example, the frequency of the 1 metal beam is 511 Hz, and it is shifted to 490 Hz after attaching the beams. InIn thethe same way, thethe resonant frequency of the the silicone silicone rubber rubberbeams beamstotothe thetip tipofofthe themetal metal beams. same way, resonant frequency of the 2nd metal beam shifted from 569 to 525 Hz, the 3rd shifted from 612 to 580 Hz, and the 4th shifted from 664 to 620 Hz. Unfortunately, the bandwidth becomes broader after attaching silicone rubber beams. It is found that cutting the silicon rubber with some increments can affect the frequency shift and the bandwidth of the beam vibration. Multimodal Technol. Interact. 2019, 3, 38 9 of 13 2nd metal beam shifted from 569 to 525 Hz, the 3rd shifted from 612 to 580 Hz, and the 4th shifted from 664 to 620 Hz. Unfortunately, the bandwidth becomes broader after attaching silicone rubber beams. It is found that cutting the silicon rubber with some increments can affect the frequency shift and the bandwidth of the beam vibration. 4. Vibration Response Depending on the Decrease of Silicone Rubber Beam Length To better understand the effect of the silicone rubber beams on the metal beam bandwidth and amplitude, further experiments are conducted. In this experiment, a total of 46 cases with the silicone Multimodal Technol. Interact. 2019, 3, x FOR PEER REVIEW 9 of 13 rubber beam length changes are investigated, and 434 iterations of a high-speed camera experiment are are recorded recorded in in order order to to find find the the relationship relationship between between the the dimensions dimensions of of the the silicon silicon rubber rubber beams beams and and the the vibrational vibrational response response of of the the metal metal beams. beams. 4.1. The Setup for Vibration Response Depending on the Decrease Of Silicone Rubber Beam Length 4.1 The Setup for Vibration Response Depending on the Decrease Of Silicone Rubber Beam Length Silicone rubber beams manually cut are attached to the first five thinnest metal beams because Silicone rubber beams manually cut are attached to the first five thinnest metal beams because these metal beams have larger vibrational amplitudes than the other metal beams. Only the 2nd metal these metal beams have larger vibrational amplitudes than the other metal beams. Only the 2nd metal beam is resonated, and its vibration response is recorded. Since the resonant frequency of the 2nd metal beam is resonated, and its vibration response is recorded. Since the resonant frequency of the 2nd beam is different, depending on the length of the silicone rubber beams, the resonant frequency of the metal beam is different, depending on the length of the silicone rubber beams, the resonant frequency beam must be found with eye observation after cutting the silicon rubber beams with an approximate of the beam must be found with eye observation after cutting the silicon rubber beams with an 1mm increment. Then, about ten frequencies including the resonant frequency of the metal beam alone approximate 1mm increment. Then, about ten frequencies including the resonant frequency of the are recorded with the high-speed camera to find the resonant frequency of the metal beam with the metal beam alone are recorded with the high-speed camera to find the resonant frequency of the silicone rubber beam attached. Figure 9 shows the silicone rubber beams attached to the first five metal beam with the silicone rubber beam attached. Figure 9 shows the silicone rubber beams thinnest metal beams. attached to the first five thinnest metal beams. Figure 9. The silicone rubber beams first five thinnest metal beams. Figure 9. The silicone rubber beams attached toattached first fiveto thinnest metal beams. 4.2. The Result of Vibration Response Depending on the Decrease of Silicone Rubber Beam Length 4.2. The Result of Vibration Response Depending on the Decrease of Silicone Rubber Beam Length The recorded vibration response of the 2nd metal beam is analyzed by an optical flow algorithm The recorded vibration response of the 2nd metal beam is analyzed by an optical flow algorithm implemented in Python. The vibration response of the 2nd beam is analyzed depending on the length implemented in Python. The vibration response of the 2nd beam is analyzed depending on the length change of the silicone rubber beams. For example, Figure 10 shows the amplitude of the 2nd metal change of the silicone rubber beams. For example, Figure 10 shows the amplitude of the 2nd metal beam depending on the forcing frequency provided by the surface transducer when the silicone rubber beam depending on the forcing frequency provided by the surface transducer when the silicone beam is 3.4mm long. It is shown that when the silicone rubber beam length is 3.4 mm, the 2nd beam’s rubber beam is 3.4mm long. It is shown that when the silicone rubber beam length is 3.4 mm, the 2nd resonant frequency is 498 Hz. beam’s resonant frequency is 498 Hz. The recorded vibration response of the 2nd metal beam is analyzed by an optical flow algorithm implemented in Python. The vibration response of the 2nd beam is analyzed depending on the length change of the silicone rubber beams. For example, Figure 10 shows the amplitude of the 2nd metal beam depending on the forcing frequency provided by the surface transducer when the silicone rubber beam is 3.4mm long.3,It38is shown that when the silicone rubber beam length is 3.4 mm, the 2nd Multimodal Technol. Interact. 2019, 10 of 13 beam’s resonant frequency is 498 Hz. Figure 10. Amplitude of the 2nd beam when silicone rubber beam length is 3.4mm. Figure 10. Amplitude of the 2nd beam when silicone rubber beam length is 3.4mm. 1. 2. 3. 4. 5. 6. 7. 8. After all video files are analyzed, several observations are noted from this experiment: After all video files are analyzed, several observations are noted from this experiment: There are certain lengths of the silicone rubber beams for which the 2nd metal beam vibrates with much lower amplitude. For example, when the silicone rubber beam length is about 10.3~10.7 mm, 5.6~6.7 mm, or 2.1 mm, the amplitude of the beam vibration is greatly decreased. These lengths at which the metal beam vibration is decreased will be referred to here as ‘diminished vibration lengths’. Between two diminished vibration lengths, the amplitude of metal beam vibration becomes larger and then becomes smaller after a diminished vibration length. Table 1 shows the amplitude difference of the 2nd metal beam depending on the silicone rubber beam lengths. The resonant frequency of the 2nd metal beam trends towards the resonant frequency of the beam without the silicone rubber beam, as the silicone rubber beam length becomes shorter. However, when the amplitude of vibration of the metal beam is very small due to the length of the silicone rubber beam, the resonant frequency becomes very different than it was without the rubber beam. When the silicone rubber beam length is longer than 10mm, the maximum amplitude and resonant frequency of the metal beam cannot be determined, and when the silicone rubber beam length is shorter than 2mm, the vibration of the beam becomes more difficult to perceive by touch. Silicone rubber beams may absorb the vibration from other metal beams. For example, there are 4 silicone rubber beams attached to the 2nd to 5th metal beams, and there is no silicone rubber beam attached to the 1st beam. When a silicone rubber beam is attached to the 1st beam, the amplitude of 2nd metal beam decreases significantly. If the length of the 2nd rubber beam is different than the lengths of the other rubber beams, then the frequency response of the 2nd metal beam is very different than it is when every rubber beam has the same length. Three experiment sets are performed to find the silicone rubber beam length that maximizes the vibrational amplitude of the 2nd metal beam. In these three experiments, the optimal rubber length is found to be 4.3, 4.6, 3.4 mm. If the difference in length between the 2nd rubber beam and other rubber beams is small, and the length of the 2nd rubber beam is around the length with the maximum amplitude, the vibration result is similar with the case of all silicone rubber beams with the same length. For example, if the difference in the length between the 2nd rubber beam and other rubber beams is around 0.6mm, and the length of the 2nd rubber beam is 3.4 mm, the maximum amplitude and resonant frequency of the 2nd metal beam in both cases are about the same. But the bandwidth is smaller in the case of every beam having same length of silicone rubber beams. Based on these results, it is hypothesized that the silicone rubber beam has its own resonant frequency, so when the resonant frequency of the silicone rubber beam is similar to the resonant frequency of the metal beam, the amplitude of metal beam vibration is smaller. To verify this Multimodal Technol. Interact. 2019, 3, 38 11 of 13 assumption, the resonant frequencies of the 2nd silicone rubber beam in 1st and 2nd mode vibration are calculated, as shown in Table 2. Table 1. The amplitude and resonant frequency of the 2nd metal beam difference depending on the silicone rubber beam lengths in an experiment set. Five Rubber Beam Lengths (mm) Amplitude of the 2nd Metal Beam (mm) Resonant Frequency of the 2nd Metal Beam (Hz) (12.2, 12.2, 12.2, 12.2, 12.2) (10.7, 10.7, 10.7, 10.7, 10.7) (9.6, 9.6, 9.6, 9.6, 9.6) (9.6, 8.5, 9.6, 9.6, 9.6) (8.5, 8.5, 8.5, 8.5, 8.5) (8.5, 7.6, 8.5, 8.5, 8.5) (7.6, 7.6, 7.6, 7.6, 7.6) (7.6, 6.7, 7.6, 7.6, 7.6) (5.6, 5.6, 5.6, 5.6, 5.6) (5.6, 4.6, 5.6, 5.6, 5.6) (4.6, 4.6, 4.6, 4.6, 4.6) (4.6, 4.0, 4.6, 4.6, 4.6) (3.4, 3.4, 3.4, 3.4, 3.4) 0.0946 0.0276 0.0854 0.2490 0.2518 0.1509 0.1467 0.0437 0.0278 0.1505 0.1332 0.2601 0.2850 494 Not observable 480 496 495 502 502 505 Not observable 485 484 490 498 Table 2. The calculated resonant frequency of the 2nd silicone rubber beam in 1st and 2nd mode vibration based on the lumped mass approximation. The 2nd Silicone Beam Lengths (mm) The Resonant Frequency in the 1st Mode (Hz) The Resonant Frequency in the 2nd Mode (Hz) 12.2 10.7 9.6 8.5 7.6 6.7 5.6 4.6 4.0 3.4 83 108 135 172 215 277 396 587 776 1074 523 680 845 1078 1348 1734 2482 3679 4866 6734 By comparison of Tables 1 and 2, it is found that when the resonant frequency of the 2nd silicone rubber beam in the 1st or 2nd mode vibration is similar with the resonant frequency of the 2nd metal beam, the metal beam vibration is not observable since the frequencies of two different materials are compensated and offset. However, the calculated resonant frequency of the silicone rubber beam is not exactly the same as the resonant frequency of the metal beam, which was obtained from the experiment, and there is a frequency difference between them. There might be several possible reasons. First, other silicone rubber beams attached to other metal beams could affect the resonant frequency of the 2nd silicone rubber beam since all metal beams are connected by the beam base part. Second, the size effect in micro-mesoscale depending on a silicone rubber beam length difference could affect the resonant frequency of silicone rubber beams, which causes the frequency difference. In microscale, the elastic modulus and damping ratio are changeable due to the size effect [19]. The further research will be needed to figure out the exact reason for the frequency difference between the frequency of the 2nd metal beam obtained from this experiment and the calculated frequency of the 2nd silicone rubber beam. In order to utilize silicone rubber beams as a compliant mechanism to increase beam vibration during contact between metal beams and skin, the vibrational characteristics of the silicone rubber beams will need to be incorporated into the metal beam design. Multimodal Technol. Interact. 2019, 3, 38 12 of 13 5. Conclusion In this paper, the haptic display using the resonant vibrotactile microbeam is presented. The concept of the resonant vibrotactile microbeam haptic display is briefly reviewed. All the parameters are considered under the three criteria: maximizing amplitude, minimizing frequency, and minimizing damping ratio of the materials. To solve the micro-meso scale beam vibration stop problem by skin contact, the human skin contact experiments are presented: dry skin contact, wet and soaped skin contact, skin contact to a constraint and metal beam tips, normal force measurement, and skin contact to silicone rubber beam tips attached to metal beam tips. In the skin contact experiment with silicone rubber beams, beam vibration can be perceived when a finger contacts the silicone rubber beams that is attached to the vibrating metal beam. This shows it is beneficial to attach silicone rubber beams to the metal beam tips to solve the user perception problem, in which a vibrating beam stops due to the normal force exerted by a finger when a finger contacts the beam tips. For more information about the effect of the silicone rubber beams on the metal beam vibration depending on the length of silicone rubber beams, forced response experiments for the metal beam are conducted by cutting the silicone rubber beams with some increments. Further research will be needed to analyze the relationship between beam vibration and silicon rubber vibration numerically. This result can be used to define the efficiency of silicone rubber beam attachment to the metal beam tips in order to solve the user perception problem. Author Contributions: The authors have contributed equally to the research contained in this paper as follows: conceptualization, D.W. and A.A.S.; methodology, D.W. and A.A.S.; software, D.W. and A.A.S.; validation, D.W.; formal analysis, D.W.; investigation, D.W.; resources, A.A.S.; data curation, D.W.; writing—original draft preparation, D.W.; writing—review and editing, D.W. and A.A.S.; visualization, D.W.; supervision, A.A.S.; project administration, A.A.S.. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Ruspini, D.C.; Kolarov, K.; Khatib, O. The haptic display of complex graphical environments. In Proceedings of the 24th Annual Conference on Computer graphics and Interactive Techniques, Los Angeles, CA, USA, 3–8 August 1997; pp. 345–352. Kilgour, A.R.; de Gelder, B.; Lederman, S.J. Haptic face recognition and prosopagnosia. Neuropsychologia 2004, 42, 707–712. [CrossRef] [PubMed] Ur Réhman, S. Expressing Emotions through Vibration for Perception and Control. Ph.D. Thesis, Department of Applied Physics and Electronics, Umeå University, Umeå, Sweden, 2010. McDaniel, T.; Bala, S.; Rosenthal, J.; Tadayon, R.; Tadayon, A.; Panchanathan, S. Affective haptics for enhancing access to social interactions for individuals who are blind. In Proceedings of the Universal Access in Human-Computer Interaction, Crete, Greece, 22–27 June 2014; pp. 419–429. D’Angio, P.C. Adaptive and Passive Non-Visual Driver Assistance Technologies for the Blind Driver Challenge® . Ph.D. Thesis, Virginia Tech, Montgomery County, VA, USA, 2010. Bach-y-Rita, P. Neurophysiological basis of a tactile vision-substitution system. IEEE Trans. Man-Machine Sys. 1970, 11, 108–110. [CrossRef] Bach-Y-Rita, P.; Collins, C.C.; Saunders, F.A.; White, B.; Scadden, L. Vision Substitution by Tactile Image Projection. Nature 1969, 221, 963–964. [CrossRef] [PubMed] Bach-y-rita, P. Tactile vision substitution: Past and future. Int. J. Neurosci. 1983, 19, 29–36. [CrossRef] [PubMed] Grant, P.; Spencer, L.; Arnoldussen, A.; Hogle, R.; Nau, A.; Szlyk, J.; Nussdorf, J.; Fletcher, D.C.; Gordon, K.; Seiple, W. The Functional performance of the brainport v100 device in persons who are profoundly blind. J. Vis. Impair. Blind. 2016, 110, 77–88. [CrossRef] Danilov, Y.; Tyler, M. Brainport: An alternative input to the brain. J. Integr. Neurosci. 2005, 4, 537–550. [CrossRef] [PubMed] Multimodal Technol. Interact. 2019, 3, 38 11. 12. 13. 14. 15. 16. 17. 18. 19. 13 of 13 Nau, A.; Bach, M.; Fisher, C. Clinical tests of ultra-low vision used to evaluate rudimentary visual perceptions enabled by the BrainPort vision device. Transl. Vision Sci. Technol. 2013, 2, 1. [CrossRef] [PubMed] Bach-y-Rita, P.; Kercel, S.W. Sensory substitution and the human–machine interface. Trends Cogn. Sci. 2003, 7, 541–546. [CrossRef] [PubMed] Wacker, P.; Wacharamanotham, C.; Spelmezan, D.; Thar, J.; Sánchez, D.A.; Bohne, R.; Borchers, J. VibroVision: An on-body tactile image guide for the blind. In Proceedings of the 2016 CHI Conference Extended Abstracts on Human Factors in Computing Systems; San Jose, CA, USA, 7–12 May 2016; pp. 3788–3791. Dakopoulos, D.; Bourbakis, N. Towards a 2D tactile vocabulary for navigation of blind and visually impaired. In Proceedings of the IEEE International Conference on Systems, Man and Cybernetics, San Antonio, TX, USA, 11–14 October 2009; pp. 45–51. Wi, D.; Sodemann, A. Vibration analysis for the development of resonant microbeam high-resolution vibrotactile haptic display. J. Vib. Control 2018, 25, 362–372. [CrossRef] Wi, D.; Sodemann, A.; Chicci, R. Vibratory haptic feedback assistive device for visually-impaired drivers. In Proceedings of the 2017 IEEE SmartWorld, Ubiquitous Intelligence Computing, Advanced Trusted Computed, Scalable Computing Communications, Cloud Big Data Computing, Internet of People and Smart City Innovation (SmartWorld/SCALCOM/UIC/ATC/CBDCom/IOP/SCI), San Francisco, CA, USA, 4–8 August 2017; pp. 1–5. Bolanowski, S.J., Jr.; Gescheider, G.A.; Verrillo, R.T.; Checkosky, C.M. Four channels mediate the mechanical aspects of touch. J. Acoust. Soc. Am. 1988, 84, 1680–1694. [CrossRef] O’Meara, D.M.; Smith, R.M. Static friction properties between human palmar skin and five grabrail materials. Ergonomics 2001, 44, 973–988. [CrossRef] Abazari, A.M.; Safavi, S.M.; Rezazadeh, G.; Villanueva, L.G. Modelling the size effects on the mechanical properties of micro/nano structures. Sensors 2015, 15, 28543–28562. [CrossRef] [PubMed] © 2019 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/). 

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Pak Heart J 2022;55(Supplement1) 51st CARDIOCON 2022: ABSTRACT COMPARATIVE ANALYSIS OF HEPATOTOXIC POTENTIAL OF SIMVASTATIN WITH ROSUVASTATIN AND REVERSAL VIA MONTELUKAST & COENZYME Q10 IN BALB/C MICE Manzoor Khan1, Sarwat Jahan1, Amber Ashraf1 1 Cardiology Division, Department of Medical Specialities, KMC, KTH - MTI, Peshawar, Pakistan Objectives: Statins cause elevations of AST and ALT, cholestatic hepatotoxicity, fulminant hepatitis, autoimmune hepatitis, and cirrhosis. (2) Various mechanisms are hypothesized including reduced levels of Coenzyme Q10, inflammation and oxidative stress. Coenzyme Q10 is anti-apoptotic and possess antioxidative potential whereas Montelukast can reduce the inflammation via inhibition of leukotrienepathway. Hence both these drugs may have a protective effect against statin induced hepatotoxicity. This study will include the comparison of hepatotoxic potential of simvastat in and rosuvastatin and assess the hepatoprotective efficacy of montelukast and coenzyme 10. Methodology: An experimental study of 2 weeks was conducted in the department of Pharmacology on a sample of 35 mice, randomly divided into 7 groups of 5 mice each for 2 weeks. Group 1 was used as control. Group 2 received simvastatin 50mg/kg/day of simvastatin intraperitoneally (I/P). Group 3 received I/P 50mg/kg/day rosuvastatin. Group 4 received 50mg/kg/day I/P simvastatin+3mg/kg of Montelukast. Group 5 received 50mg/kg/day of rosuvastatin +3mg/kg of Montelukast. Group 6 received 50mg/kg/day of simvastatin+10mg/kg of coenzyme Q10. Group 7 received 50mg/kg/day of rosuvastatin+10mg/kg of coenzyme Q10 I/P. Results: Both the statins showed hepatotoxicity, however Simvastatin showed higher liver function derangements. Montelukast did not reduce hepatotoxicity significantly. A slightly lower rise in liver parameters was however observed. Coenzyme Q10 did not show any protective potential against the statins, rather higher liver function derangements were observed. Conclusion: Simvastatin is more hepatotoxic as compared to Rosuvastatin. Motelukast does have a slight protective effect but is not significant enough. Coenzyme Q10 was not only not protective but infact indicated a hepatotoxic rend either by itself or via potentiating of statin induced hepatotoxicity. Keywords: Hepaotoxicity, Hepatoprotection, Oxidative stress, Inflammation Citation: Khan M, Jahan S, Ahmad F, Ashraf A. Comparative Analysis of Hepatotoxic Potential of Simvastatin with Rosuvastatin and Reversal via Montelukast & Coenzyme Q10 in BALB/C MICE. Pak Heart J. 2022;55(Supplement1):S21. https://doi.org/10.47144/phj.v55iSupplement1.2437 Corresponding Author: Manzoor Khan, Cardiology Division, Department of Medical Specialities, KMC, KTH MTI, Peshawar. http://www. pakheartjournal.com S21 

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E3S Web of Conferences 150, 010 0 6 (20 20) EDE7-2019 https://doi.org/10.1051/e3sconf/20201500100 6 Comparative Study of Groundwater Pollution of M’nasra and Sfafaa zones (Gharb, Morocco) by Nitrates Najib EL KHODRANI*1,4, Serine OMRANIA2, Abdrrahim NOUAYTI3, Abdelmajid ZOUAHRI1, Ahmed DOUAIK1, Hamza IAAICH1, Mbark LAHMAR1, and Mohammed FEKHAOUI4 1 Research Unit on Environment and Conservation of Natural Resources, Regional Center of Rabat, National Institute of Agricultural Research (INRA),Rabat, Morocco. 2 Laboratory of Spectroscopy, Molecular Modeling, Materials, Nanomaterials, Waters and Environment, CERNE2D, Faculty of Sciences, Mohammed V University, Rabat, Morocco. 3 Laboratory of Applied Geosciences, Faculty of Sciences, University Mohamed First Oujda 60000, Morocco. 4 Scientific Institute, Mohammed V University, Rabat, Morocco. Abstract. The quality of water resources in Morocco is degraded by pollution from agriculture, industry and urbanization. In this work we have realized a comparative study of two groundwater pollution zones: M’nasra (Kenitra) and Sfafaa (Sidi Slimane). In general, the results of this study showed that most water wells are strongly mineralized as the conductivity far exceeds the standard of drinking water. Sodium chloride (NaCl) concentration is between 33.9 and 1184.6 mg / l and shows some of the wells are loaded with salts. The pH is generally basic in all wells. Pollution by nitrates two regions showed that the ground water is strongly affected M’nasra zone terms of the permeability of the soil of the city. The concentration of nitrate can reach high values compared to the results obtained on the ground water at Sfafaa zone whose nitrate concentrations around 20 mg/l. Further pollution of groundwater in the two regions of the shows values of organic matters that go far beyond the standards of drinking. 1 Introduction Groundwater contamination by nitrates (NO3-) represents a massive environmental problem. From today, NO3- is the most ubiquitous groundwater contaminant in the world and agricultural activities are mainly blamed for these problems. Groundwater is a strategic water source for human activities. In addition to being often of large volumes, they generally provide better quality water than surface water. These waters constitute 23% of the planet's freshwater resources [1] which must be conserved and protected from all kinds of pollution. In fact, the natural levels of nitrates in groundwater are generally very low (typically less than 10 mg/l), but their concentrations increase with human activity such as agriculture (nitrogen fertilizers), domestic effluents, leachates from landfills, etc. This increase is the main cause of degradation of groundwater quality and mainly of more vulnerable water tables. * In general, nitrates move relatively slowly in soil and groundwater: there is a lag time of approximately 20 years between pollution activity and detecting the pollutant in groundwater. For this reason, it is anticipated that routine pollution activities will continue to affect nitrate concentrations for several decades. However, if the pressure in the aquifer is high, transport can be very fast in the saturation zone. In the Gharb region of Morocco, nitrate concentration in some wells reach 130 mg/l due to intensive agricultural practices [2]. Vulnerable aquifers are frequently located in zones of high water demand, such as coastal areas (Algeria, Italy, France, Morocco, Tunisia, etc). Besides, irrigated farming accounts for a large share of total water withdrawals: 83% in Greece, 68% in Spain, 57% in Italy, and 52% in Portugal [3]. The drip irrigation system is more and more encouraged by the Moroccan government via 80% of subvention. The mismanagement of irrigation can result in water misuse and also in strong negative impacts on the environment, such as nitrate pollution and eutrophication. Corresponding author: najibelkhodrani@gmail.com © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). E3S Web of Conferences 150, 010 0 6 (20 20) EDE7-2019 https://doi.org/10.1051/e3sconf/20201500100 6 The objective of this work is to evaluate and compare the groundwater pollution of two zones of M'nasra and Sfafaa (plain of Gharb) by nitrates. This study will subsequently raise the awareness of leaders to implement a plan of action to minimize the vulnerability of groundwater in Morocco. Water samples were collected in 1l clean plastic bottles and stored in the freezer (-10°C) until their analysis. Geology Concerning the pedo-landscape, our study zone is a part of the Vertisol domain of the Gharb region, with alluvial deposits from the Quaternary, mainly of the Late Pleistocene and Holocene. In general, the Gharb plain is a depression between two major structural domains: the Primary Meseta to the South and the Rif mountains to the North and the East. This plain is mainly marked by geological subsidence of the Middle and Late Miocene. The Gharb load is contemporary to the Rif mountains uprising, which caused enormous landslides. The East of the Gharb plain, corresponding to our area of study, is an accumulation zone of Quaternary continental sediments, alternately coarse (cobble and gravel) and fine (silts and clays), following climate cycles. The distribution of these deposits is very heterogeneous both laterally and vertically. Description of the study area Geographic location The surface area of the Gharb region is 600 Km2. The region of M’nasra is dominated by sandy and clayey soils. The climate is Mediterranean with annual precipitations ranging between 480 and 600 mm, and the average temperature is 27°C in summer and 13°C in winter (Office Régional de Mise en Valeur Agricole du Gharb, 2017). Eighteen water samples were collected from 18 wells (less than 50 m depth) installed in 19 sites located on the coastal zone of M’nasra in Gharb region, Northwest of Morocco (Figure. 1). The levy has been carried out in the periods the summer during 2017. The Sfafaa zone belongs to the Province of Sidi Slimane, located around the geographic coordinates 34° 15′0'' and 6° 9′36''. It is limited to the North by the Province of Sidi Kacem , in the South-east by the rural commune of Boumaiz (Province of Sidi Slimane), and to the West by the rural commune of Kecybia (Province of Sidi Slimane). The zone of Sfafaa extends over approximately 197km2. The samples of water wells were collected during the summer2014.The mean annual precipitation of 386.4 mm. We have prospected 17 wells, belonging to the Sfafaa zone. (a) Piezometric map of the M'nasra zone. 2 E3S Web of Conferences 150, 010 0 6 (20 20) EDE7-2019 https://doi.org/10.1051/e3sconf/20201500100 6 (b) Piezometric map of the Sfafaa zone. Fig. 1. Piezometric maps of the two regions chosen for the study. The main crops groun in the study area are field crops, vegetable crops (potatoes, tomatoes, etc) wheat etc (M'nasra and Sfafaa). Table 1 summarizes the main crops and fertilizer doses applied at the national level [4]. Table 1. Applied Fertilizer Doses for Main Crops Crops Area (Ha) IRRIGATED * Sugar Crops 33.423 7.400 + Sugar Cane + Sugar Beet * Cereals 5.200 2.200 5.556 * Vegetable 8.265 Quantity of fertilizer (Kg/Ha) P2O5 K2O N Frequency (%) * N P2O5 K2O 5.853,82 2.949,87 3.274,3 175-200 155 67-97 55 126 42-56 35-102 63 21-28 100 - 910-1.040 341 372,2-539 286 277,2 233,4-311,1 182-530,5 138,6 116,6-155,7 216,8-556,8 805-1.631,4 259,8-641,6 99 111,2-138,8 283,4-396,7 136-693,3 162,4-227,4 46,2 97,5-192,4 428-623,5 293-951,6 113,7-251,7 23 153,9-188,1 1.263,1-1.977,1 878,7 103,6-253,3 86,35-103,6 933,6 180,4-184,2 + Industrial tomato +Potato + Strawberry +Melon -Watermelon + Niora 1.417 2.719 1.624 1.650 8.55 153-400 296-600 160-400 60 130-160 200-250 50-255 100-140 28 114-225 288-440 110-350 70-155 14 180-220 100 - *Peanut * Citrus 10.283 1.919 115-180 54-132 0-80 45-54 0-85 94-96 - RAINFED 99.252 * Sugar Crops 2.075 155 126 63 100 * Cereals * Leguminous 59.067 7.744 60-67 14 28-42 28 14-21 14 60 10 * Forages * Vegetable (Melon) * Sunflower * Olive tree 9.376 6.092 6.847 8.051 60 60 60 14 28 28 28 28 14 14 14 14 30 60 60 10 Total Area Total fertilizer quantity (Tonne) 132.675 (%)*: Frequency of use by farmers. 3 3.227,4 1.841,64 907,24 321,6 261,45 130,72 2.126,4-2.374,5 992,3-1.488,5 496,2-744,2 10,85 21,7 10,85 168 219,3 246 11,2 78,4 102,34 114,8 22,54 39,2 51,17 57,4 11,2 9.131,22 4.791,51 4.181,54 E3S Web of Conferences 150, 010 0 6 (20 20) EDE7-2019 https://doi.org/10.1051/e3sconf/20201500100 6 2 Material and Methods 2.1 Characteristics of groundwater sampling points The present study is conducted on a network of 35 stations spread over two regions in the Gharb Plain M'nasra (Kenitra) and Sfafaa (Sidi slimane) (Figure 2). The stations were chosen according to their representativity in relation to the urban perimeter of the two cities, agricultural zones (M'nasra and Sfafaa) and their proximity to the sources of pollution (industrial zones or Ouled Berjal waste discharge). The geographical distribution is as follows: - PM12, PM13, PM14 and PM15 stations located in the North East of the M'nasra zone (upstream of Sebou). - PS1, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10 and PS11 stations located in the center of the Sfafaa zone. - PS12, PS13 and PS14 stations located upstream of Oued Bhet (Sidi Slimane). - PS15, PS16 and PS17 stations downstream of Oued Bhet (Sidi Slimane). -PM1, PM2, PM3, PM4, PM5, PM6, PM11 and PM16 stations located between the Sebou River and the Atlantic Ocean. - Stations PM7, PM8, PM9, PM10, PM11, PM17, PM18 located after the public landfills (Ouled Berjal). Fig. 2. Map of the M’nasra and Sfafaa zones with the sampling points. 4 located E3S Web of Conferences 150, 010 0 6 (20 20) EDE7-2019 https://doi.org/10.1051/e3sconf/20201500100 6 2.2 Sampling and laboratory analysis retains. The PCA was performed in this study using SPSS 20. The measurement of electrical conductivity (EC) and hydrogen ion concentration (pH) were performed in situ using the Jenway 3510 pH-meter. Chloride (Cl-) was measured by the titrimetric method. Nitrates (NO3-) was determined using Jasco V-530 UV/VIS spectrophotometer. Sodium (Na+) was determined using the Jenway Clinical PFP 7 flame photometer and oxidizability by KMnO4.Those measurements were performed according to the AFNOR standards [5]. 3 Results and discussion 3.1 Parameters Physicochemical Human activities exercise much polluting pressures on surface water, then deferred to the water table. This deterioration in the quality of certain nitrate wells in the aquifer could be attributed to point and dispersal releases of livestock products and wastewater that has not been previously treated. Certain factors seem to favor this contamination such as the shallow depth, the permeability of the soil, the speed of infiltration, the proximity of public landfills, industrial zones, etc. Deep aquifers indirectly bound to the soil surface are therefore better protected against nitrate leaching, in contrast to shallow aquifers, which are more vulnerable because of their shallow depth which facilitates contact between leached nitrates and groundwater [8]. Contamination of the water table could cause many diseases in the population using groundwater as a source of drinking water. The effects on human health of acute poisoning by nitrates are now known, which is not the case of long-term effects. 2.3 Statistical analysis First of all, a matrix of Pearson correlation coefficients between any two variables was computed and the coefficients were tested for their statistical significance. We then used Principal Component Analysis [6], a multivariate analysis technique that provides an excellent means for gaining useful information from data sets with many variables [7]. In particular, PCA can aid in the compression and classifcation of data. The purpose is to reduce the dimensionality of a data set by finding a new set of variables, smaller than the original set of variables, which nonetheless retains most of the sample’s variance. Success relies on the presence of correlations among at least some of the original variables; otherwise, the number of new variables will be almost the same as the number of original variables. The new variables, called principal components, are uncorrelated, and are ordered by the fraction of the total variance each The results obtained showed that the pH varies between 7.4 and 8.1 for the aquifer of the M'nasra zone, whereas for the 17 wells in the Sfafaa zone, the pH admits a minimum value of 6.6 and 7.4 as the maximum value. The pH values thus measured for the two zones chosen for the study are safe for the consumer (Figure 3). Fig. 3. Variation of pH values. Electrical conductivity is used to evaluate the rate of mineralization of the environment. The higher the conductivity, the more water is strongly mineralized. The results of this study show that some wells in the Sfafaa zone (PS16, PS15 and PS12) are highly mineralized. The values are 5 E3S Web of Conferences 150, 010 0 6 (20 20) EDE7-2019 https://doi.org/10.1051/e3sconf/20201500100 6 between 370 and 6110 μS / cm. In addition, the groundwater conductivity at the M'nasra zone shows a maximum value of 1269.5 μS / cm. At the Sfafaa zone, well water is mainly used for irrigation in the region. This is accompanied by a high salinity, in particular the PS16 well which has a concentration of 1184.62 mg / l in NaCl (Figure 4) while the other wells have concentrations varying between 33.93 and 477.95 mg / l. In addition, the salinity for the wells in the M'nasra zone varies between 53.31 and 319.4 mg / l. Fig. 4. Variation in the concentration of NaCl. ingestion, could increase the risk of cancer and fetal malformation (teratogenicity) in humans [1011].The results obtained for the nitrate analysis for the two study zones showed that the concentrations for 18 sampling points in the M'nasra vary between 49.6 and 198.4 mg / l whereas those detected at the wells of the Sfafaa zone range from 0 to 40.9 mg / l (Figure 5). 3.2 Nitrate pollution Domestic and urban discharges contribute locally to nitrate contamination of aquifers, but fertilization by the application of chemical or organic nitrogen fertilizers and intensive animal husbandry (animal waste) constitute the main cause. Excess nitrates in soil are driven every year a little deeper, mainly by winter rains. The rate of migration of nitrates to the aquifers is slow, on average 1 to 2 m / year, but varies according to the nature of the subsoil. The excess of nitrogen spread on the surface therefore takes on average 10 to 20 years to reach a water table 20 m deep [9]. On the surface of the soil, organic and mineral elements alter cycles of water, carbon and nitrogen on a global scale. These transformations involve multiple risks to human health, aquatic spaces, biodiversity and agricultural productivity. The challenge for agriculture is therefore to produce more respectfully less using water resources, with respect for biodiversity and greenhouse gas emissions [10]. For the M'nasra zone, it is noted that the wells near the Ouled Berjal landfill and the Kenitra industrial zone are loaded with nitrates with values that go well beyond drinking water standards. However, all the wells in the Sfafaa zone, far from pollution sources, have nitrate concentrations that are below the WHO standard (50 mg / l) [10]. However, in the highly fissured geological formations, the rains reach the aquifer in a few hours to a few months, this is the case of the M'nasra zone whose soil has cracks, whereas in the less permeable formations it is will take several tens or even hundreds of years. Nitrates and nitrites are not carcinogenic if they do not exceed WHO standards, but their possible transformation into N-nitroso compounds in water or food before ingestion, or in the body after 6 E3S Web of Conferences 150, 010 0 6 (20 20) EDE7-2019 https://doi.org/10.1051/e3sconf/20201500100 6 Nitrogen inputs, if they are higher than plant exports, represent surpluses likely to flow to rivers and aquifers. In 2010, nitrogen surpluses ranged from 5 to 145 kg / ha of utilized agricultural area (UAA), with an average of 32 kg / ha [4]. In addition, it has been shown that the risk of pollution of the Tadla water table is aggravated, for vegetable crops, by the regular application of manure at doses up to 80 T / ha [12]. This shows that the mismanagement of fertilizer products can contribute to the increase in groundwater pollution and subsequently the infiltration of soluble substances not retained by vegetation: excess cases in relation to its needs. The regions most affected by nitrogen pollution were those of intensive farming with a high load of organic nitrogen due to animal effluents, those of heavily fertilized field crops and those with shallow water resources (basement areas with low permeability basements, alluvial aquifers, karst aquifers) [13]. Fig. 5 . Variation of nitrate concentration. Although, the water table in the M'nasra zone has higher levels compared to the Sfafaa zone, the nitrate pollution is high this may be due to contamination by the largely developed industrial activities in the city of Kenitra, but also by leachates from the city's uncontrolled Ouled Berjal landfill[14]. deficit. The city of Larache landfill is a typical model of deterioration of water resources through a leachate contamination from the landfill and agricultural practices [19]. The results obtained on the pollution of the water table of the city of Taza showed a more or less strong degradation especially downstream of the city discharge [20]. The analysis of ground and surface waters revealed that nitrate ions have a mean concentration of less than 50 mg / l with temporal variations of remarkable amplitude (standard suggested by WHO) [14,21]. The vulnerability of groundwater depends mainly on the countries and regions because in reality the essential variable to be considered is the renewable resource and, that is to say the volume that recharges the groundwater each year [15]. In addition, the results of the physicochemical analysis in the leachate of several discharges have shown a high contamination of organic salts and heavy metals at Egypt [16]. In addition, it has been shown that in Romania landfills can represent potential sources of pollution for surface water and groundwater [17, 18]. It is for this reason that the development of new disposal areas is of utmost importance. Leachate percolation from landfill waste is a source of pollution of groundwater resources by infiltration, thus constituting a threat as important as that related to the increasing water Organic matter by oxidizability by KMnO4 This parameter is used to evaluate the organic matter content in the well water. In fact, the organic matter present in the wells varies considerably from one well to another. In the well waters of M'nasra zone, it varies between 2.2 and 10.6 mg / l whereas those in the Sfafaa zone have a minimum of 0 mg / l and a maximum of 9.9 (Figure 6). However, these values show that 57.1% of well water cannot be used as a source of drinking water. 7 E3S Web of Conferences 150, 010 0 6 (20 20) EDE7-2019 https://doi.org/10.1051/e3sconf/20201500100 6 Fig. 6 . Variation of Organic Matter. 3.3 Multivariate statistical analysis of physicochemical parameters Physicochemical parameters (variables) and water points (individuals) were projected separately in the factorial plane (PC1xPC2) of the principal component analysis (PCA) to better understand their representative distributions with respect to the factorial axes. The interpretation of the statistical analysis by the PCA has been done only on two axes PC1 and PC2 because they alone contain more than 70% of the information. PC1 axis presents 47.8% and the PC2 axis presents 23.4% of the total inertia (Figure 7). The PCA allowed us to characterize 2 groups of chemical elements each corresponding to a set of groundwater samples with the same characteristics. A C B Fig. 7 .Loading plot for the principal component analysis: groundwater properties in the PC1–PC2 plan. Groups A: This group consists essentially of NaCl and the electrical conductivity (EC) opposed to pH and nitrate along the PC1 axis. These parameters correspond to the set of wells consisting of PS12, PS15 and PS16 (Group 1) and which has a very 8 E3S Web of Conferences 150, 010 0 6 (20 20) EDE7-2019 https://doi.org/10.1051/e3sconf/20201500100 6 high conductivity and salinity and very low pH, nitrates (Figure 7). This group is formed by organic matter. It is placed in negative position on the PC2 axis. This setting characterizes the PS1, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11, PS13, PS14, and PS17 wells (Group 2). Groups B: 1 2 Fig. 8 . Score plot for the principal component analysis: water samples in the PC1–PC2 plan. In order to avoid ecological and health problems, it is necessary to take concrete measures to limit the pollution of the Gharb water table (M'nasra) and to protect the health of the population in rural areas. 4. Conclusion The results obtained have shown that parameters such as salinity, conductivity, and nitrates far exceed drinking water standards. The concentration of nitrates in the well waters of the M'nasra zone varies around 150 mg / l whereas those detected in the wells of the Sfafaa zone are close to 20 mg / l. Based on investigations and analysis, some suggestions for solving the problem, such as best management practices for nitrogen fertiliser and improving environmental awareness, are discussed. Groundwater in the M'nasra zone is more contaminated with nitrates than Sfafaa. This pollution detected at the level of the wells is mainly due to their situations in relation to the landfill and also in relation to the industry. In addition, the waters of the Sfafaa zone are more saline compared to the waters of M'nasra, especially the PS16 point which is downstream of Oued Beht. To avoid pollution of the water table, it is necessary to strike a balance between the misuse of nitrates sources and the total prohibition of them. With regard to organic matter determined by KMnO4 oxidability, 57.1% of well water cannot be used as a source of drinking water. References 1. 2. 3. 4. 5. 9 M. Laaouan , M.A.Aboulhassan , S. Bengamra, A.Taleb , S. Souabi , M. Tahiri , J. Mater. Environ. Sci. 7 ,4 (2016) 1298-1309 B. Marouane , A. Dahchour ,S. Dousset ,S. El Hajjaji , Mor. J. Chem. 2 ,4 (2014) 375-382 J. Berbel , J. Calatrava , A. Garrido , The Gap Between Theory and Practice (Eds: F. Molle, J. Berkoff ), Biddles Ltd, King’s Lynn press, UK (2007), p. 295 M. Achkar , Rationalisation de l'utilisation des engrais et pesticides au Maroc, Revue H.T.E, 123 (2002) 5 F. Laurent, Pollutions des ressources en eau et agriculture : l'ouest de la France. In Conférence E3S Web of Conferences 150, 010 0 6 (20 20) EDE7-2019 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. https://doi.org/10.1051/e3sconf/20201500100 6 du Monde Diplomatique "L'eau, source de vie, source de conflits", Le Mans, Presses Universitaires de Rennes, (2005) 143-150 Joliffe, I.T. Principal component analysis. 2nd ed. Springer, New York (2002). H. Vereecken, and M. Herbst. Statistical regression. Dev. Soil Sci.30 (2004) 3–18. E. Hassoune , A. Bouzidi,Y. Koulali , D. Hadarbach , Bulletin de l’Institut Scientifique, Rabat, section Sciences de la Vie, 28 (2006) 61-71 Commissariat Général au Développement Durable., L'eau, source de vie, source de conflits, Conférence du Monde Diplomatique, (http://www.developpementdurable.gouv.fr/ IMG/pdf/LPS161. pdf, 161(2013) 2-4 World Health Organization., Guidelines for drinking water quality, 2nd Recommendations. Genève, (1993) P. Levallois, D. Phaneuf , La contamination de l'eau potable par les nitrates : analyse des risques à la santé, Revue canadienne de santé publique, 85, 3(1994) 192-196 H. Berdai, B. Soudi, A. Bellouti , Contribution à l'étude de la pollution nitrique des eaux souterraines en zones irriguées, Revue HTE, 128 (2004) 65-87 F. Laurent, Modélisation des processus et analyse des dynamiques territoriales. Habilitation à Diriger des Recherches en Géographie UMR ESO (Espaces et Sociétés) Université du Maine : Agriculture et pollution de l’eau, (2012) 1-186 S. Omrania, N. El Khodrani , A. Zouahri , A. Douaik , H. Iaaich , M. Lahmar , and S. El Hajjaji,. Materials Today: Proceedings 13(2019) 1092-1101. I. Giroux , Contamination de l’eau souterraine par les pesticides et par les nitrates dans les régions en culture de pommes de terre, Campagne d’échantillonnage de 1999-20002001 Ministère de l’Environnement Gouvernement du Québec, (2003) M. Magda , M. Abd El-Salam , I. Gaber , G. Abu-Zuid , Journal of Advanced Research, 6 (2015) 579-586 P. Casen, Agriculture and Agricultural Science Procedia; 6(2015) 500-505 A-M. Şchiopu, B.M. Robu , I. Apostol , M. Gavrilescu ,. Environmental Engineering and Management Journal. 8 , 5 (2009) 1155-1164 H. Er-Raioui , S. Bouzid , S. Khannous , M.A. Zouag , Int. J. Biol. Chem. Sci. 5,3(2011) 1118-1134 M. Ben Abbou , M. El Haji , M. Zemzami , F. Fadil F., Afrique SCIENCE 10 ,1 (2014) 171. 21. X. Huang , H. Deng , C. Zheng , G. Cao,. Science of the Total Environment.543 (2016) 357-372. 10 

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International Electronic Journal of Algebra Volume 27 (2020) 218-236 DOI: 10.24330/ieja.663075 INDECOMPOSABLE NON UNISERIAL MODULES OF LENGTH THREE Gabriella D’Este Received: 2 May 2019; Revised: 8 October 2019; Accepted: 21 October 2019 Communicated by Christian Lomp Abstract. We investigate a particular class of indecomposable modules of length three, defined over a K–algebra, with a simple socle and two non isomorphic simple factor modules. These modules may have any projective dimension different from zero. On the other hand their composition factors may have any countable dimension as vector spaces over the underlying field K. Moreover their endomorphism rings are K–vector spaces of dimension ≤ 2. Mathematics Subject Classification (2010): 16G20, 16D99 Keywords: Pseudo projective module, quasi projective module, quiver and representation 1. Introduction The aim of this note is to construct left R–modules M , where R is a K–algebra over some field K, with the following properties: (1) M has exactly three non zero proper submodules N1 , N2 and N1 ∩ N2 . (2) M /N1 is not isomorphic to M /N2 . (3) End M /Ni is isomorphic to K for i = 1, 2. Given a module M , we say that M is quasi projective (resp. pseudo projective) if, for any submodule X of M , any morphism (resp. any epimorphism) f ∶ M → M /X is of the form π ○ g, where π ∶ M → M /X is the canonical epimorphism and g is an endomorphism of M . By [4, Lemma 2.1] any module M satisfying (1) and (2) is not quasi projective. Moreover, if (1) and (2) holds, then M is pseudo projective if and only if it satisfies (3) with K = Z2 . In the sequel we shall say, for short, that M satisfies (123) if M satisfies conditions (1), (2) and (3). If x is a vertex of a quiver Q, and R is the K–algebra given by Q, then x also denotes the simple module (Vi , fj ), with Vx = K, Vi = 0 for i ≠ x and fj = 0 for any j. Moreover, P (x) and I(x) denote the projective and injective module corresponding to the vertex x. In the examples constructed in [4] R is always a K–algebra given by quivers with at least 2 vertices and at most 4 vertices, and the modules satisfying (123) are three dimensional INDECOMPOSABLE NON UNISERIAL MODULES OF LENGTH THREE 219 vector spaces. In this note we consider algebras given by quivers with n vertices for any n ≥ 1. By making one point extensions or coextensions, or by changing the relations, we obtain modules satisfying (123) of any injective dimension (Proposition 2.1) and of any projective dimension ≥ 1 (Proposition 2.2). We also show that modules satisfying (123) are very far from being selforthogonal (Examples 2.3, 2.4 and 2.5), and that they cannot be faithfully balanced (Proposition 3.8). Moreover their endomorphism ring is a rather small commutative algebra (Theorem 3.6). As we shall see, the free algebra K < x, y > admits modules of dimension three satisfying (123) (Example 2.5, Proposition 2.6, Corollaries 3.10 and 3.11). On the other hand, the free algebra K < x, y, z > admits modules satisfying (123) of any countable dimension ≥ 3 (Theorem 3.2). Finally, suitable matrix algebras admit modules satisfying (123) of any infinite dimension over K (Proposition 3.4 and Corollary 3.5). This paper is organized as follows. In Section 1 we recall some definitions, we fix some conventions and we describe the main results. In Section 2 we investigate homological dimensions and extensions of modules satisfying (123). Finally, in Section 3, we describe composition factors and endomorphism rings of these modules. For more background on generalizations of projective modules we refer to [3] and [5]. For more background on quivers and their representations we refer to [1] and [2]. 2. Homological dimensions and extensions We begin with two results on homological dimensions. Proposition 2.1. Any d ∈ N ∪ {∞} is the injective dimension of a module M satisfying (123). Proof. If d = 0, 1, ∞, then the assertion follows from [4, Example 2.3 and Theorem 2.8]. Assume now 2 ≤ d < ∞. Let n = d + 3 and let R be the K-algebra given by the 220 GABRIELLA D’ESTE following quiver with relations ab = 0 for all arrows a and b. ● n  ● ● ● ● 1 2 4    ● 3 Next let M be the module 0 / M 12 3 . Then the injective coresolution of M is / 5 / 124 3 4 / ... Hence n − 3 = d is the injective dimension of M . / n n−1 / n / 0 .  Proposition 2.2. Any d with 1 ≤ d ≤ ∞ is the projective dimension of a module M satisfying (123). Proof. The modules 12 3 and 12 2 in [4, Examples 2.3 and 2.4] have projective dimension 1 and ∞, respectively. Assume now 1 < d < ∞ and let n = d + 2. Next let R be the K–algebra given by the following quiver with relations ab = 0 for all INDECOMPOSABLE NON UNISERIAL MODULES OF LENGTH THREE 221 arrows a and b. 1 2 ● ●  3  ●  4 ● ● n Let M be the module I(3) = / n−1 / n 0 12 3 . Then the projective resolution of M is / ... n / 3 4 / M / 1 ⊕ 2 3 3 / 0 . Consequently the projective dimension of M is n − 2 = d.  Propositions 2.1 and 2.2 suggest that we investigate the groups of selfextensions of the modules satisfying (123). Example 2.3. There is a module M satisfying (123) such that Extn (M, M ) ≃ K for any n ≥ 1. Construction. As in [4, Theorem 2.8], let R be the K–algebra given by the quiver a ● 1 / ● d b with relations ba = 0 and b3 = 0. Next let M be the module 2 Then I(1) = 1 and I(2) = 12 2 . 2 12 are the indecomposable injective modules. From 2 the exact sequence 0 → M → I(2) → 2 → 0 we obtain the long exact sequence 0 0 Ð→ Hom(M, M ) Ð→ Hom(M, I(2)) Ð→ Hom(M, 2) Ð→ Ext1 (M, M ) Ð→ 0 Ð→ ≃ ≃ . . . Ð→ 0 Ð→ Exti (M, 2) Ð→ Exti+1 (M, M ) Ð→ 0 . . . . . . . Consequently we have Ext1 (M, M ) ≃ Hom(M, 2) ≃ K (1) and Extn+1 (M, M ) ≃ Extn (M, 2) for any n ≥ 1. (2) 222 GABRIELLA D’ESTE 2 On the other hand, from the exact sequence 0 Ð→ 2 Ð→ I(2) Ð→ 1 ⊕ 2 Ð→ 0, we obtain the long exact sequence 0 Ð→ Hom(M, 2) Ð→ Hom(M, I(2)) Ð→ 2 Hom (M, 1 ⊕ ) Ð→ Ext1 (M, 2) Ð→ 0 . . . Ð→ 0 Ð→ Exti (M, 1 ⊕ 2 2 2 ) Ð→ Exti+1 (M, 2) Ð→ 0 Ð→ . . . . Hence, comparing dimensions and using the fact that 1 is an injective module, we have Ext1 (M, 2) ≃ K (3) and Extn+1 (M, 2) ≃ Extn (M, 2 2 ) for any n ≥ 1. (4) Combining (2) and (3), we get Ext2 (M, M ) ≃ K. Finally, from the exact sequence 0 Ð→ 2 2 the long exact sequence 0 Ð→ Hom (M, 2) Ð→ Ext1 (M, 2 2 (5) Ð→ I(2) Ð→ 1 ⊕ 2 Ð→ 0, we obtain 2 2 ) Ð→ Hom(M, I(2)) Ð→ Hom(M, 1 ⊕ ) Ð→ 0 Ð→ . . . Ð→ 0 Ð→ Exti (M, 1 ⊕ 2) Ð→ Exti+1 (M, 2 2 ) Ð→ 0 → . . . . Therefore we have Ext1 (M, 2 2 )≃K (6) and Extn+1 (M, 2 2 ) ≃ Extn (M, 2) for any n ≥ 1. (7) Putting (2), (4) and (6) together, we get Ext3 (M, M ) ≃ K. (8) Combining (4) and (7), we obtain Exti+2 (M, 2) ≃ Exti (M, 2) for any i ≥ 1. (9) This result and (2) imply that Exti+3 (M, M ) ≃ Exti+1 (M, M ) for any i ≥ 1. (10) Putting (1), (5), (8) and (10) together, we conclude that Extn (M, M ) ≃ K for any n ≥ 1.  As the next examples show, we may have Ext1 (M, M ) ≠ 0 also when M is defined over hereditary algebras. INDECOMPOSABLE NON UNISERIAL MODULES OF LENGTH THREE 223 Example 2.4. There is a module M satisfying (123) such that dim Ext1 (M, M ) = 2. Construction. Let R be the infinite dimensional K–algebra given by the quiver a : ● 1 b / ● . Let M be the module described by the following picture 2 8 a v1 v2 ● ● b   b ● v3 where {v1 , v2 , v3 } is a basis of M and av1 = v1 , av2 = 0, bv1 = v3 = bv2 . Then Rv1 , Rv2 and Rv1 ∩ Rv2 = Rv3 are the three non zero proper submodules of M . On the other hand the three composition factors of M are one dimensional. Let U1 = M /Rv1 an let U2 = M /Rv2 . Then aU1 = 0 and aU2 ≠ 0. Hence M satisfies (123). From the exact sequence 0 Ð→ Rv3 ≃ P (2) Ð→ M Ð→ U1 ⊕ U2 Ð→ 0 (1) we get the long exact sequence 0 0 = Hom(U1 ⊕ U2 , M ) Ð→ Hom(M, M ) Ð→ Hom(Rv3 , M ) Ð→ ≃ Ð→ Ext1 (U1 ⊕ U2 , M ) Ð→ Ext1 (M, M ) Ð→ 0. (2) ≃ Let e1 be the path of length zero around the vertex 1. Then the definition of U1 and U2 guarantees the existence of epimorphisms f ∶ P (1) Ð→ U1 and g ∶ P (1) Ð→ U2 such that Ker f = Ra ⊕ Rb and Ker g = R(e1 − a) ⊕ Rb. Consequently, there are exact sequences of the form 0 Ð→ P (1) ⊕ P (2) Ð→ P (1) Ð→ Ui Ð→ 0 for i = 1, 2. (3) Hence also the following long sequences are exact 0 = Hom(Ui , M ) Ð→ Hom(P (1), M ) Ð→ Hom(P (1) ⊕ P (2), M ) Ð→ Ð→ Ext1 (Ui , M ) Ð→ 0 for i = 1, 2. (4) Since Hom(P (1), M ) ≃ K 2 and Hom(P (1) ⊕ P (2), M ) ≃ K 3 , we deduce from (4) that Ext1 (Ui , M ) ≃ K for i = 1, 2. Consequently we deduce from (2) that dim Ext1 (M, M ) = 2. In the next example we use a quiver with one vertex.  224 GABRIELLA D’ESTE Example 2.5. The free algebra A = K < x, y > admits a module M satisfying (123) such that dim Ext1 (M, M ) = 10. Construction. Let M be the A–module described by the following picture x 8 v1 v2 ● ● y f y x   ● v3 More precisely, let {v1 , v2 , v3 } be a basis of M and assume that xv1 = v1 , yv2 = v2 , yv1 = v3 = xv2 and xv3 = 0 = yv3 . Also in this case Av1 , Av2 and Av1 ∩ Av2 = Av3 are the three non zero proper submodules of M . Moreover the three composition factors S1 = Av1 /Av3 , S2 = Av2 /Av3 and S3 = Av3 are pairwise non isomorphic. Hence M satisfies (123). From the exact sequence 0 Ð→ A2 Ð→ A Ð→ Si Ð→ 0 for i = 1, 2, 3, (1) we obtain the long exact sequences 0 Ð→ Hom(Si , M ) Ð→ Hom(A, M ) Ð→ Hom(A2 , M ) Ð→ Ð→ Ext1 (Si , M ) Ð→ 0 for any i. Since Hom(Si , M ) = 0 for i = 1, 2 and Hom(S3 , M ) ≃ K, we obtain dim Ext1 (Si , M ) = 3 for i = 1, 2 (2) and dim Ext1 (S3 , M ) = 4. (3) Since the sequence 0 Ð→ S3 Ð→ M Ð→ S1 ⊕ S2 Ð→ 0 is exact and A is hereditary, we obtain the long exact sequence 0 = Hom(S1 ⊕ S2 , M ) Ð→ Hom(M, M ) Ð→ Hom(S3 , M ) Ð→ ≃ Ð→ Ext1 (S1 ⊕ S2 , M ) Ð→ Ext1 (M, M ) Ð→ Ext1 (S3 , M ) Ð→ 0. (4) Putting (2), (3) and (4) together, we conclude that dim Ext1 (M, M ) = 10.  In the next statement we compare the dimensions of certain subspaces of small modules satisfying (123). Proposition 2.6. Let A be the free K–algebra K ⟨x, y⟩, and let M be a module satisfying (123) with dim M = 3. Then, among others, the following cases are possible: INDECOMPOSABLE NON UNISERIAL MODULES OF LENGTH THREE 225 (i) dim xM = dim yM = 2; (ii) dim xM = dim yM = 1; (iii) dim xM = 2, dim yM = 1. Proof. (i) This follows from Example 2.5, where we have xM = ⟨v1 , v3 ⟩ and yM = ⟨v2 , v3 ⟩. (ii) Let M be the A–module, obtained in an obvious way from Example 2.4, with basis {v1 , v2 , v3 } described by the following picture. 8 x v1 v2 ● ● y y   ● v3 Then we have xM = ⟨v1 ⟩ and yM = ⟨v3 ⟩. (iii) Let M be the A–module with basis {v1 , v2 , v3 } described by the following picture. 8 x v1 v2 ● ● y x   ● v3 then we have xM = ⟨v1 , v3 ⟩ and yM = ⟨v3 ⟩.  3. Composition factors and endomorphism rings In the sequel we denote by N+ the set of positive integers. In order to construct modules satisfying (123) of infinite dimension, we need the following lemma. Lemma 3.1. Let A = K ⟨x, y⟩. Then for any d ∈ N+ ∪{ℵ0 } there is a simple module S with dim S = d and End S ≃ K. Proof. If d = 1, the assertion is obvious. Assume d is finite and d > 1. Let S be the module with basis {v1 , . . . , vd } described in an obvious way by the following picture. v1 ● d x / v2 ● ● y x / vd ● 226 GABRIELLA D’ESTE If 0 ≠ v = ∑di=1 ki vi , then we have yxi v = kd−i v1 for i = 0, . . . , d − 1. Consequently Av = Av1 = S. Hence S is a simple module of dimension d. Assume now f ∈ End S and f (v1 ) = ∑di=1 ki vi . Since yxi v1 = 0 for i = 0, . . . , d−2, we have kd−i v1 = yxi f (v1 ) = f (yxi v1 ) = 0 for i = 0, . . . , d − 2. Hence kd = kd−1 = ⋅ ⋅ ⋅ = k2 = 0, and so f is the multiplication by k1 . Assume d = ℵ0 . Let S be the module with basis {vn ∣n ≥ 1} described in obvious way by the following picture. x y v1 x ) i ● ● v2 x ) i y i ● y v3 ) ● v4 If 0 ≠ v = ∑ni=1 ki vi with kn ≠ 0, then we have y n−1 v = kn v1 . Consequently Av = Av1 = S, and so S is a simple module of dimension ℵ0 . Assume now f ∈ End S and f (v1 ) = ∑ni=1 ki vi for some n ≥ 2. Since yv1 = 0, it follows that ∑n−1 i=1 ki+1 vi = yf (v1 ) = f (yv1 ) = 0. Hence we have k2 = ⋅ ⋅ ⋅ = kn = 0, and so f is the multiplication by k1 . The lemma is proved.  By dealing with reasonably large free K–algebras it is easy to construct big modules satisfying (123). Theorem 3.2. If di ∈ N+ ∪ {ℵ0 } for i = 1, 2, 3 and A = K ⟨x, y, z⟩, then there is an A–module M , satisfying (123), such that the three composition factors of M have dimensions d1 , d2 , d3 and d3 = dim soc M . Proof. Let M be the A–module with basis B1 ∪ B2 ∪ B3 with ∣Bi ∣ = di for any i described by the following picture. ● v1 ● i ● y u1 ● i z y z  ● ω1 ● i y More precisely we have B1 = {vi }i≥1 , B2 = {ui }i≥1 , B3 = {ωi }i≥1 and the following conditions hold. If d1 = 1, then yv1 = v1 , xv1 = 0 and zv1 = ω1 . If d1 > 1, then B1 generates the K ⟨x, y⟩–module of dimension d1 constructed in Lemma 3.1, and we have zv1 = ω1 and zvi = 0 for any i > 1. If d2 = 1, then zu1 = u1 , xu1 = 0 and yu1 = ω1 . If d2 > 1, then B2 generates the K ⟨x, z⟩–module of dimension d2 (with z INDECOMPOSABLE NON UNISERIAL MODULES OF LENGTH THREE 227 instead of y) constructed in Lemma 3.1, and we have yu1 = ω1 and yui = 0 for any i > 1. If d3 = 1, then yω1 = ω1 and xω1 = 0 = zω1 . If d3 > 1, then B3 generates the K ⟨x, y⟩–module of dimension d3 constructed in Lemma 3.1, and we have zωi = 0 for any i. Let now N1 = Av1 , N2 = Au1 . Then we have N1 ∩ N2 = Aω1 , and so dim N1 ∩N2 = d3 . On the other hand dim M /N1 = d2 and dim M /N2 = d1 . Moreover, by Lemma 3.1, K is the endomorphism ring of M /N1 , M /N2 and N1 ∩ N2 . To end the proof, take any 0 ≠ m ∈ M . If m ∈ N1 ∩N2 , then we have Am = Aω1 = N1 ∩N2 . If m ∈/ N1 , then there exists a ∈ A and k ∈ K such that zam = u1 + kω1 . Consequently there is some b ∈ A such that zb(u1 + kω1 ) = u1 , and so N2 = Au1 ⊆ Am. Suppose now m ∈/ N2 . Then there exist a ∈ A and s ∈ N1 ∩ N2 such that yam = v1 + s. Consequently ω1 = zyam ∈ Am, and so N1 ∩ N2 = Aω1 ⊆ Am. It follows that v1 = yam − s ∈ Am; hence N1 = Av1 ⊆ Am. Putting things together, we conclude that Am = N1 if m ∈ N1 ∖ N2 , Am = N2 if m ∈ N2 ∖ N1 , and Am = N1 + N2 = M if m ∈/ N1 ∪ N2 . Hence M satisfies (123) and the proof is complete.  Corollary 3.3. Let R be the K–algebra given by the quiver 1 a1 : ● 2 d c ● d b2 . d  a3 : a2 b1 : 3 ●  d b3 If di ∈ N+ ∪ {ℵ0 } for i = 1, 2, 3, then there is an R–module V = (Vi , fi ) such that V satisfies (123) and dim Vi = di for any i. Proof. By Lemma 3.1 we can define a simple K ⟨x, y⟩–module (Vi ; ai , bi ) with dimension di and endomorphism ring K for any i = 1, 2, 3. Next we fix a non zero map c ∶ V1 Ð→ V3 and a non zero map d ∶ V2 Ð→ V3 . In this way we obtain an R–module V = (V1 , V2 , V3 ; fj ) with the following properties: ● The three non zero proper submodules of V are N1 = (V1 , 0, V3 ; fj ), N2 = (0, V2 , V3 ; fj ) and N1 ∩ N2 = (0, 0, V3 ; fj ). ● V /N1 is not isomorphic to V /N2 . ● End V /Ni is isomorphic to K for i = 1, 2. Consequently V satisfies (123) and dim Vi = di for any i.  By hypothesis, two non isomorphic composition factors belonging to the top of a module satisfying (123) have the smallest possible endomorphism ring. However 228 GABRIELLA D’ESTE it is easy to see that the socle of a module satisfying (123) may have a large endomorphism ring. Proposition 3.4. Let E be an extension field of K with [E ∶ K] > 1. Then there are a K–algebra R and a faithful R–module M , satisfying (123), such that End(soc M ) ≃ E and M is not injective. ⎛K ⎜ Proof. Let R be the matrix algebra ⎜ 0 ⎜ ⎝E 0⎞ ⎟ . Next let P be the left ideal K 0⎟ ⎟ E E⎠ ⎧ ⎫ ⎪ ⎪ ⎛K 0 0 ⎞ ⎛0 0 0⎞RRRR ⎪ ⎪ ⎪ ⎪ R ⎪ ⎪ R ⎪ ⎪ ⎜ ⎟ ⎜ ⎟RR ⎜ 0 K 0⎟, and let Q be the left ideal ⎨ ⎜0 0 0⎟RR a ∈ E ⎬. Finally let M = R ⎜ ⎟ ⎜ ⎟ ⎪ ⎪ RRR ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ RR ⎝ E E 0⎠ ⎝ ⎠ a −a 0 ⎪ ⎪ R ⎩ ⎭ ⎛K 0 0⎞ ⎛ 0 0 0⎞ ⎜ ⎟ ⎜ ⎟ P /Q, L1 = ⎜ 0 0 0⎟, L2 = ⎜ 0 K 0⎟, N1 = L1 /Q and N2 = L2 /Q. Then N1 , ⎜ ⎟ ⎜ ⎟ ⎝ E E 0⎠ ⎝E E 0 ⎠ N2 and N1 ∩ N2 = soc M are the unique proper submodules of M . Moreover M /N1 0 and M /N2 have dimension one over K and we have M /N1 ≃/ M /N2 . Hence M satisfies (123), and we clearly have End(soc M ) ≃ E. Moreover it is easy to check ⎛E 0 0⎞ ⎟ ⎜ that M is a faithful R–module. Finally, let P̃ be the left R–module ⎜ 0 E 0⎟. It ⎟ ⎜ ⎝E E 0⎠ remains to check that M = P /Q is an essential submodule of P̃/Q. To see this, take ⎛x ⎜ any p̃ = ⎜ 0 ⎜ ⎝z 0 y t ⎛0 0⎞ ⎟ ̃ ⎜ ∈ P ∖ P . Then ⎜0 0⎟ ⎟ ⎜ ⎝1 0⎠ 0 0 0 0⎞ ⎟ p̃ = 0⎟ ⎟ 0⎠ ⎛0 ⎜ ⎜0 ⎜ ⎝x ⎛0 0⎞ ⎟ ⎜ and ⎜0 0 0⎟ ⎟ ⎜ ⎝0 0 0⎠ 0 0 0 1 0⎞ ⎟ p̃ = 0⎟ ⎟ 0⎠ ⎛0 0 0 ⎞ ⎟ ⎜ ⎜0 0 0⎟. Consequently R(p̃+Q)∩P /Q ≠ 0, and so P /Q is an essential submodule ⎜ ⎟ ⎝0 y 0 ⎠ of P̃/Q, as claimed.  Corollary 3.5. Any infinite cardinal d is the dimension over K of a faithful module satisfying (123). Proof. If E is an extension field of K with [E ∶ K] = d, then the claim follows from the proof of Proposition 3.4.  As the next result shows, only two K–algebras occur as the endomorphism ring of a module satisfying (123) and defined over a K–algebra. INDECOMPOSABLE NON UNISERIAL MODULES OF LENGTH THREE 229 Theorem 3.6. Let M be a module satisfying (123) and defined over a K–algebra. Let N1 and N2 be the two maximal submodules of M . The following facts hold: (i) If N1 ∩ N2 ≃/ M /Ni for any i, then End M is isomorphic to K. (ii) If N1 ∩ N2 ≃ M /Ni for some i, then End M is isomorphic to K[x]/(x2 ). Proof. Let π ∶ M Ð→ M /(N1 ∩ N2 ) be the canonical morphism. Since N1 ∩ N2 is fully invariant in M , for any f ∈ End M there is a unique morphism f ⋆ ∈ End (M /(N1 ∩ N2 )) such that π ○f = f ⋆ ○π. Let ρ ∶ End M → End (M /(N1 ∩ N2 )) ≃ K ⊕ K be the morphism sending f to f ⋆ for any f . We first note that End (M /(N1 ∩ N2 )) has non trivial idempotents. (1) We next observe that f2 = 0 for every f ∈ Ker ρ. (2) Since M is indecomposable and idempotents lift modulo nil ideals, we conclude that ρ is not surjective. (3) Suppose first N1 ∩ N2 ≃/ M /Ni for any i. In this case ρ is injective. This remark and (3) imply that End M is isomorphic to K. Hence (i) holds. Assume now N1 ∩ N2 ≃ M /Ni for some i. In this case there is some 0 ≠ f ∈ Ker ρ. Since End (M /Ni ) ≃ K, it follows that Ker ρ is the vector space generated by f . Putting (2) and (3) together, we conclude that End M is isomorphic to K[x]/(x2 ). Thus also (ii) holds.  Corollary 3.7. Let R be a K–algebra, let M be a module satisfying (123), and let E = End M . The following facts hold: (i) R is not commutative and E is commutative. (ii) M is a cyclic R–module and M is not a cyclic E–module. Proof. (i) We know from [4, Remark 2.2] that R is not commutative, while we know from Theorem 3.6 that E is commutative. (ii) Let N1 and N2 be the maximal submodules of M . Then any m ∈ M ∖(N1 ∪N2 ) generates M as an R–module. By Theorem 3.6 we have dim E ≤ 2. On the other hand we clearly have dim M ≥ 3. Hence M is not a cyclic E–module.  As usually, we say that a bimodule A XB is faithfully balanced if the canonical morphisms A Ð→ End XB and B Ð→ EndA X, sending any a ∈ A and b ∈ B to the corresponding multiplications, are isomorphisms. 230 GABRIELLA D’ESTE Proposition 3.8. Let R, M, N1 , N2 and E = End M be as in Corollary 3.7. Let T be the algebra of all endomorphisms of M as an E–module. The following facts hold: (i) M is not a faithfully balanced R − E bimodule. (ii) If R is a finite dimensional algebra, then we may have dim T = dim R + 1. Proof. We first note that M is not a simple R–module. (1) Assume first E ≃ K. Then T ≃ EndK (M ) and M is a simple T –module. This remark and (1) prove that M is not faithfully balanced. Assume now E = K[f ] for some endomorphism f of M such that f (N1 ) = 0 and f (N2 ) = N1 ∩ N2 . (2) Let now B be a basis of M of the form B1 ∪ B2 ∪ B3 such that B1 ∪ B3 and B2 ∪ B3 are bases of N1 and N2 respectively, while B3 is a basis of N1 ∩ N2 . Next let s ∶ M Ð→ M be a K–linear map such that s(b) = 0 for any b ∈ B1 ∪ B3 , (3) s(b) ∈ B1 for any b ∈ B2 . (4) and Consequently we deduce from (2), (3) and (4) that (s ○ f )(b) = 0 = (f ○ s)(b) for any b ∈ B. It follows that s ∈ T . On the other hand we deduce from (4) that s(N2 ) ⊆/ N2 . Hence s does not act as the multiplication by an element of R, and so (i) holds. To prove (ii), we choose R and M as in [4, Example 2.4]. More precisely, let R be the K –algebra given by the quiver ● 1 and let M be the module 12 2 a / ● d b with relations ba = 0 and b2 = 0, 2 . We first note that ⎛a ⎜ R is isomorphic to the algebra of all matrices over K of the form ⎜0 ⎜ ⎝c 0⎞ ⎟ . b 0⎟ ⎟ d b⎠ 0 (5) INDECOMPOSABLE NON UNISERIAL MODULES OF LENGTH THREE 231 Consequently E is isomorphic to the algebra of all matrices over K of the form ⎛x 0 0 ⎞ ⎜ ⎟ ⎜ 0 x 0 ⎟. Therefore it is easy to check that ⎜ ⎟ ⎝ 0 y x⎠ ⎛a ⎜ T is isomorphic to the algebra of all matrices over K of the form ⎜0 ⎜ ⎝c 0⎞ ⎟ . b 0⎟ ⎟ ⎠ d b e (6) Hence, by (5) and (6), we have dim R = 4 and dim T = 5. Hence also (ii) holds.  In the next statement we collect some results on dimensions and generators. Proposition 3.9. Let R be a K–algebra and let M be an R–module satisfying (123). The following facts hold: (i) R is generated by at least two elements. (ii) dim R ≥ 4. (iii) There is an algebra B, having a B–module satisfying (123), such that B is generated by two elements and dim B = 4. Proof. (i) This follows from the fact that R is not commutative (Corollary 3.7). (ii) We first note that M is a cyclic R–module which is not projective (Corollary 3.7 and [4, Lemma 2.1]). Consequently either dim R = ∞ or 3 ≤ dim M < dim R < ∞. (iii) Let B be the K–algebra given by the quiver ● a 1 / ● d b with relations 2 ba = 0 and b2 = 0, used to prove condition (ii) of Proposition 3.8. Next let e1 and e2 be the paths of length zero around 1 and 2. Then {e1 , e2 , a, b} is a basis of B, and we clearly have e2 = 1 − e1 , a = (a + b)e1 and b = (a + b)e2 = (a + b)(1 − e1 ). Hence B is generated by e1 and a + b.  The proof of Proposition 2.1 shows that many modules of dimension 3, satisfying (123), are injective modules over suitable algebras. We finally show that also the previous K ⟨x, y⟩–modules of dimension 3 have this property. Corollary 3.10. Let B be the K–algebra used to prove Proposition 3.9 (iii), and let M be the K ⟨x, y⟩–module used to prove Proposition 2.6 (ii) (resp. (iii)). Then M is a faithful injective B–module. 232 GABRIELLA D’ESTE Proof. By hypothesis M is described by the following picture 8 x v1 v2 ● ● y ⎛ ⎜ ⎜ ⎜ ⎜ ⎜ ⎜resp. ⎜ ⎜ ⎜ ⎜ ⎜ ⎝ y   ● v3 8 x v1 v2 ● ● y x   ● v3 ⎞ ⎟ ⎟ ⎟ ⎟ ⎟ ⎟. ⎟ ⎟ ⎟ ⎟ ⎟ ⎠ Let f and g be the endomorphisms of M induced by x and y. Then we clearly have f 2 = f , g 2 = 0, f g = 0 and gf ≠ 0 (resp. f 3 = f 2 , g 2 = 0, f g = 0 and gf = g). On the other hand, by Proposition 3.9, we have dim K ⟨f, g⟩ ≥ 4. Consequently {1, f, g, gf } (resp. {1, f, f 2 , g}) is a basis of K ⟨f, g⟩. Moreover the K–linear map B Ð→ K ⟨f, g⟩ such that e1 ↦ f , e2 ↦ 1 − f , a ↦ gf , b ↦ g − gf (resp. e1 ↦ f 2 , e2 ↦ 1 − f 2 , a ↦ g, b ↦ f − f 2 ) is an algebra isomorphism. Since 1 2 , 2 2 , 12 2 , 1 , 2 , are the indecomposable B–modules, we conclude that the B–module M is the injective module 12 2 .  Corollary 3.11. Let C be the K–algebra given by the Dynkin diagram a 1 / 3 o b 2 , and let M be the K ⟨x, y⟩–module constructed in Example 2.5. Then M is a faithful injective C–module. Proof. By hypothesis M is described by the following picture. x 8 v1 v2 ● ● y f y x   ● v3 Let f and g be the endomorphisms of M induced by x and y. Then we clearly have f 3 = f 2 , g 3 = g 2 , f g = f g 2 = f − f 2 , gf = gf 2 = g − g 2 , and f 2 g = g 2 f = 0. Moreover {1, f, f 2 , g, g 2 } is a basis of K ⟨f, g⟩. As always, let ei be the path of length zero around the vertex i. Then the K–linear map C Ð→ K ⟨f, g⟩ such that e1 ↦ f 2 , e2 ↦ g 2 , e3 ↦ 1 − f 2 − g 2 , a ↦ g − g 2 , b ↦ f − f 2 is an algebra isomorphism. Hence the C–module M is the injective module 12 3 .  INDECOMPOSABLE NON UNISERIAL MODULES OF LENGTH THREE 233 In all the previous examples of faithful modules M , satisfying (123) and defined over finite dimensional K–algebras R, we always have dim R − dim M ≤ 2. To see that this is not always true, it suffices to consider the following example. Example 3.12. There are a K–algebra R and a faithful R–module M , satisfying (123), such that dim M = 4, End M ≃ K and dim R = 10. Construction. Let M be the K ⟨x, y, z⟩–module described by the following picture. y 8 v1 v2 ● ● f z y z   x i ● y v3 ) ● v4 Then we deduce from Theorems 3.2 and 3.6 that M is a K ⟨x, y, z⟩–module satisfying (123) with End M ≃ K and dim M = 4. Let f, g, h be the endormorphisms of M induced by x, y, z respectively, and let R = K ⟨f, g, h⟩. Next let e = 1−g 2 −h2 −gf and ` = ge. Then the linear maps g 2 , h2 , hg, gh, gf, `, f h, f gh, f, e are described by ten different matrices (with entries in K) with one entry equal to 1 and the remaining ones equal to 0, as suggested by the following matrix ⎛ g2 ⎜ ⎜0 ⎜ ⎜ ⎜ hg ⎜ ⎝f h 0 0 2 h 0 gh gf f gh f 0⎞ ⎟ 0⎟ ⎟. ⎟ `⎟ ⎟ e⎠ This remark and the shape of M imply that R is isomorphic to the matrix algebra ⎛K ⎜ ⎜0 ⎜ ⎜ ⎜K ⎜ ⎝K 0 0 K 0 K K K K 0⎞ ⎟ 0⎟ ⎟. ⎟ K⎟ ⎟ K⎠ Consequently we have dim R = 10, as claimed.  As the next example shows, the dimension of a faithful module satisfying (123) and that of its endomorphism ring do not determine the dimension of the algebra. Example 3.13. There are a K–algebra A and a faithful A–module L, satisfying (123), such that dim L = 4, End L ≃ K and dim A = 9. 234 GABRIELLA D’ESTE Construction. Let L be the K ⟨x, y, z⟩–module described by the following picture. v1 ● x ) i y v2 v3 ● ● f z y z   ● v4 Then we have dim L = 4. Proceeding as in Theorem 3.2 we conclude that the K ⟨x, y, z⟩–module L satisfies (123). Hence we deduce from Theorem 3.6 that End L ≃ K. Next let f, g, h be the endomorphisms of L induced by x, y, z respectively, and let A = K ⟨f, g, h⟩. Finally let e = 1 − gf − f g − h2 . Then the elements gf, gf g, f, f g, h2 , hgf, hg, gh, e are described by nine different matrices (with entries in K) with one entry equal to 1 and the remaining ones equal to 0, as suggested by the following matrix ⎛ gf ⎜ ⎜ f ⎜ ⎜ ⎜ 0 ⎜ ⎝hgf 0⎞ ⎟ 0 0⎟ ⎟. ⎟ h2 0⎟ ⎟ gh e⎠ gf g 0 fg 0 hg This observation and the shape of M imply that A is isomorphic to the matrix algebra ⎛K ⎜ ⎜K ⎜ ⎜ ⎜0 ⎜ ⎝K K 0 K 0 0 K K K 0⎞ ⎟ 0⎟ ⎟. ⎟ 0⎟ ⎟ K⎠ Hence we have dim A = 9.  Example 3.14. There are a K–algebra B and a faithful B–module U , satisfying (123), such that dim U = 4, End U ≃ K[x]/(x2 ) and dim B = 8. Construction. Let U be the K ⟨x, y, z⟩–module described by the following picture. v1 ● x ) i y v2 v3 ● ● y z   ● v4 Let f, g, h be the endomorphisms of U induced by x, y, z respectively, and let B = K ⟨f, g, h⟩. Then U is a faithful B–module with dim U = 4 and, proceeding as in INDECOMPOSABLE NON UNISERIAL MODULES OF LENGTH THREE 235 Theorem 3.2, it is easy to see that the B–module U satisfies (123). Finally, by Theorem 3.6, we have End U ≃ K[x]/(x2 ). Let e = 1 − gf − f g and let ` = g − gf g. Then the elements gf, gf g, f, f g, e, h, hg, ` are described by matrices with one or two entries equal to 1 and the remaining ones equal to 0, as suggested by the following matrix ⎛gf ⎜ ⎜f ⎜ ⎜ ⎜0 ⎜ ⎝h gf g 0 fg 0 0 e hg ` 0⎞ ⎟ 0⎟ ⎟. ⎟ 0⎟ ⎟ e⎠ This remark and the shape of U imply that B is the K–algebra of all matrices (with entries in K) of the form ⎛⋆ ⎜ ⎜⋆ ⎜ ⎜ ⎜0 ⎜ ⎝⋆ ⋆ 0 ⋆ 0 0 t ⋆ ⋆ 0⎞ ⎟ 0⎟ ⎟. ⎟ 0⎟ ⎟ t⎠ Hence we have dim B = 8, as claimed.  Corollary 3.15. There exist two finite dimensional factor algebras A and B of K ⟨x, y, z⟩ and two faithful modules A L and B U , satisfying (123), such that dim L = dim U , dim soc L = dim soc U , dim A + dim End L = dim B + dim End U and dim A ≠ dim B. Proof. Let A, B, L and U be as in Examples 3.13 and 3.14. Then we have dim L = dim U = 4, dim soc L = dim soc U = 1, dim A = 9, dim B = 8, dim End L = 1 and dim End U = 2.  References [1] I. Assem, D. Simson and A. Skowronski, Elements of the Representation Theory of Associative Algebras, Vol. 1, London Mathematical Society Student Texts, 65, Cambridge University Press, Cambridge, 2006. [2] M. Auslander, I. Reiten and S. O. Smalø, Representation Theory of Artin Algebras, Cambridge Studies in Advanced Mathematics, 36, Cambridge University Press, Cambridge, 1995. [3] J. Clark, C. Lomp, N. Vanaja and R. Wisbauer, Lifting Modules, Supplements and Projectivity in Module Theory, Frontiers in Math., Boston, Birkhäuser, 2006. [4] G. D’Este and D. Keskin Tütüncü, Pseudo projective modules which are not quasi projective and quivers, Taiwanese J. Math., 22(5) (2018), 1083-1090. 236 [5] GABRIELLA D’ESTE P. A. Guil Asensio, D. Keskin Tütüncü, B. Kalebog̃az and A. K. Srivastava, Modules which are coinvariant under automorphisms of their projective covers, J. Algebra, 466 (2016), 147-152. Gabriella D’Este Department of Mathematics University of Milano Via Saldini 50 20133 Milano, Italy e-mail: gabriella.deste@unimi.it 

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WITHDRAWN: An empirical survey on the effects of broaching pre-cracking process on fracture behavior of materials Research Article Keywords: Posted Date: July 8th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1558794/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License EDITORIAL NOTE: The full text of this preprint has been withdrawn by the authors while they make corrections to the work. Therefore, the authors do not wish this work to be cited as a reference. Questions should be directed to the corresponding author. Page 1/2 Abstract The full text of this preprint has been withdrawn, as it was submitted in error. Therefore, the authors do not wish this work to be cited as a reference. Questions should be directed to the corresponding author. Full Text The authors have withdrawn this preprint from Research Square. Page 2/2 

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Biodivers Conserv (2018) 27:1619–1638 https://doi.org/10.1007/s10531-016-1268-6 ORIGINAL PAPER Offshore renewable energy and nature conservation: the case of marine tidal turbines in Northern Ireland John R. Haslett1 • Marina Garcia-Llorente2,3 • Paula A. Harrison4,5 • Sen Li5 • Pam M. Berry5 Received: 30 August 2016 / Revised: 18 November 2016 / Accepted: 24 November 2016 / Published online: 5 December 2016 Ó The Author(s) 2017. This article is published with open access at Springerlink.com Abstract The global demand for renewable energy continues to increase rapidly and with it the necessity to develop and test new technologies to deliver the power. Offshore renewable energy sources that harness wind, wave or tidal power are of major interest. Technological advances in these directions have not been matched by a clear understanding of the environmental impacts of the new devices, with most existing research concentrated on the impacts of offshore wind farms. Decisions often continue to be made without the support of a clear evidence base. Here we use an underwater tidal turbine, SeaGen, constructed and operated within the Strangford Lough marine protected area in Northern Ireland, as a case study to explore the potential impacts of the turbine as points of concern and argumentation in the decision-making processes. We use information obtained from official documents and one-to-one interviews with the main stakeholders. Our results demonstrate that during the construction and operation of the turbine the perceptions and views of different stakeholders sometimes disagreed but were often surprisingly similar in relation to both likelihood and intensity of the potential impacts of the turbine on marine Communicated by Rob Bugter, Paula Harrison, John Haslett and Rob Tinch. This article is part of the special issue on the BESAFE project. & John R. Haslett john.haslett@sbg.ac.at 1 Department of Cell Biology and Physiology, Division of Animal Structure and Function, University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria 2 Department of Applied Research and Agricultural Extension, Madrid Institute for Rural, Agricultural and Food Research and Development (IMIDRA), Ctra. Madrid-Barcelona (N-II), KM. 38.200, 28802 Alcalá De Henares, Madrid, Spain 3 Social-Ecological Systems Laboratory, Department of Ecology, Universidad Autónoma de Madrid, c. Darwin 2, Biology, 28049 Madrid, Spain 4 Present Address: Centre for Ecology and Hydrology, Lancaster Environment Centre, Library Avenue, Bailrigg, Lancaster LA1 4AP, UK 5 Environmental Change Institute, University of Oxford, South Parks Road, Oxford OX1 3QY, UK 123 1620 Biodivers Conserv (2018) 27:1619–1638 biodiversity, ecosystem services and human well-being in general. The overall consensus of views was refined and evolved under an adaptive management approach over the 10 years of the discussions and decision-making processes. The results are discussed in relation to cumulative gains in knowledge, future arrays of many underwater turbines and multiple use of oceans within social ecological systems to maintain the conservation of marine biodiversity. Keywords Adaptive management
Decision-making
Ecosystem service
Marine protected area
Stakeholder views
Trade-off Introduction The global demand for renewable energy continues to increase rapidly (most recently accelerated by the United Nations 2015 Paris Agreement to combat climate change) and with it the necessity to develop and test new technologies to deliver renewable/clean power. Offshore renewable energy sources that harness wind, wave or tidal power are of major interest. Wind farms in the sea are now commonplace in many regions, but advances in wave and tidal power technology have also been rapid during their relatively short history (Lawrence et al. 2013). The Ocean Energy status Report of the EC for 2014 cites an estimate of 100 tidal energy companies worldwide, with more than half located within the EU and other strong representation in Canada, Australia, USA and eastern Asia. Most of these deployment activities are single devices or small, pre-commercial arrays (Magagna and Uihlein 2015). Unfortunately, the technological advances have not been matched by a clear understanding of the environmental impacts of the new machinery, and this remains a major concern and focus of research (Inger et al. 2009; Bailey et al. 2014). Most existing research has concentrated on the impacts of offshore wind farms on biodiversity (e.g., Bergström et al. 2014) and/or ecosystem service provision (e.g., Mangi 2013). However, even for wind farms, our overall knowledge is still very incomplete and the urgent call for more data and new research by Inger et al. (2009) remains valid, echoed by Bailey et al. (2014). Further complications arise from the relatively recent recognition that biodiversity conservation in Europe and also globally, is embedded within complex, dynamic socialecological systems that encompass the provision of ecosystem services, wider societal needs and human well-being, all with the need for appropriate and integrated sectoral policy and environmental management (Haslett et al. 2010; Garcı́a-Llorente et al. 2015). In the context of marine environments, offshore renewable energy has already been noted as a new human activity entering the complexity of marine social-ecological systems (Burkhard and Gee 2012). How these systems respond has now begun to be directly addressed, but again the focus has been largely on wind farms (Busch et al. 2011; Burkhard and Gee 2012; Mangi 2013). Marine tidal turbines that use tidal currents to turn large, submerged rotors have many parallels with wind turbines; indeed they function rather like ‘‘submerged windmills’’, but there are also many unique aspects of this still very young family of devices that can impact upon marine biodiversity and different components of social-ecological systems. These unique aspects arise mainly from the rotor blades moving under water rather than in the air above sea level, thus impacting on different types of organisms and parts of marine 123 Biodivers Conserv (2018) 27:1619–1638 1621 social-ecological systems. The large gaps in knowledge that exist create uncertainties in decision-making processes concerning siting, construction, operation and eventual decommissioning of the turbine devices (Inger et al. 2009). This is true even at the level of testing full size operational prototype devices, including ‘‘SeaGen’’, the turbine that is the subject of this study. SeaGen is ‘‘the world’s first’’ commercial scale open stream underwater tidal turbine, constructed and operated in the strong tidal flow at the entrance to Strangford Lough on the east coast of County Down, Northern Ireland. Strangford Lough is a shallow, tidal sea lough about 30 km long and up to approximately 8 km wide. The Lough is almost landlocked, but is connected to the open sea by a narrow 8 km long channel known as the Strangford Narrows, where the SeaGen turbine is sited. The Lough supports a recognised wealth of marine and coastal biodiversity, and the entire area has many different designations of nature conservation status, at national and EU levels. These include Special Protection Area (SPA), Special Area of Conservation (SAC), Natura 2000 site and RAMSAR status and most recently Marine Conservation Zone under the Marine Act (Northern Ireland) 2013. Also, the area is important for its landscape, recreational use, some commercial fishing and a diversity of other ecosystem services (Strangford Lough and Lecale Partnership 2014). This marine turbine project is important for the Northern Ireland and UK Governments’ requirement to achieve renewable energy targets set by the EU (Department of the Environment, Northern Ireland 2009), but at the same time, the impacts throughout all stages of the turbine development were not well understood. The dual importance of the necessity to increase renewable energy output, with the associated extra benefits of hi-tech and industrial advances, while at the same time ensuring the continued essential role of the Protected Area for marine biodiversity conservation and the provision of a wide range of ecosystem services, created a spectrum of potential conflicts of stakeholder and policy interests that was new and complex. Decision-making processes under these potentially antagonistic circumstances were guided by the adoption of an adaptive management approach, agreed by the stakeholders during the early stages of the negotiations. In the present context, adaptive management refers to an iterative process in which uncertainty surrounding the environmental effects of a human activity is reduced progressively by carefully managed, science-led monitoring of agreed indicators of environmental impacts. From the very beginning, an adaptive approach offers a middle way, so that risks and the needs of the different interest groups are continually re-assessed in the light of new information and balanced within an agreed management framework. This is clearly explained by Savidge et al. (2014), who also provide a useful general summary of adaptive management in the environmental context, with details of how these principles have been applied to the SeaGen situation. These authors also provide a primarily biological account of the environmental monitoring studies undertaken in Strangford Lough in relation to the turbine, including information on the findings, some of the resulting adaptations and also pointing out the lessons learned. In the present paper we address the stakeholder argumentation and decision-making aspects of the turbine development, thus complementing more biological or technical perspectives. Particularly, we explore the use of the potential impacts of the turbine as points of concern and argumentation. We examine the levels of agreement between the main stakeholders involved and then follow the temporal dynamics of the discussions as the turbine development progressed. We differentiate between the negative impacts on both biodiversity itself and on humans—including ecosystem services, as well as the positive potential benefits on each of these. We also distinguish between the likelihood and 123 1622 Biodivers Conserv (2018) 27:1619–1638 intensity of the impacts. The information obtained should aid future argumentation and decision-making to protect biodiversity in offshore renewable energy development and also in wider social-ecological system contexts. Methods Information on the turbine development and its impacts was obtained from selected official written reports and from one-to-one meetings and interviews with a variety of the central stakeholders. Reports examined and timeline of events The reports examined represent important strategic events along the timeline of the discussions and decision-making surrounding the turbine development, as depicted in Fig. 1. They were chosen as significant and clearly documented markers along the entire chain of events that conveniently bring the different relevant aspects of the study together. They were: Event 1 an initial Independent Environmental Statement, which was an impact assessment undertaken in the period April 2004–June 2005, before any construction work was undertaken (Royal Haskoning 2005). One of the general conclusions of this report was that the potential impact of the SeaGen marine current turbine on some of the designated features was uncertain. Because of this uncertainty of impact, an adaptive management approach to construction and deployment, with integrated mitigation and monitoring was proposed by the turbine company and was accepted by the Department of Environment, Northern Ireland and became the basis for progressing the project. Event 2 EU Habitats Directive Article 6 Report, covering the period 15 December 2005–2 February 2008 (Department of the Environment (Northern Ireland) 2008). To enable the turbine to be constructed and allowed to operate, a government Marine Licence was required. For this, particularly in relation to modifications (variations) to the conditions of the Licence and Natura 2000 site requirements, an assessment was necessary under Article 6 of the EU Habitats Directive. A Stage One Screening Assessment was undertaken. The report was favourable to granting the Licence and indicated that a Stage two Assessment was not required. Event 1 Independent Environment al Statement 2004 Event 2 Licensing variations and Habitats Directive Article 6 Report by the Department of the Environment (DOE) Event 4 Future scenario 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2015 Fig. 1 Timeline and the four events identified 123 Event 3 Result of Final Report of Environmental Monitoring Programme Biodivers Conserv (2018) 27:1619–1638 1623 Event 3 Environmental Monitoring Programme Final Report (Royal Haskoning 2011). As a condition of the Marine Licence and following from the 2005 Environmental Impact Assessment report, it was required that a detailed Environmental Monitoring Programme and associated suite of mitigation measures be established, covering the entire duration of the turbine development. Monitoring data collection began preinstallation in 2005 and a final report was delivered in January 2011. Event 4 a future scenario of multiple turbines (arrays) developed at other sites, considered during the stakeholder interviews. (See ‘‘Methods’’ section on data gathering from target stakeholders below). Target stakeholders The central stakeholders that were consulted for the present study were identified from information gained from a number of preliminary stakeholder scoping meetings with governmental and NGO contacts, conducted in the years 2012 and 2013. These preliminary discussions revealed that over 50 interested parties, covering a wide spectrum of stakeholder types were included in the dialogue of discussion and consultation during the 10 years and more of the turbine project. However, many of the individuals only contributed for a portion of the proceedings due to changes in job situation or political changes or discontinuities, while others were not actively involved. To provide continuity within the present study and to ensure the highest possible degree of consistency over the full time period, we chose to focus on a small, central group of individuals who had been active and closely aware of all developments and discussions throughout the entire time of the turbine project. Each of the five persons consequently interviewed represents a major stakeholder group and each has been involved in the process from its beginning in 2004 to the end of the present investigation (end 2013). The stakeholders were: (A) (B) (C) (D) (E) Government (Environment) Government (Agri-Food) Consortium of conservation NGOs in Northern Ireland Academic research Local residents and cultural heritage NGO It may be noted that the industrial company responsible for the turbine development initiative relied upon the information provided by the other stakeholders in the decisionmaking processes and so was not included in the design of the present study. Data gathering from target stakeholders In a first round of one-to-one discussion interviews with the above-listed key stakeholders, the interviewees were asked to comment generally on their perspective of the potential impacts of the turbine and the relative importance of these to the decision-making processes surrounding the turbine development. From these discussions, in combination with the information available in the official reports examined, a total of 21 potential negative impacts of the turbine on biodiversity, ecosystem services or directly affecting humans were clearly identified relevant to the aims of the present study. Each impact was a possible point of argument to be considered during the negotiations and their usage could be followed over the turbine development. In addition, direct questioning during the 123 1624 Biodivers Conserv (2018) 27:1619–1638 discussions with the interviewees identified 10 possible positive impacts of the turbine. However, these did not appear in the documentation and did not form any part of the official discussions and decision-making process. All impacts identified, both negative and positive, are listed in Table 1. A further round of interviews with the stakeholders centred on a ‘‘questionnaire’’ in which each of the interviewees was asked to populate a simple table reflecting their perceived importance attached to the impacts of the turbine. The table questionnaire was presented in two parts, covering first the negative impacts and then the positive impacts as listed in Table 1. Both parts had the same structure, so that for each impact listed, two aspects of importance, ‘‘likelihood to occur’’ and ‘‘intensity of impact’’ were scored using a Likert-style scale of 1–5, defined respectively as 1 = very unlikely/very weak; 2 = unlikely/weak; 3 = neutral/medium; 4 = likely/strong; 5 = very likely/very strong. This Likert scale is similar to that used by Jones and Eiser (2010) to investigate wind farm impacts. The interviewees were required to provide separately scored assessments for each of the events defined along the timeline. Note that the table consisted of only a list with parallel blank cells and did not include any statements, sentences or other text. As all of the interviewees had been actively involved with the turbine throughout its history, this provided a strong temporal continuity and provides a valuable insight into the role of cumulative gains in knowledge in the discussion process. All selected interviewees had clear recall of the sequence of events and had no difficulties in differentiating along the timeline. In addition to the three historical events, a fourth ‘‘event’’ of a future scenario (2013 and beyond, see the timeline of Fig. 1) was defined for similar scoring within the questionnaire. This required the interviewed stakeholders to extend their views on the listed potential impacts of the single turbine to the consideration of future large arrays of many turbines at other coastal locations. Previous discussions with the stakeholders had revealed that such arrays were already being planned. It is most important to note that this future scenario referred only to arrays at other sites and did not include any reference to the future of the existing turbine. The interviews with the selected stakeholders were all conducted during the year 2013. The views expressed by all selected interviewees were based on their professional experience and judgements, though possible influences of personal views cannot be completely excluded. The information obtained from the questionnaires was visualised in polar (radial) diagrams and analysed using simple descriptive statistical techniques to provide information on similarities and differences between stakeholders and also along the temporal axis. The Kappa statistic (or coefficient) was calculated as a measure of the level of agreement between different individuals, taking into account that agreement (or disagreement) can occur by chance (Viera and Garrett 2005). Results Differences and similarities in stakeholder perspectives The likelihood and intensity of occurrence scores were averaged across the four events on the timeline and analysed separately for each stakeholder (Figs. 2, 3). The results show considerable variation in stakeholder views on both likelihood and intensity of both negative and positive impacts of the turbine. 123 Biodivers Conserv (2018) 27:1619–1638 1625 Table 1 Broad classification of the potential environmental impacts of the marine turbine that are considered in the case study, as identified from the reports examined and consultations with stakeholders Potential negative impacts on biodiversity Protected habitats (general) Protected species (general) Birds Marine mammals Sharks/other elasmobranchs Teleost fish Shellfish (molluscs, crustaceans) Benthic communities Plankton communities Cabling to land—electric fields, abrasion Noise/vibration marine animals Potential negative impacts on humans, including ecosystem services Regulating Nutrient cycling/food web dynamics Water quality (sediment, waste remediation) Cultural Nature watching Recreational boating/fishing Landscape/seascape quality Boating navigation/access Provisioning Commercial fishing—teleosts Commercial fishing (pot fishing) Human well-being Noise/vibration humans Tidal hydrodynamics Potential positive impacts Regulating New marine habitat Improved biodiversity protection from access and fishing prevention Water quality Air quality (reduction in CO2, etc.) Cultural New biological research initiatives Educational value Cultural heritage addition Tourist attraction Human well-being Power economic value Local employment Note that some of the categories can overlap 123 1626 Biodivers Conserv (2018) 27:1619–1638 Fig. 2 Polar diagrams showing the differences between stakeholder groups of the scores for likelihood (a) and intensity (b) of occurrence of negative impacts, averaged across all four events Fig. 3 Polar diagrams showing the differences between stakeholder groups of the scores for likelihood (a) and intensity (b) of occurrence of positive impacts, averaged across all four events Particularly Stakeholder C, the conservation NGO consortium, tended to give higher ratings for likelihood and intensity of negative impacts than the other stakeholders. Ratings by this stakeholder of positive impacts were less extreme. The difference is borne out by calculation of Kappa coefficients to measure the degree of agreement of each stakeholder with the others, for all four events combined (Table 2). Again, stakeholder C is shown to 123 Biodivers Conserv (2018) 27:1619–1638 1627 Table 2 The degree of agreement/disagreement between stakeholders’ scores of likelihood and intensity of impacts as measured by Kappa coefficients Stakeholder B (Government) Stakeholder C (NGO) Stakeholder D (Academic research) Stakeholder E (NGO) 0.111 0.250 0.352 0.372 0.038 0.152 0.114 Likelihood Stakeholder A (Government) Stakeholder B (Government) Stakeholder C (NGO) 0.140 Stakeholder D (Academic research) 0.214 0.229 Intensity Stakeholder A (Government) Stakeholder B (Government) Stakeholder C (NGO) Stakeholder D (Academic research) 0.208 0.210 0.295 0.403 0.145 0.144 0.198 0.065 0.126 0.34 Data from four different events were combined together. Likelihood/intensity values were categorised as: low (1); medium (2–3); high (4–5). Kappa Interpretation: \0 poor agreement; 0.0–0.20 slight agreement; 0.21–0.40 fair agreement; 0.41–0.60 moderate agreement; 0.61–0.80 substantial agreement; 0.81–1.00 almost perfect agreement. Highlighted in the table are kappa [0.2 in bold representing a certain degree of agreement and kappa \0.1 in italics representing more disagreement between stakeholders disagree most with the other stakeholders for both the likelihood and intensity of impact occurrence. The Kappa coefficients also highlight that there are various instances of at least fair agreement between stakeholders, indicating that some general trends do occur at the level of the entire time span covered by this study. In Figs. 2 and 3, these areas of general agreement may be seen where the coloured lines representing the different stakeholders follow similar patterns. For example, the negative impacts on sharks and other elasmobranch fish, marine mammals, birds, protected species and protected habitats are rated similarly in relative terms by the stakeholders for both likelihood and intensity, although the values assigned by each stakeholder may be different. Those impacts that were scored most highly (the ‘‘top three’’) by each stakeholder for likelihood and intensity of occurrence also reveal general similarities. For instance, the likelihood of negative impacts on landscape/seascape quality, boating navigation and access, and the impacts of noise/vibrations on marine animals were scored highly by 3 of the 5 stakeholders, while the intensity of the effects of noise on marine animals was also considered to be high by 3 of the stakeholders, and intensity of impact on recreational boating/fishing was scored highly by 4 of the 5 stakeholders. Equivalent patterns can be seen for positive impacts, but these are not so clear. For example, the similarity in the patterns for cultural heritage, educational value and local employment, and the high scores given by 4 of the 5 stakeholders to the likelihood of positive impacts from power economic value and to local employment. Also, for intensity, 123 1628 Biodivers Conserv (2018) 27:1619–1638 higher scores for the benefits through educational value and the creation of new biological research were given by 3 of the 5 stakeholders. To examine more closely where areas of greatest and least differences of stakeholder opinion occur in relation to the different impacts considered, standard deviations of the means of ‘‘likelihood’’ and ‘‘intensity’’ values were calculated for each impact, pooled over all four events on the timeline (Fig. 4). Larger standard deviations indicate greater differences in stakeholder rating values. The ranking order for the negative impacts clearly indicates that opinions on ecosystem services and other human impacts are the most strongly divided. Conversely, the ratings given to impacts on particular aspects of Fig. 4 Levels of disagreement between stakeholder ratings of negative (a) and positive (b) impacts using standard deviations of the means from all stakeholders over all four events and ranked by likelihood of impact (grey bars) 123 Biodivers Conserv (2018) 27:1619–1638 1629 biodiversity and conservation issues are much more consistent between stakeholders—they share similar opinion values. For the positive impacts there is no such clear interpretation. Opinions on the turbine being a source of providing improved biodiversity protection, or benefitting air quality or creating new marine habitat are the most variable, but there is more general consensus on the other positive impacts, particularly in providing local employment and the education value. Temporal dynamics: constancy and change in perceived impacts along the timeline Examination of the impacts information separately for each of the four events along the timeline, but with stakeholder views combined, identifies some general patterns in likelihood and intensity of the influences of the turbine on both biodiversity and ecosystem services/human well-being. Amongst the negative impacts (Fig. 5), those acting on parts of biodiversity, including benthic communities, sharks, marine mammals and protected species and habitats in general, and also impacts from cabling to land and from noise/vibration effects on animals, are all classified as having medium to strong intensities, as well as being relatively likely to occur within all or most of the events. Thus these may be regarded as, in the stakeholders‘ combined consensus, the greatest threats to biodiversity, taken over the entire time period. This is in contrast to the negative impacts on, for example, birds, teleost fish, shellfish, plankton commercial fishing and nature watching, all of which are considered to be rather unlikely to occur, and when they do, rather weak in their intensity across most of the events. These are still considered by the stakeholders to represent potential threats to biodiversity and ecosystem services, but at lower levels. Note that there appears to be a Fig. 5 Polar diagrams showing the differences between stakeholder groups of the scores for likelihood (a) and intensity (b) of occurrence of negative impacts, averaged across all stakeholders 123 1630 Biodivers Conserv (2018) 27:1619–1638 general positive correlation between likelihood and intensity of impacts, reflected in the overall similarities of the shapes of the diagrams Fig. 5a, b). For the positive impacts (Fig. 6), although a number of these were considered likely to occur within the time periods of most or all of the events, such as educational value, local employment, power economic value and new biological research, only the last of these, new research, was considered to be of strong intensity. Again, as noted with the negative impacts, the results for the positive impacts also appear to exhibit an overall correlation between likelihood and intensity (Fig. 6a, b). The similarities in the patterns of likelihood and intensity of impacts are supported by examining the mean intensity values in the different likelihood categories (1–5), for both negative and positive impacts. These show that mean intensity generally increases as likelihood increases (Table 3). These results also show that overall, stakeholders seem more cautious in rating intensity, especially when they rated likelihood with high scores. In other words, an impact may be expected to occur, but its impact intensity may not be as strong as its likelihood. This is also to be seen in both Figs. 5 and 6, where the plots of intensity tend to show lower scores (closer to the centre of the circles) than the likelihood scores. This is particularly pronounced among the positive impacts (Fig. 6). The patterns described so far have referred to general trends exhibited by the events along the timeline considered overall. However, the results also permit changes in the importance of the different impacts to be followed more specifically as the turbine project evolved along the timeline of events, as well as consideration of the future scenario of arrays of turbines. In considering the results below, it should be remembered that of the four events defined in the timeline of Fig. 1, the first three refer to the documented history of the turbine project and to a single construction in Strangford Lough, while the fourth event refers to a scenario situation with arrays of many turbines at other locations along the coast of Northern Ireland, that are already being planned. Thus the future scenario event 4 must be interpreted rather differently to the other three events. Fig. 6 Polar diagrams showing the differences between stakeholder groups of the scores for likelihood (a) and intensity (b) of occurrence of positive impacts, averaged across all stakeholders 123 Biodivers Conserv (2018) 27:1619–1638 1631 Table 3 Relationship between likelihood and intensity of impacts Impacts Mean intensity value (SD) Likely = 1 Likely = 2 Likely = 3 Likely = 4 Likely = 5 Intensity of negative impacts 1.06 (0.28) 2.09 (0.63) 2.95 (0.57) 3.53 (0.88) 4.18 (0.98) Intensity of positive impacts 1.13 (0.34) 2.07 (0.36) 2.89 (0.81) 2.91 (0.85) 3.70 (1.22) Mean intensity values (standard deviation in parenthesis) within different likelihood score categories (1–5) for both negative and positive impacts Throughout the consecutive events 1 to 3, there is a clear, strong trend that the negative impacts were perceived in the early stages (the initial Environmental Statement, event 1) as more likely to occur and with noticeable intensity, but the assessment of the importance of the same impacts decreases with time up to and including event 3, when the Environmental Monitoring Report was completed in 2011. This is indicated by the progressive inward retreat of the concentric patterns in the polar diagrams of Fig. 5, so that the results from event 3 are mostly depicted as tighter, inner rings. Impacts that conform to this pattern include most of the impacts on both biodiversity and ecosystem services/human wellbeing. The only (sometimes only partial) exceptions are influences on plankton communities, teleost fish, commercial fishing for teleosts and impacts of noise/vibration on humans. Intensity and likelihood patterns are very similar, but not identical. In the future scenario of event 4, the scores then show a distinct and strong reverse trend, with values of intensity and likelihood increasing again, usually very strongly, to attain levels above those of event 3 (the only exception is the impact of noise/vibration on humans, where a strong negative impact was reported on a house during the operation of the turbine). This strong reverse trend reflects the differences between the single turbine situation where there has been concentrated effort to mitigate the negative effects, and the consideration of arrays of many turbines together, where environmental risks may be amplified and are not yet understood. The temporal dynamics of the positive impacts are rather different. Indeed, in many instances the trend is the opposite to that of the negative impacts—there is often an increase in likelihood and intensity ratings from event 1 to event 3, and this trend of increase usually extends to encompass the future scenario event 4 (Fig. 6). Although the changes in score values between events are generally not as marked as in the negative impacts, there is still clear indication that the stakeholders give more credence to the potential positive impacts of the turbine, or even arrays of turbines, as the turbine project progressed. These relative changes over time are described qualitatively with reference to the polar diagrams. A supporting quantitative analysis is provided in Table 4, in which differences in mean score values between consecutive events are used to quantify the relative changes between events. These statistics verify the conclusions drawn from the polar diagrams. For example, the negative impacts are seen initially as more likely to occur and with higher intensities but the assessments often decline with time over the events 1–3 (highlighted in italics in Table 4) and for the scenario event 4 the values of likelihood and intensity increase again (results highlighted in bold in Table 4). Also, the positive impacts often exhibit a trend for likelihood and intensity ratings to increase over time from events 1 to 3 and this may extend to include the scenario event 4 (highlighted in bold in Table 4). 123 1632 Biodivers Conserv (2018) 27:1619–1638 Table 4 Differences in mean score values of each impact, compared between consecutive events Intensity P0 PA Likely PB PC P0 PA PB PC Negative impacts on biodiversity Protected habitats (general) 3.6 -1 -1 1.6 3.8 -0.2 -1.6 1.2 Protected species (general) 4 -0.6 -1.4 1.4 4 -0.2 -2 1.4 Birds 2 -0.2 -0.4 0.4 2.2 -0.2 -0.4 1 Marine mammals 4.4 -0.6 -1.4 1.4 4.2 -0.4 -1.8 1.4 Sharks/other elasmobranchs 4.4 -0.6 -1.4 1.4 4.4 -0.8 -1.2 1.2 Teleost fish 2 -0.4 -0.2 0.8 2.6 0 -0.4 1.2 Shellfish (molluscs, crustaceans) 2 0 -0.6 1 2.2 -0.2 -0.2 1 Benthic communities 3.4 0 -1 0.8 3.6 -0.2 -1.2 1.6 0.4 Plankton communities 1.4 0 0 0.4 1.4 0 0 Cabling to land—electric fields, abrasion 3.2 -1.4 -0.6 2 4.4 -0.8 -0.8 2.6 Noise/vibration marine animals 4.4 0 -0.6 1 4.2 0.4 -2 1.4 Negative impacts on humans (including ecosystem services) Nutrient cycling/food web dynamics 1.6 0 -0.2 0.8 1.6 0 -0.2 0.4 Water quality (sediment, waste remediation) 2.2 0.4 -0.6 1.2 2.2 0 -0.6 1.2 Nature watching 2.4 -0.2 -0.8 1.2 2.8 0 -0.6 0.8 Recreational boating/fishing 4.4 0 -0.4 0 4.2 -0.2 -0.8 0.2 Landscape/seascape quality 3.6 -0.2 -0.2 0.6 4.2 -0.6 -0.8 1.8 Noise/vibration humans 2.8 -0.2 0.8 -0.2 3 -0.4 1.2 -0.8 1 Tidal hydrodynamics 3.2 -0.4 -0.6 0.8 3.8 -0.2 -0.4 Boating navigation/access 3.6 -0.2 -0.4 0.6 4.4 -0.6 -0.4 1.8 Commercial fishing—teleosts 1.4 0 0 1.6 1.6 0 0 1.4 Commercial fishing (pot fishing) 1.8 0 -0.4 1.6 2 0.2 -0.4 1.2 Positive impacts Power economic value 3 -0.4 0.2 0.6 4 -0.4 0.4 1.2 Local employment 2.2 0.2 -0.4 0.2 4.2 0.2 -0.2 1.6 Educational value 2.8 0 0.6 0 3.6 0 0 0.4 Cultural heritage addition 2.4 0.2 0.8 -0.4 2.4 0.2 0 0.6 Tourist attraction 1.8 0.6 0.4 -0.8 2.6 0.8 0 -0.6 New marine habitat 2.2 0 0 1.4 2.6 0 0.2 1.4 Improved biodiversity protection from access and fishing prevention 2.2 0 0 1.2 2.4 0 0 1.4 0.6 Water quality 1.4 0.2 0 0.6 1.4 0.2 0 Air quality (reduction in CO2 etc.) 2.2 0 0 1 3.4 0 0 1.8 New biological research 2.4 1.4 0.2 0.2 2.4 1 0.2 0.4 P0 averaged values in ‘‘2004–2006’’; PA averaged values in ‘‘2005–2008’’ minus averaged values in ‘‘2004–2006’’, PB averaged values in ‘‘2011’’ minus averaged values in ‘‘2005–2008’’; PC averaged values in ‘‘2013?’’ minus averaged values in ‘‘2011’’. Highlighted in the table values are relative changes [0 in bold and relative changes \0 in italics 123 Biodivers Conserv (2018) 27:1619–1638 1633 Discussion This study has demonstrated that during the construction and operation of the marine tidal turbine in Strangford Lough, the perceptions and views of different stakeholders sometimes disagreed but were often surprisingly similar in relation to both likelihood and intensity of the potential impacts of the turbine on marine biodiversity, ecosystem services and human well-being in general. While stakeholders maintained their own profiles in having differences in opinion on the overall degree of importance of the impacts (some being more conservative in their judgements than others), there was a general consensus of views that was honed/refined and evolved over the entire timeline of the discussions and decision-making processes. This underlines the paramount importance of establishing and maintaining effective stakeholder engagement throughout the negotiations, as has been previously stressed most recently in regard to marine protected area policy in Scotland (Hopkins et al. 2016a) and also in regard to the development of marine renewable energy (Inger et al. 2009). Such evolution of stakeholderś perspectives, specifically their assessments of the negative impacts, is particularly relevant to understanding the full dynamics of this case study. This is because the progressive changes observed between events 1, 2 and 3 on the timeline can be understood as reflecting stakeholderś increased knowledge, particularly the cumulative gain in scientific information that was obtained from the continuous monitoring programme and associated academic research (Savidge et al. 2014). This cumulative gain in knowledge also informed on potential points for consideration in the argumentation processes relating to future arrays of turbines at other sites, as referenced by the scenario of event 4. It is interesting that all the different stakeholders displayed an overall appreciation of the many gains in scientific knowledge as the monitoring results became available. Thus no large differences were observed between the responses of stakeholder D, representing academic biological research and the other stakeholders, as might have been otherwise expected had the new scientific knowledge been appreciated only by this stakeholder group (Figs. 2, 3). The clearly higher ratings of likelihood and intensity of negative impacts assigned by stakeholder C, representing the consortium of conservation NGOs, is most likely to be a reflection of a slightly stronger tendency towards application of the precautionary principle by this stakeholder. These findings conform with the results of Berry et al. (2016, this issue) who showed that different stakeholder groups share some common views on biodiversity and its conservation, particularly in relation to moral, intrinsic and ecological values. There are clear patterns in the temporal dynamics of the use and perceived importance and effectiveness of arguments based on the individual negative impacts during the negotiations and decision-making process surrounding the turbine development. Most of the listed negative impacts were used as arguments which were repeated in the events over the entire time course of the turbine development. Some of these exhibited increased and broadening use along the timeline of events. Particularly, the arguments of risks to marine mammals, notably seal populations with some focus on the EU Habitats Directive protected species status of the harbour seal, and the risks of any type of large animal collisions with the turbine increased their status as centres of attention during the course of the case (Savidge et al. 2014). However, arguments centred on Protected Area status, including benthic and Habitats Directive listed habitats, and acoustic/vibration disturbance to animals also maintained higher profiles. Arguments of negative impacts on cetaceans (dolphins and 123 1634 Biodivers Conserv (2018) 27:1619–1638 whales), sharks and other elasmobranchs (cartilaginous fish), including risks from electromagnetic fields and cables scraping the seabed, showed lost impetus over the timeline. Risks to birds maintained a low profile throughout the study. All of these temporal patterns of argumentation using particular impacts are also the same as, or very similar to, those reflected in the official report documents examined. From the very broad spectrum of concerns and uncertainties initially identified in the early Environmental Impact statement (Royal Haskoning 2005), focus on the different specific impacts was progressively narrowed and concentrated through the Habitats Directive Article 6 Report (Department of the Environment (Northern Ireland) 2008) and the Final Report of the monitoring programme (Royal Haskoning 2011), in the manner reported above. One important point here is that the above examples and the present results overall clearly show that concerns and discussions along the timeline of the decision-making processes were very strongly oriented to impacts that centred on aspects of biodiversity or included aspects of biodiversity as a component. The negative impacts of the turbine on ecosystem services and human well-being are noticeably absent from active argumentation. All stakeholders were aware of the full spectrum of impacts, including those affecting ecosystem services and human well-being, and they provided judgements on these in the questionnaire used. Equally, the human-directed influences were identified and considered within the formal Environmental Impact Assessment (event 1) in the early stages of the turbine project (Royal Haskoning 2005). However, after this assessment, the primary documentation up to and including the Monitoring Programme Report of event 3 (Royal Haskoning 2011), is mainly focussed on the potential threats to biodiversity and biodiversity protection policy affecting the Protected Area. Other impacts appear to have been regarded as much more peripheral, even though there can be direct positive links between biodiversity and ecosystem service provision. For example, in the review of biodiversity and ecosystem service relationships by Harrison et al. (2014), positive relations were found to be frequently reported between commercial fishing and the attributes of biodiversity of species richness, abundance and size of individuals. Also, species-based recreation was shown to have a positive relation with both abundance and size of the species present. Concerns about negative impacts on the seal populations in the Lough maintained a particularly high profile throughout the timeline of the project. Seals may appear simply as charismatic species capable of attracting public attention. However, and most importantly, in addition to their strong aesthetic appeal, the seals are the subjects of a more complex set of arguments involving the logistics of combining environmental policy with changing levels of scientific knowledge. Particularly, the risks of collisions with the rotating blades of the turbine, together with the fact that the harbour seal is listed in the Annexes of the EU Habitats Directive, so that protection of the species is legally obligatory. This is in contrast to all the other organisms present: no other species or community or habitat type offered such a strong combination of arguments (sometimes referred to as ‘‘bundles’’ of arguments). For example, it might have been expected that the bird fauna and bird habitats, which have a strong public profile and are commonly used in argumentation in biodiversity conservation matters, would play a pivotal role in discussions of the turbine development. However, despite the EU Habitats and Birds Directives listings of a few species and recognition that diving bird species potentially could be at risk of colliding with the turbine rotors whilst in operation, arguments involving birds remained at a low profile, becoming less important as the project progressed. This is because, as research and monitoring continued and the associated levels of scientific knowledge increased, it became clear that apart from some early concerns about disturbances to birds caused by the construction of the turbine (mitigated by undertaking the work outside the overwintering and breeding 123 Biodivers Conserv (2018) 27:1619–1638 1635 seasons), birds and bird habitats were at negligible risk from the turbine project (Department of the Environment, Northern Ireland 2008). The potential positive impacts of the turbine on biodiversity and human well-being, beyond the provision of renewable energy (including both the economic value of the power and the inferred improvement of air quality), were not included in the argumentation and decision-making processes. The positive impacts listed in Table 1 were identified and obtained from discussions during meetings with the stakeholders for this study and were not included in the official reports or other documentation. However, a number of these potential positive impacts have been recently widely discussed in the literature in relation to offshore wind farms. Two of the more obvious examples that are particularly pertinent to the present study may be expanded upon here. First, there is the recognition of the creation of new habitat provided by the anchoring structures of the turbine (often referred to as ‘‘foundations’’) on the seabed. The advantages of such new, artificial substrate have been well advertised in the wind farm literature as being available for colonisation by benthic organisms, to form new reef communities (Inger et al. 2009; Busch et al. 2011; Lacroix and Pioch 2011; Mangi 2013; Bergström et al. 2014; Hammar et al. 2016) and also as structures that may encourage the aggregation of fish (Bergström et al. 2014; Gilbert et al. 2015; Hammar et al. 2016). Taking the idea a step further, it has been suggested that new designs of the underwater portions of wind turbines could incorporate structural features specifically intended to enhance colonisation and shelter opportunities for marine organisms and even to accommodate aquaculture needs. Thus offshore wind farms could become an element in the creation of a green infrastructure in marine environments (Lacroix and Pioch 2011). Second, boating navigation and fishing restriction/exclusion zones designated around offshore wind farms for reasons of safety and damage to fishing tackle can provide protection for biodiversity. In this context, offshore wind farms have been widely suggested as capable of fulfilling functions very similar to those of marine protected areas (Inger et al. 2009; Mangi 2013; Hammar et al. 2016). In addition, protection could extend to boosting commercial fish stocks, with spillover of populations that could be harvested in adjacent fishing grounds (Busch et al. 2011; Mangi 2013). But all these issues are complex, not always observable and relationships are still not well understood (Bergström et al. 2014; Vandendriessche et al. 2014). The extent to which these and other recognised potential positive influences could apply to the rather different situation of tidal turbines with their submerged rotors impacting on the underwater environment remains unclear. Both of the above examples serve to highlight the importance of considering possibilities for trade-offs between different impacts that may affect biodiversity protection, provision of various ecosystem services and human well-being, involving different sectors and stakeholder interests, all within the framework of entire marine social-ecological systems. Such trade-offs were not addressed or attempted during the tidal turbine negotiations of the present study, even though the potential clearly exists. But with the focus on a single turbine in the Strangford Lough Protected Area, all such trade-offs are small and of only minor importance. Throughout the turbine development as documented by events 1–3, the central concerns of all the major stakeholders were clearly the maintenance of marine and coastal biodiversity protection, including the integrity of the protected area and local cultural landscape, so no major trade-off situations arose. However, when considered in the light of the future scenario of event 4, large arrays of turbines at other locations (with the parallels to wind farms), the potential importance of various trade-offs between the different impacts begins to take on a new and much greater significance. 123 1636 Biodivers Conserv (2018) 27:1619–1638 Indeed, from the social-ecological system perspective, such arrays will be a major new addition to multi-sectoral ocean use and will further stimulate trade-offs already documented as being partly catalysed by wind farms. For example, trade-offs identified among offshore wind energy, commercial fishing and whale watching as explored by White et al. (2012) in Massachusetts coastal waters. This type of thinking has led to a new recognition of the importance of considering how we use and divide ocean space and the need for marine spatial planning that must also keep the maintenance of marine environmental quality and conservation at the forefront (White et al. 2012; Gilbert et al. 2015; Hammar et al. 2016; Hopkins et al. 2016a). Of course the confinement of the present study to a single working prototype turbine within a marine protected area is also a limitation to the generality of the results—the findings cannot be used to directly extrapolate to situations of arrays of turbines as envisaged at other coastal sites. Such extrapolation would be potentially extremely dangerous where much larger areas of seabed are involved. Such need for caution is clearly reflected by the results of the stakeholderś responses to the questionnaire presented here and future understanding will require continued new research effort with an emphasis on marine spatial planning that includes biodiversity protection as discussed above. Also, some may argue that the restriction of the study to only five stakeholders limits the amount of data obtained and the ensuing analyses. However, focusing on a small number of reliable experts that had key roles throughout the entire duration of the project ensured that the information captured was of a consistent and high quality, avoiding the need for more complicated statistical analysis of a highly patchy albeit larger dataset. One potential bureaucratic complication to this study is that Northern Ireland has experienced a complex set of governmental changes over the years. Issues of environmental policy and its governance within Northern Ireland have been the responsibility of a variety of governmental entities, mainly associated with the Department of the Environment, but under a variety of different names, during the time course of the SeaGen turbine project. This includes some periods when the local Government, the Northern Ireland Assembly, was suspended, during which times responsibility was at the national level, handled directly from the UK Government in London. However, such internal changes do not appear to have interfered with the decision-making processes related to the development of the turbine project and indeed reflects the reality of politically unstable situations that could arise in any country. Undoubtedly, the innovative ‘‘adaptive management approach’’ to the turbine development as reported here and described by Savidge et al. (2014) with integrated mitigation and monitoring, adopted from the early stages, has played a crucial role in the success of the entire turbine project. Adaptive management has been acknowledged as a useful approach in other studies involving marine protected areas in various contexts, including management for the provision of ecosystem services (Rees et al. 2012) or for resilience to climate change (Hopkins et al. 2016b). However, even though there is a general perception of the need for processes to be adaptive, the reality is that currently, adaptive management has rarely been demonstrated, nor the legal or scientific capability to carry it out (Hopkins et al. 2016b). The present study joins the work of Savidge et al. (2014) as one of the first to provide such evidence. The present study has clearly demonstrated the importance of establishing and maintaining effective stakeholder engagement and that the flexibility created among the engaged stakeholders permitted a strong adherence to the precautionary principle of biodiversity protection throughout. It may be hoped that continued high levels of stakeholder engagement and agreement will allow such an approach to be maintained when arrays of underwater turbines are developed in the near future. 123 Biodivers Conserv (2018) 27:1619–1638 1637 Acknowledgements Open access funding provided by Paris Lodron University of Salzburg. We thank all the stakeholders who took part in this study and gave freely of their time, thoughts and on occasion fast access to documented information. We also thank two reviewers for providing helpful and constructive comments. This work was supported by the European Union, under FP7 project BESAFE (FP7ENV.2011.282743). MGL was also funded by a postdoctoral grant from the Spanish National Institute for Agriculture and Food Research and Technology (INIA) which is co-funded by the European Social Fund. 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. References Bailey H, Brookes KL, Thompson PM (2014) Assessing environmental impacts of offshore wind farms: lessons learned and recommendations for the future. Aquat Biosyst 10:8. http://www. aquaticbiosystems.org/content/10/1/8 Bergström L, Kautsky L, Malm T, Rosenberg R, Wahlberg M, Capetillo NA, Wilhelmsson D (2014) Effects of offshore windfarms on marine wildlife—a generalised impact assessmen. Environ Res Lett 9:034012. doi:10.1088/1748-9326/9/3/034012 Berry PM, Fabók V, Blicharska M, Bredin YK, Garcia-Llorente M, Kovács E, Geamana N, Stanciu A, Termansen M, Jääskejäinen T, Haslett JR, Harrison PA (2016) Why conserve biodiversity? A multinational exploration of stakeholderś views on the arguments for biodiversity conservation. Biodivers Conserv. doi:10.1007/s10531-016-1173-z Burkhard B, Gee K (2012) Establishing the resilience of a coastal-marine social-ecological system to the installation of offshore wind farms. Ecol Soc 17(4):32. doi:10.5751/ES-05207-170432 Busch M, Gee K, Burkhard B, Lange M, Stelljes N (2011) Conceptualizing the link between marine ecosystem services and human well-being: the case of offshore wind farming. Int J Biodivers Sci Ecosyst Serv Manag 7:190–203 Department of the Environment (Northern Ireland) (2008) Marine Current Turbine (MCT) Article 6 Assessment, Stage One Screening. Environment and Heritage Service, Northern Ireland, February 2008. Unpublished report Department of the Environment (Northern Ireland) (2009) Planning Policy Statement 18 ‘‘Renewable Energy’’. Planning and Environmental Policy Group. http://www.planningni.gov.uk/index/policy/ planning_statements_and_supplementary_planning_guidance/planning_policy_statement_18__renewable_ energy.pdf Garcı́a-Llorente M, Iniesta-Arandia I, Willaarts B, Harrison PA, Berry P, del Mar Bayo M, Castro A, Montes C, Martı́n-López B (2015) Biophysical and sociocultural factors underlying spatial trade-offs of ecosystem services in semiarid watersheds. Ecol Soc 20(3):39. doi:10.5751/ES-07785-200339 Gilbert AJ, Alexander K, Sardá R, Brazinskaite R, Fischer C, Gee K, Jessopp M, Kershaw P, Los HJ, Morla DM, ÓMahony C, Pihlajamäki M, Rees S, Varjopuro R (2015) Marine spatial planning and good environmental status: a perspective on spatial and temporal dimensions. Ecol Soc 20(1):64. doi:10. 5751/ES-06979-200164 Hammar L, Perry D, Gullström M (2016) Offshore wind power for marine conservation. Open J Mar Sci 6:66–78. doi:10.4236/ojms.2016.61007 Harrison PA, Berry PM, Simpson G, Haslett JR, Blicharska M, Bucur M, Dunford R, Egoh B, GarciaLlorente M, Geamana N, Geertsema W, Lommelen E, Meiresonne L, Turkelboom F (2014) Linkages between biodiversity attributes and ecosystem services: a systematic review. Ecosyst Serv 9:191–203. doi:10.1016/j.ecoser.2014.05.006 Haslett JR, Berry PA, Bela G, Jongman RHG, Pataki G, Samways MJ, Zobel M (2010) Changing conservation strategies in Europe: a framework integrating ecosystem services and dynamics. Biodivers Conserv 19:2963–2977 Hopkins CR, Bailey DM, Potts T (2016a) Scotland́s Marine Protected Area network: reviewing progress towards achieving commitments for marine conservation. 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JRC Science and Policy Reports, Joint Research Centre—Institute for Energy and Transport. European Commission. Luxembourg Publications Office of the European Union. Mangi SI (2013) The impact of offshore windfarms on marine ecosystems: a review taking an ecosystem services perspective. Proc IEEE 101:999–1009 Rees SE, Austen MC, Attrill MJ, Rodwell LD (2012) incorporating indirect ecosystem services into marine protected area planning and management. Int J Biodivers Sci Ecosyst Serv Manag 8:273–285 Royal Haskoning(2005) Strangford Lough Marine Current Turbine: Environmental Statement. Final Environment Impact Assessment. Report produced for Marine Current Turbines Ltd. Royal Haskoning, Edinburgh, June 2005. Unpublished report Royal Haskoning (2011) SeaGen Environmental Monitoring Programme. Final Report produced for Marine Current Turbines Ltd. Royal Haskoning, Edinburgh, January 2011. 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Cellular and Molecular Life Sciences (2021) 78:1233–1261 https://doi.org/10.1007/s00018-020-03656-y Cellular and Molecular Life Sciences REVIEW TLR4 and CD14 trafficking and its influence on LPS‑induced pro‑inflammatory signaling Anna Ciesielska1 · Marta Matyjek1 · Katarzyna Kwiatkowska1 Received: 10 June 2020 / Revised: 25 August 2020 / Accepted: 22 September 2020 / Published online: 15 October 2020 © The Author(s) 2020 Abstract Toll-like receptor (TLR) 4 belongs to the TLR family of receptors inducing pro-inflammatory responses to invading pathogens. TLR4 is activated by lipopolysaccharide (LPS, endotoxin) of Gram-negative bacteria and sequentially triggers two signaling cascades: the first one involving TIRAP and MyD88 adaptor proteins is induced in the plasma membrane, whereas the second engaging adaptor proteins TRAM and TRIF begins in early endosomes after endocytosis of the receptor. The LPS-induced internalization of TLR4 and hence also the activation of the TRIF-dependent pathway is governed by a GPIanchored protein, CD14. The endocytosis of TLR4 terminates the MyD88-dependent signaling, while the following endosome maturation and lysosomal degradation of TLR4 determine the duration and magnitude of the TRIF-dependent one. Alternatively, TLR4 may return to the plasma membrane, which process is still poorly understood. Therefore, the course of the LPS-induced pro-inflammatory responses depends strictly on the rates of TLR4 endocytosis and trafficking through the endo-lysosomal compartment. Notably, prolonged activation of TLR4 is linked with several hereditary human diseases, neurodegeneration and also with autoimmune diseases and cancer. Recent studies have provided ample data on the role of diverse proteins regulating the functions of early, late, and recycling endosomes in the TLR4-induced inflammation caused by LPS or phagocytosis of E. coli. In this review, we focus on the mechanisms of the internalization and intracellular trafficking of TLR4 and CD14, and also of LPS, in immune cells and discuss how dysregulation of the endo-lysosomal compartment contributes to the development of diverse human diseases. Keyword CD14 · Endocytosis · Endotoxin · Endosome · LPS · TLR4 Abbreviations AD Alzheimer’s disease ALS Amyotrophic lateral sclerosis AOHA Acyloxyacyl hydrolase Arf6 ADP-ribosylation factor 6 BMDC Bone marrow-derived dendritic cells BMDM Bone marrow-derived macrophages CF Cystic fibrosis CFTR Cystic fibrosis transmembrane conductance regulator CHS Chédiak–Higashi syndrome CLIC Clathrin-independent carrier Dab2 Disabled-2 DAMP Damage-associated molecular pattern * Anna Ciesielska a.ciesielska@nencki.edu.pl 1 Laboratory of Molecular Membrane Biology, Nencki Institute of Experimental Biology of Polish Academy of Sciences, 3 Pasteur St., 02‑093 Warsaw, Poland DC Dendritic cells ERC Endosomal recycling compartment GAG​ Glycosaminoglycan GEEC GPI-AP-enriched early endosomal compartment GMFG Glia maturation factor-γ GPI-AP Glycosylphosphatidylinositol-anchored proteins HMGB1 High mobility group box protein 1 IP3 Inositol 1,4,5-trisphosphate IFN Interferon IKK IκB kinase IL Interleukin IRAK Interleukin-1 receptor-associated kinase IRF Interferon regulatory factor LBP LPS-binding protein LPS Lipopolysaccharide LSD Lysosomal storage disorders MHC Major histocompatibility complex MPS Mucopolysaccharidosis 13 Vol.:(0123456789) 1234 MVB Multivesicular bodies NPC Niemann–Pick’s type C disease OMV Outer membrane vesicles oxPAPC Oxidized 1-palmitoyl-2-arachidonyl-snglycero-3- phosphorylcholine PAMP Pathogen-associated molecular pattern PD Parkinson’s disease PI Phosphatidylinositol PI(4,5)P2 Phosphatidylinositol 4,5-bisphosphate PI(3,4,5)P3 Phosphatidylinositol 3,4,5-trisphosphate PIP5KI Phosphatidylinositol 4-phosphate 5-kinase type I PLCγ2 Phospholipase Cγ2 PRR Pattern recognition receptor PS Phosphatidylserine RIPK1 Receptor-interacting serine/threonine-protein kinase 1 sCD14 Soluble form of CD14 TAK1 Transforming growth factor-beta-activated kinase 1 TBK1 TANK binding kinase 1 TIR Toll/IL-1R homology domain TLR Toll-like receptor TNF-α Tumor necrosis factor-α Vps Vacuolar protein sorting-associated protein Introduction The mammalian family of Toll-like receptors (TLR) consists of thirteen members with TLR4 being the most extensively studied one. TLRs are representatives of pattern recognition receptors (PRR), so named for their ability to recognize evolutionarily conserved components of microorganisms, including bacteria, viruses, fungi and parasites, collectively called pathogen-associated molecular patterns (PAMPs). The recognition of a PAMP by a PRR triggers rapid inflammatory reactions essential for the innate immunity, as discussed in several previous exhaustive reviews [1–5]. TLR4 is activated by lipopolysaccharide (LPS, endotoxin), a major component of the outer membrane of Gram-negative bacteria. During infection, TLR4 responds to the LPS present in tissues and the bloodstream and triggers pro-inflammatory reactions facilitating eradication of the invading bacteria [6]. It has been indicated that TLR4 can also be activated by endogenous compounds called damage-associated molecular patterns (DAMPs), including high mobility group box protein 1 (HMGB1) and hyaluronic acid. These compounds are released during tissue injury and can activate TLR4 in non-infectious conditions to induce tissue repair [7, 8]. Altogether, apart from LPS and its derivatives, up to 30 naturally occurring agonists of TLR4 with various chemical structures have been postulated. However, only three of them, 13 A. Ciesielska et al. ­ i2+, the plant secondary metabolite paclitaxel, and disulfide N HMGB1 have been demonstrated to be direct activators of TLR4, while the others can act as chaperones for TLR4 or promoters of LPS internalization [7, 9, 10]. Nevertheless, the impact of endogenous DAMPs on the TLR4 activity broadens the spectrum of pathophysiological conditions involving the TLR4-induced pro-inflammatory responses far beyond infectious diseases. TLR4 binds LPS with the help of LPS-binding protein (LBP) and CD14, and an indispensable contribution of the MD-2 protein stably associated with the extracellular fragment of the receptor (Fig. 1). The requirement for MD-2 for TLR4 activation by LPS was established shortly after the identification of TLR4 as the LPS receptor, since virtually no responses to LPS were detected in macrophages derived from MD-2−/− mice [11, 12]. The binding of an LPS molecule to the TLR4/MD-2 complex involves acyl chains and phosphate groups of lipid A, the conserved part of LPS and the main inducer of pro-inflammatory responses to LPS [13, 14]. Hexa-acylated and diphosphorylated LPS, like Escherichia coli LPS (O111:B4), is one of the most potent agonists of TLR4 whereas under-acylated LPS and dephosphorylated LPS species have a weaker pro-inflammatory activity especially in human cells [15]. Structural determinants of this phenomenon are found in the TLR4/MD-2 complex and also in CD14 protein [13, 16], as discussed in the following sections. Under-acylated and dephosphorylated LPS is synthesized by commensal bacteria which colonize human intestines, like Bacteroides thetaiotaomicron, which evade recognition by PRR [17] and are crucial for the maintenance of the intestinal immune balance [15]. In addition, the impermeability of the intestine epithelium to LPS is an important factor preventing its egress into the bloodstream [18]. On the other hand, during infection, deacylation and dephosphorylation of bacterial LPS is important for the termination of inflammatory responses, as discussed in detail below in the section concerning LPS detoxification. An exaggerated and uncontrolled pro-inflammatory signaling triggered by TLR4 during infection can lead to sepsis, septic shock, and death [19]. Infections with Gram-negative bacteria, including E. coli and Pseudomonas aeruginosa, are the prevailing cause of severe sepsis in humans [20]. In addition, low doses of LPS derived from the gut microbiota can in certain conditions enter the bloodstream and evoke so-called metabolic endotoxemia leading to a chronic low-grade TLR4-dependent inflammation which contributes to the development of metabolic diseases, such as type 2 diabetes [18, 21–23]. Prolonged activation of TLR4 is also linked with several human hereditary and neurodegenerative diseases and also with autoimmune diseases and cancer [24, 25]. On the other hand, an experimental exclusion of TLR4triggered signaling during low-grade polymicrobial sepsis TLR4 and CD14 trafficking and its influence on LPS‑induced pro‑inflammatory signaling 1235 Fig. 1  Pro-inflammatory signaling pathways of TLR4. TLR4 activates the MyD88-signaling pathway at the plasma membrane and after a CD14-dependent endocytosis initiates the TRIF-dependent cascade. Via activated NF-κB TLR4 also contributes to the activation of the cytosolic NLRP3 inflammasome. See text for details resulted in an impaired bacterial clearance and thereby worsened organ injury leading to a higher mortality of mice [26, 27]. Thus, the cellular level of TLR4 and its signaling activity have to be tightly regulated to be beneficial rather than harmful to the host. TLR4 is expressed in immune cells mainly of myeloid origin, including monocytes, macrophages and dendritic cells (DC), and also in some non-immune cells, like endothelial cells [28]. Most myeloid cells express also high amounts of plasma membrane-anchored CD14 [29, 30], which facilitates the activation of TLR4 by LPS and controls the subsequent internalization of the LPS-activated TLR4 important for receptor signaling and degradation. With the help of CD14, TLR4, unlike all the other TLRs, triggers two signaling pathways called the MyD88-dependent and the TRIF-dependent one after the adaptor proteins involved in their induction [31–34]. These signaling pathways lead to the production of two sets of pro-inflammatory cytokines which only partially overlap [35–39]. The MyD88- and TRIF-dependent signaling pathways are triggered consecutively and linked with the redistribution of the LPS-activated TLR4 from the plasma membrane to endosomes [40]. The translocation of TLR4 to the cell interior and its further lysosomal degradation facilitate termination of the inflammatory response [41]. In this review, we discuss the mechanisms of the internalization and intracellular trafficking of TLR4 and CD14, and also of LPS in immune cells. We emphasize the mechanisms regulating the trafficking of TLR4 and CD14 and thereby affecting the TLR4-induced pro-inflammatory signaling, its magnitude, duration and eventually also TLR4 degradation. Finally, we discuss data pointing to how disturbances in those processes contribute to the development of several human diseases. Signaling pathways triggered by TLR4 Activation of TLR4 by LPS is preceded by a chain of reactions which aim at converting LPS aggregates, derived from bacteria, into LPS monomers concentrated at the cell surface in the vicinity of the receptor (Fig. 1). These reactions are initiated by the serum LBP protein which binds to LPS aggregates (micelles) and, in the most typical scenario, facilitates subsequent extraction of LPS monomers 13 1236 by CD14 and the delivery of the LPS to the TLR4/MD-2 complex [42–44]. In agreement, blocking of LBP with an anti-LBP antibody inhibited the LPS-induced TLR4 signaling and endocytosis of the receptor [45]. Recently, the whole process of LPS transfer from micelles via LBP, CD14 to TLR/MD-2 was visualized at a single-molecule resolution [46]. That study indicated that one LBP molecule bound to an LPS micelle mediates several rounds of LPS transfer to CD14 molecules [46]. CD14 is a glycosylphosphatidylinositol (GPI)-anchored protein localized in nanodomains of the plasma membrane enriched in cholesterol and sphingolipids, so-called rafts, which are, therefore, considered as sites of TLR4 activation [3]. CD14 is detected predominantly on the surface of myeloid-lineage cells; however, low amounts are also found in non-myeloid cells, e.g., hepatocytes, adipocytes, corneal, and intestinal epithelial cells [29, 30, 47–51]. These latter cells produce mainly a soluble form of CD14 lacking the GPI anchor (sCD14) [47, 48]. However, mechanisms of the release of sCD14 were studied mainly in immune cells, where limited proteolysis of the membrane form of CD14 and also proteolysis-independent sCD14 formation have been detected [52]. The proteolysis of the membrane-bound CD14 can be carried out on the cell surface (so-called shedding) or intracellularly, after phagocytosis of bacteria when a following secretion of a truncated 13-kDa form of sCD14, called presepsin, has also been detected [53, 54]. The release of sCD14 that is independent of its membrane-bound form should not be neglected since patients suffering from paroxysmal nocturnal hemoglobinuria with defects in GPI anchor synthesis, who do not express membrane-anchored CD14, have normal levels of serum sCD14 [55, 56]. Both membrane-bound and soluble CD14 can transfer the LPS molecule to the TLR4/MD-2 complex [44, 46, 57]. LPS is bound in the N-terminal hydrophobic pocket of CD14 which differs in some details of structure between human and murine CD14 [16, 58]. The CD14 hydrophobic pocket probably accommodates up to five acyl chains of the endotoxin while the remaining one can facilitate the association of the CD14-LPS complex with MD-2 [16]. It has been found recently that the transfer of LPS from CD14 to MD-2 in the TLR4/MD-2 complex is facilitated by TLR4 which probably forms a transient intermediate with CD14, LPS or both [46]. A crystallographic analysis of the human TLR4/MD-2 complex with LPS bound has revealed that five of the six acyl chains of LPS are buried in the hydrophobic pocket of MD-2 while the sixth one interacts with TLR4 of another TLR4/MD-2 complex, and that the dimerization of the TLR4/MD-2 complexes is strengthened by ionic bonds between the phosphate group of lipid A and the neighboring TLR4 molecule [13]. Accordingly, removing of one or two acyl chains from the LPS molecule converts it 13 A. Ciesielska et al. from an agonist to an antagonist of human TLR4, as was shown using LPS of Neisseria meningitidis H44/76, Pseudomonas aeruginosa, and Yersinia pestis [59–61]. Interestingly, murine TLR4 is activated with similar efficiency by tetra-, penta- and hexa-acylated LPS from the same bacteria [59–61]. A contrasting ability to activate human and murine TLR4/MD2 complex has also been shown for tetraacylated lipid IVa which turned out to be an antagonist for the former and a weak agonist for the latter [62]. The agonistic activity of lipid IVa toward murine TLR4 is due to its unique ionic interactions at the dimerization interface of the murine receptor that cannot be formed with human TLR4 [63]. Moreover, the length of the acyl chains as well as their saturation also seem important for the pro-inflammatory activity of LPS, as has been postulated based on studies of the immunomodulatory activity of lipid A and cardiolipin analogues [64, 65]. Accordingly, penta-acylated diphosphorylated LPS with one unsaturated acyl chain from Rhodobacter spheroides and its synthetic tetra-acylated lipid A derivative, Eritoran, are antagonists for both human and mouse TLR4 [66–68]. The interaction of TLR4/MD-2 with two molecules of an agonistic LPS species induces dimerization of the ectodomains of two TLR4 molecules which acquire an “M-shape” with their intracellular fragments put in juxtaposition [12, 13, 69]. Each intracellular fragment contains a Toll/IL-1R homology (TIR) domain prone to homotypic interactions with TIR domains of four adaptor proteins. When in the plasma membrane, TLR4 interacts with the first adaptor pair, TIRAP (also called Mal [70]) and MyD88 [71, 72]. Apart from the TIR domain, TIRAP also carries a domain enriched in basic and aromatic residues that interacts with phosphatidylinositols (PIs) and phosphatidylserine (PS) [72–74]. In the plasma membrane, TIRAP can bind both PS and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) but only the latter interaction is required for TLR4 signaling [72, 73]. The TLR4-bound TIRAP recruits MyD88 which further binds interleukin-1 receptor-associated kinase (IRAK) 1 and 2 and a submembrane signaling complex called the myddosome is formed [72, 75, 76]. The assembled myddosome recruits E3 ubiquitin ligase TRAF6, which triggers a signaling cascade involving TAK1 kinase and leading, through the phosphorylation and activation of IκB kinases α/β (IKKα/β), to nuclear translocation of the NF-κB transcription factor. In addition, downstream of TRAF6 and TAK1, MAP kinases are phosphorylated to activate transcription factors AP-1 and CREB [77]. The signaling complex of TLR4 containing TIRAP and MyD88 also activates type I PI3-kinase which phosphorylates PI(4,5)P 2 to phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) and triggers activation of Akt [74, 78]. Collectively, the MyD88-dependent signaling pathway induces expression of genes encoding pro-inflammatory mediators, such as tumor necrosis factor α (TNF-α), TLR4 and CD14 trafficking and its influence on LPS‑induced pro‑inflammatory signaling interleukin (IL)-6, cyclooxygenase 2, and type III interferons (IFNλ1/2), the latter contributing to epithelial barrier integrity, which is crucial for host defense [35, 79–81]. In addition, the MyD88-dependent signaling pathway also participates in the production of anti-inflammatory mediators, like IL-10 helping to terminate the inflammation [35, 82]. It is worth mentioning that several factors negatively affect the MyD88-depenedent signaling cascade to prevent an exaggerated production of pro-inflammatory mediators. These include A20 ubiquitin-modifying enzyme, the BCAP-PI3kinase–Akt axis, and Lyn tyrosine kinase [83–86]. Recent studies indicate that the LPS-induced MyD88dependent signaling triggers and also modulates the cell metabolism. It was shown that TRAF6 interacts with TBK1, the kinase which in turn activates Akt kinase leading to a rapid enhancement of glycolysis [87, 88]. The LPS-stimulated glycolysis and subsequent synthesis of acetyl-CoA and de novo synthesis of fatty acids can facilitate, respectively, histone acetylation required for gene transcription and expansion of the endoplasmic reticulum and Golgi apparatus necessary for intense production and secretion of cytokines [87, 89]. The MyD88-dependent signaling is followed by internalization of TLR4. Simultaneously, TIRAP and MyD88 dissociate from the membrane allowing TLR4 to bind in the endosome a second set of TIR-containing adaptor proteins, TRAM and TRIF [90]. In this tandem, TRAM is a bridging adaptor facilitating the interaction of TRIF with TLR4 [91]. In unstimulated cells, both TLR4 and TRAM localize to the plasma membrane, endosomes, endocytic recycling compartment (ERC), and the Golgi apparatus [40, 92–95]. However, the presence of TRAM and TLR4 in the same cellular compartment is not sufficient for their interaction. Detailed studies have revealed that in the plasma membrane, TRAM localizes to regions enriched in CD14 rather than TLR4 and stimulation of cells with LPS induces the interaction between TLR4 and TRAM in endosomes [94]. TRAM binds to membranes via a myristoyl residue attached to Gly2 (after the removal of the N-terminal methionine) and via a neighboring polybasic domain that mediates its interaction with various PIs and phosphatidic acid [40]. Both regions are required for the targeting of TRAM to the plasma membrane; however, only the N-myristoylation governs its location to endosomes [40]. Furthermore, only the N-myristoylation is indispensible for the TRAM involvement in the endosomal signaling pathway of TLR4 [40, 93]. Nonmyristoylated Gly2Ala TRAM acted as a dominant negative mutant interfering with this signaling pathway of TLR4 [93]. The TRIF-dependent signaling pathway of TLR4 includes activation of the ubiquitin ligase TRAF3 followed by the activation of non-canonical IKK kinases: TANK binding kinase 1 (TBK1) and IKKε. TBK1 phosphorylates the pLxIS consensus motif of TRIF that is necessary to recruit 1237 interferon regulatory factor (IRF) 3. IRF3 is also phosphorylated by TBK1 and then dissociates from TRIF, dimerizes and translocates to the nucleus [96]. Finally, IRF3 and to a lower extent also TBK1-activated IRF7 induce expression of genes encoding type I IFN, the chemokine CCL5/ RANTES and interferon-regulated genes as that encoding the chemokine CXCL10/IP-10 [35, 80, 91, 97]. This pathway also participates in the production of the anti-inflammatory interleukin IL-10 [98]. In addition, TRIF induces late activation of NF-κB via recruitment and activation of TRAF6 [99] or receptor-interacting serine/threonineprotein kinase 1 (RIPK1) [100]. RIPK1 interacts with the RIP-homotypic motif (RHIM) present in the C-terminus of TRIF, which is also recognized by RIPK3 [101]. RIPK1 and RIPK3 are engaged in LPS-induced ERK1/2 activation and cytokine production but in certain conditions can also mediate necrotic cell death [102–104]. Ultimately, TLR4 is degraded in lysosomes [41]. It has recently been established that TLR4 is also involved in so-called canonical activation of the NRLP3 inflammasome. The LPS-induced activation of this inflammasome via the canonical and also via a non-canonical, hence not involving TLR4, pathways has been intensively studied and discussed in excellent reviews [105–108]. In brief, the NLRP3 inflammasome is a multimeric complex of NRLP3, ASC, NEK7 and pro-caspase-1, which ultimately serves to promote autoproteolysis and activation of caspase-1. In turn, caspase-1 cleaves pro-IL-1β and pro-IL-18 to their secreted forms. Caspase-1 also catalyzes the proteolysis of gasdermin D which binds to the inner surface of the plasma membrane and forms pores which can lead to cell death via pyroptosis or release of IL-1β from living cells [109]. Activation of TLR4 in human and murine macrophages serves as a priming signal inducing expression of NRLP3 and pro-IL-1β and posttranslational modifications of NRLP3. The assembly of a fully active inflammasome is triggered by a second stimulus provided by diverse PAMPs and DAMPs. The exact mechanism leading to the inflammasome formation in response to these structurally different molecules is not known, but they all seem to induce cellular stress manifested by, e.g., ­K+ efflux, mitochondria dysfunction, lysosome damage, which is then somehow detected by NLRP3. Since the expression of NRLP3 is triggered in an NF-κB-dependent manner, both signaling pathways of TLR4 can in principle be involved. Accordingly, caspase-1 cleavage and activation occurred in LPS-stimulated macrophages derived from MyD88- or TRIF-knock-out mice while the double knock-out did not support it [110, 111]. Activation of the non-canonical inflammasome does not require TLR4 participation, but is induced by LPS present in the cytosol. LPS directly binds and activates caspase-11 (human caspase-4 and -5) which next activates the primed NLRP3 inflammasome and induces IL-1 release 13 1238 and pyroptosis. It is unclear how LPS enters the cytosol; however, recent studies indicate that it can escape from endosomes which are formed during internalization of LPS-rich outer membrane vesicles (OMV) derived from Gram-negative bacteria or from phagosomes enclosing the bacteria [112]. It can also be internalized by the receptor for advanced glycation end-products (RAGE) after binding to HMGB1 [113]. In addition, in human and porcine monocytes (but not in murine cells), LPS induces NLRP3 activation and IL-1β release without the second stimulus [114]. Gaidt et al. called this phenomenon the “alternative inflammasome” and found that it is induced by TLR4-dependent activation of the TRIFRIPK1-capsase-8 axis upstream of NLRP3 and caspase-1. While it ultimately leads to IL-1β secretion, no concomitant pyroptosis of monocytes occurs [115]. Recently, the alternative activation of inflammasome by apolipoprotein C3 and TLR2/TLR4 heterodimer has been described, pointing to its involvement in sterile inflammation triggered by triglyceride-rich lipoproteins [116]. In addition to LPS-induced cytokine production, stimulation of macrophages and DC with the endotoxin induces of adaptive immune responses which are executed by T and B lymphocytes. The initiation of the adaptive immune responses follows activation of various TLRs and involves antigen presentation in the context of major histocompatibility complex (MHC) class I and II, as reviewed by [5, 117]. It has been established that activation of TLR4 by LPS in both murine DC and macrophages induces upregulation of costimulatory molecules (CD40, CD80, and CD86) on the cell surface which are required for antigen presentation for T lymphocytes. However, significant differences were found in the signaling pathways leading to this phenomenon. Namely, in macrophages, the upregulation depends strictly on the TRIF-dependent pathway, whereas in DC both the MyD88- and TRIF-dependent ones are involved [118–120]. The increased cell surface presence of the costimulatory molecules and also of MHC II is a hallmark of DC maturation required for antigen presentation by these cells. LPS upregulates the cell surface level of MHC II at various steps of the formation of antigen-MHC II complexes, including enhanced lysosomal acidification, proteolysis yielding antigens, and transport of the complexes to the surface of DC [121, 122]. Beside the contribution to the upregulation of MHC II and costimulatory molecules, the TLR4-triggered MyD88-dependent signaling in DC also induces production of cytokines leading to Th1 cell polarization and also facilitates fusion of MHC I-bearing recycling endosomes with phagosomes to allow cross-presentation of antigens during infection [123]. The signaling pathways triggered by TLR4 described above are best characterized in monocytes, macrophages, and DC; however, it has to be emphasized that certain 13 A. Ciesielska et al. aspects of the LPS-induced responses vary between DC and macrophages. In murine DC, LPS induces activation of nuclear factor of activated T cells (NFAT) and apoptosis of fully matured DC [124]. Also in these cells, CD11b contributes significantly to TLR4 activation [125]. Additionally, murine DC vary from human ones since only selected subtypes of the latter expresses TLR4 and CD14, as described in more detail in the next chapter. A cell specificity of the LPS-induced responses is also observed in other immune cells, including human and murine granulocytes, mast cells, and lymphocytes which express low amounts of TLR4. Among those cells, CD14 is found in neutrophils and murine basophils [29, 30, 126–129]. Thus, due to a lack of CD14 and low content of TLR4/MD-2 and TRAM in mast cells, the TRIF-dependent pathway could not be detected in cells of murine origin [130]. Therefore, these cells stimulated with LPS produce TNF-α but not IFN-β [131, 132]. Activation of TLR4 in neutrophils leads to ROS generation, cytokine production and other responses which are cell specific, like autophagy and modulation of cell survival [30, 133–136]. In addition, in murine neutrophils, LPS induces production and secretion of histamine in a PI3K-dependent manner [137]. Moreover, LPS can stimulate neutrophils to form NETs (neutrophil extracellular traps), i.e., extracellular fibers composed of decondensed chromatin and granule proteins which trap bacteria allowing their extracellular killing [138–140]. In summary, TLR4 activated by LPS triggers two consecutive signaling pathways which are correlated with a redistribution of the receptor: the MyD88-dependent signaling is triggered by TLR4 localized to the plasma membrane, while the TRIF-dependent one by the TLR4 internalized in endosomes. The two pathways lead to a synchronized production of pro- and anti-inflammatory mediators, contribute to the activation of NLRP3 inflammasome, modulate cell metabolism, initiate adaptive immune responses and other cell type-specific reactions; eventually lysosomal degradation of TLR4 takes place. This course of events suggests that the TLR4-induced pro-inflammatory reactions can be regulated by the rate of its endocytosis and trafficking through the endo-lysosomal compartment. Mechanisms controlling internalization of TLR4 Endocytosis of TLR4 is required for the TRIF-dependent pro-inflammatory signaling to occur and also for the following degradation of the receptor and termination of the signaling [40, 41]. Somewhat surprising, it has been established that the LPS-induced internalization of TLR4 is independent of its signaling activity. Thus, a TLR4 mutant lacking the intracellular part, hence the TIR domain, underwent TLR4 and CD14 trafficking and its influence on LPS‑induced pro‑inflammatory signaling internalization despite being unable to trigger myddosome formation in murine bone marrow-derived macrophages (BMDM) stimulated with LPS. The amount of the truncated receptor in the plasma membrane progressively declined, similarly as in the case of wild type TLR4 [17]. On the contrary, the extracellular domain of TLR4 was indispensable for its LPS-induced endocytosis and further studies have indicated that in fact the interaction of MD-2 with CD14 drives the uptake of the receptor. Thus, the impact of MD-2 on LPS-induced signaling stems from its role in the dimerization of the TLR4/MD-2 complexes during LPS binding and the contribution to the endocytosis of TLR4 [17]. In most cases, the LPS-induced internalization of TLR4 is controlled by CD14 (Tab. 1) [32, 33]. Exceptions include TLR4 endocytosis followed by the TRIF-dependent signaling induced by a TLR4/MD-2 agonistic antibody (UT12) or a synthetic small-molecule TLR4 ligand (1Z105) [141]. And while phagocytosis of the Gram-negative E. coli did occur 1239 in DC derived from CD14-knock-out mice [33], no LPSinduced endocytosis of TLR4 took place in DC or BMDM derived from those animals. Accordingly, the TRIF-dependent signaling was abolished, while the MyD88-dependent one was not affected by the CD14 depletion especially in cells stimulated with so-called rough chemotype (devoid of the O-polysaccharide chain) of LPS [31, 32, 34]. In cells poor in CD14, such as murine splenic B lymphocytes, TLR4 does not undergo endocytosis [33]. The dependence of TLR4 internalization on CD14 has been confirmed by recent studies on CD14 glycosylation. Inhibition of CD14 core fucosylation caused by depletion of α-(1,6)-fucosyltransferase impaired CD14 and TLR4 endocytosis and thereby the TRIF-dependent signaling. It was found that the interference with CD14 fucosylation led to a reduction of its cell surface level which was considered the main reason of the impaired TLR4 endocytosis [142, 143]. Conversely, the increase of Table 1  Effects of down-regulation of selected proteins involved in TLR4 endocytosis and degradation on TLR4 internalization and signaling Proteina CD14 TRAM PLC-γ2/Ca2+ Syk PI3K p110δ Arf6 Dynaminb Dynamin Clathrinb Dab2 Hrs Lystc Vps33B GMFGd Rab7a Rab7bd Rab11 Rab10 TMED7 TAG​ Effect on TLR4 internalization MyD88-dependent pathway TRIF-dependent pathway ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ nd ↓ ↓ ↑ nd – – ↓ nd – nd ↓ nd nd ↓ nd ↑ – – ↑ ↓ – ↓ ↑ ↓ – – ↑ – ↑ ↑ nd ↑ – ↓ – – ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ nd ↓ ↓ ↑ nd ↓ ↑ ↑ ↓ ↑ ↓ ↓ ↑ ↑ Cells References macrophages, DC HEK293, macrophages macrophages DC macrophages DC J776, Raw264.7, macrophages, DC DC, macrophages, Raw264.7 J774, actrocytes HEK293 Raw264.7, J774, macrophages astrocytes Raw264.7 HEK293, monocytes macrophages, DC macrophages macrophages macrophages Raw264.7, macrophages monocytes, HEK293 macrophages, Raw264.7 HEK293, THP-1, macrophages, PBMCs HEK293, THP-1, PBMCs [17, 33, 160] [40, 93] [33] [33, 150] [33] [74] [73, 152, 153] [40, 74, 155] [159, 160] [41] [155, 160, 265] [159] [166] [41] [183] [180] [196] [183] [184] [95] [179] [182] [181] ↓ Down-regulation, ↑ upregulation, nd no data a b c d In most cases, proteins were down-regulated by knock-out or knock-down of encoding genes Inhibition of the protein caused by a drug treatment Deleterious mutation Overexpression of the protein had opposite effects on the MyD88- and TRIF-dependent pathways 13 1240 CD14 level during maturation of murine DC accelerates the LPS-induced endocytosis of TLR4 [33]. Notably, it has been established that CD14 undergos low-rate constitutive endocytosis also in unstimulated cells [17]. The decreasing pool of cell-surface CD14 is replenished by the newly synthesied protein. Binding of LPS to TLR4/MD-2 and the concomitant interaction of MD-2 with CD14 converts the TLR4/MD-2/LPS complex into a cargo of CD14. Simultanously, the rate of CD14 internalization is accelerated [17, 144]. On the basis of those data, CD14 has earned the name of “transporter associated with the execution of inflammation” (TAXI) [17]. The mechanism of the CD14-dependent internalization of TLR4 remains to be revealed, but it is likely linked with the raft localization of CD14, activation of the Syk tyrosine kinase, and local turnover of PI(4,5)P2 [33, 50, 145]. CD14 triggers biphasic generation of PI(4,5)P2 in LPS-stimulated macrophages and the newly generated PI(4,5)P2 accumulates in the raft fraction of these cells. The PI(4,5)P2 generation correlated with a biphasic activation of NF-κB and was required for maximal production of cytokines in the both signaling pathways of TLR4 [145, 146]. An increase of the PI(4,5)P2 level can be needed for the recruitment of TIRAP to the plasma membrane, while the following PI(4,5)P2 hydrolysis/phosphorylation is required for the TLR4 endocytosis to occur. The uptake of TLR4 can be initiated by the Syk kinase which binds to the immunoreceptor tyrosinebased activation motif of DAP12 which is an adaptor of CD300b receptor, and/or the γ chain of Fcε receptor [147]. Subsequently, Syk activates phospholipase Cγ2 (PLCγ2). Indeed, inhibition/knock-down of Syk or PLCγ2 abolished TLR4 internalization in LPS-stimulated murine BMDM and DC [33, 50, 147]. It deserves to be mentioned that Syk/ PLC-γ2 inhibitors blocked not only the LPS-, but also the UT12-induced endocytosis of TLR4 that proceeded without a CD14 involvement [141]. PLCγ2 cleaves PI(4,5)P2 to DAG and inositol 1,4,5-trisphosphate (­ IP3) which induces C ­ a2+ release from the endoplasmic reticulum. Inhibition of this process by 2-ABP, an antagonist of ­IP3 receptor localized predominantly in the endoplasmic reticulum, blocked TLR4 endocytosis and the TRIF-dependent signaling [148]. PI(4,5) P2 is also a substrate for class I PI3-kinase which phosphorylates it to PI(3,4,5)P3. Mutation of the p110δ catalytic subunit of PI3-kinase in murine DC slowed down the rate of LPS-induced endocytosis of TLR4 while phagocytosis of E. coli was not affected [74]. In addition, CD14 controls the influx of extracellular ­Ca2+ mediated by TRPM7, a plasma membrane cation channel with kinase activity. A knock-out of TRPM7 inhibited endocytosis of CD14/TLR4 in murine BMDM. Furthermore, phosphorylation and nuclear translocation of IRF3 and also of NF-κB were inhibited and the following production of the TRIF-dependent cytokines was reduced. These processes were depended on the influx of 13 A. Ciesielska et al. extracellular ­Ca2+ rather than on the release of ­Ca2+ from intracellular stores, although an input of the latter to the regulation of TLR4-induced signaling was not precluded [149]. Since the downstream effectors of Syk, PLCγ2 and PI3kinase, are involved in a broad spectrum of cellular processes [74, 150, 151], their activity is likely to affect various other aspects of TLR4-induced signaling in addition to the receptor endocytosis. Nevertheless, the impaired internalization of TLR4 observed after inhibition/mutation/knockdown of those enzymes correlated with a down-regulation of the TRIF-dependent signaling reflected by reduced IRF3 phosphorylation and impaired expression of genes encoding ISG54/IFIT-2, TRAIL, CCL5/RANTES, and IFN-β. The MyD88-dependent production of cytokines was not affected or was even elevated at the expense of the TRIF-dependent one [33, 74, 148]. Another important protein involved in PIs turnover taking part in the LPS-induced TLR4 endocytosis and signaling is the small GTPase ADP-ribosylation factor 6 (Arf6). Arf6 activates PI4-phosphate 5-kinase type Iα (PIP5KIα) which phosphorylates PI(4)P to PI(4,5)P2 [73]; therefore, its activity can modulate TLR4 signaling, endocytosis and trafficking. Indeed, PIP5KIα and PIP5KIγ were found to colocalize with CD14 in murine macrophage-like J774 cells, and their silencing reduced LPS-dependent production of TNF-α and CCL5/RANTES [145]. In Arf6-deficient Raw264.7 cells, the LPS-induced activation of NF-κB was lower than in their wild type counterparts suggesting a down-regulation of the MyD88-dependent signaling due to insufficient PI(4,5)P2 generation in the plasma membrane [73, 152]. Moreover, in these conditions, the cell surface level of TLR4 was also elevated and increased even further after stimulation of the cells with LPS likely due to an inhibited endocytosis of the receptor [152]. Furthermore, a depletion or inhibition of Arf6 blocked the TRIF-dependent signaling, which was reflected by a reduced phosphorylation of IRF3 and decreased expression of Ifnβ1 [152, 153]. Recent studies have indicated that the endosomal signaling of TLR4 is negatively regulated by prostaglandin E2 in thioglycollate-elicited peritoneal murine macrophages. Inhibition of prostaglandin E2 receptor EP4 or inhibition of prostaglandin E2 synthesis enhanced the LPS-induced internalization of TLR4 and Syk activation, and consequently augmented the TRIF-dependent signaling [86]. Endocytosis of TLR4 is also negatively regulated by CD13 metallopeptidase [154]. CD13 associates with TLR4 in resting murine BMDM and DC and its expression is enhanced in LPSstimulated cells. CD13 undergoes LPS-induced endocytosis together with TLR4 and CD14 and these proteins colocalizes in Rab5-positive endosomes. However, a knock-out of CD13 enhanced TLR4 endocytosis in macrophages and increased IRF3 activation and IFN-β production. Accordingly, mice lacking CD13 displayed enhanced IFN-β-triggered signaling TLR4 and CD14 trafficking and its influence on LPS‑induced pro‑inflammatory signaling leading to exacerbated ischemic muscle injury. The mechanism of the CD13 involvement in TLR4 endocytosis remains unknown, but it could be related to the raft localization of CD13 [154]. The LPS-induced internalization of TLR4 occurs via clathrin-independent and/or clathrin-dependent pathways, while E. coli bacteria undergo phagocytosis in which TLR4 is involved [3, 33, 41, 155, 156]. The TLR4 endocytosis depends on dynamin, a GTPase responsible for pinching off of clathrin-coated buds, some macro- and micropinosomes, and phagosomes [157, 158] inhibition of dynamin with dynasore prevented LPS-induced internalization of TLR4 in several cell types, including macrophages [40, 141, 155, 159]. Accordingly, in such conditions, the TRIF-dependent signaling was inhibited [40, 74, 95, 155, 159–161]. In addition, dynasore attenuated the LPS-induced cleavage of caspase-3 in murine microglia and almost completely abolished expression of IL-1β in these cells and in rat astrocytes overexpressing TLR4 [156, 159]. In HEK293 cells expressing TLR4/CD14 and a dominant negative dynamin II mutant (Dyn K44A), the NF-κB activation was upregulated in comparison to cells overexpressing wild-type dynamin, suggesting an upregulation of the MyD88-dependent signaling [41]. In contrast to the well-defined role of dynamin in LPSinduced TLR4 internalization, the role of clathrin in this process is more puzzling. Early electron microscopy studies suggested that endocytosis of LPS and CD14 is mainly clathrin-independent, yet no direct observation of TLR4 was possible at that time [162, 163] (see below). On the other hand, at least partial colocalization of TLR4 with transferrin, a typical cargo of clathrin-coated vesicles, was observed in HEK293 cells and in Ba/F3 cells transfected with TLR4/MD-2 and CD14 and stimulated with LPS for 45–60 min [41, 94]. In addition, partial colocalization of TLR4 with clathrin at the plasma membrane was observed in the HEK293 transfectants and in U373 glioma cells transfected with TLR4 and CD14 [164]. The authors claimed that in the spots of TLR4 that were positive for clathrin also CD14 was accumulated; however, it has to be noticed that they did not label CD14 directly but only observed Cy5labeled LPS. In addition, in all the studies cited above CD14 and TLR4/MD-2 transfectants were used, which warrants their cautious interpretation in view of the relatively low level of TLR4 expression in native macrophages. Nevertheless, a line of studies using inhibitors of clathrin-dependent endocytosis support its contribution to TLR4 internalization. Monodansylcadaverine and chlorpromazine inhibited the LPS-induced internalization of TLR4 in Raw264.7 cells and in rat astrocytes overexpressing TLR4 [155, 159, 165]. Chlorpromazine also reduced production of CCL5/ RANTES and IFN-β in those cells, and of IFN-β in murine DC and macrophages [155, 159, 165]. Similarly, incubation of J774 cells with another clathrin inhibitor, pitstop-2, 1241 reduced LPS-induced TNF-α and CCL5/RANTES production; however, the effect was weaker than that of dynasore [160]. Similarly, silencing of raftlin, a protein interacting with the heavy chain of clathrin and with clathrin-related adaptor protein AP-2 also reduced TLR4 internalization and negatively regulated the TRIF-dependent signaling pathway in human and murine cells poor in CD14 [165]. This additionally proves an engagement of clathrin in the internalization of TLR4 and points to the role of raftlin in this process [165]. Recent studies have revealed that disabled-2 (Dab2), an adaptor protein involved in the recruitment of some transmembrane receptors, e.g., low-density lipoprotein receptor, to clathrin-coated pits can also affect the TLR4 internalization [166]. Silencing of Dab2 in Raw264.7 cells increased the surface level of TLR4 in unstimulated cells but accelerated its LPS-induced endocytosis. In accordance, phosphorylation of IRF3 and expression of TRIF-dependent genes, including Cxcl10, Ifit1, Ccl5 and Ifnβ1, was upregulated in these cells. Since TLR4 lacks a binding site for Dab2, the authors postulated that at steady state Dab2 binds and sequesters clathrin, thereby preventing TLR4 endocytosis. Stimulation of cells with LPS induces phosphorylation of Dab2 probably causing its dissociation from the plasma membrane and the following release of clathrin, and thereby allowing the clathrin-dependent internalization of TLR4. Thus, Dab2 can act as a negative regulator of TLR4 internalization [166]. Finally, the difference in the regulation of TLR4 endocytosis between murine macrophages and DC deserves attention. It was mentioned above that murine DC but not macrophages can phagocyte E. coli without a CD14 involvement [33]. Additionally, due to the lower level of CD14 in DC than in macrophages only in DC the endocytosis of TLR4 is facilitated by CD11b integrin [125]. CD11b in DC promotes both the MyD88-dependent and TRIF-dependent signaling of TLR4 in addition to its engagement in the receptor endocytosis. CD11b is also crucial for TLR4-triggered activation of the adaptive immunity by T lymphocyte [125]. The difference in the responses to LPS between macrophages and DC can be even more pronounced in humans. Only one human conventional (myeloid) DC subtype, cDC2 (also called CD1C +), expresses TLR4 among them only subsets ­CD5low/high expresses CD14, whereas murine conventional DC express both Tlr4 and Cd14 [29, 124, 167–172]. In addition, in human DC and also monocytes, TLR4 seems to be mainly intracellular [95, 173]. This suggests that in human DC, details of CD14-TLR4 signaling and trafficking can differ from those in their murine counterparts or in human macrophages. In addition, recent studies of murine mast cells have revealed that TLR4 trafficking in these cells can be regulated by huntingtin [174]. 13 1242 In summary, although many gaps in the understanding of the mechanisms controling TLR4 endocytosis remain, the crucial roles of CD14, PIs turnover, and dynamin in this process have been firmly established. Notably, modulation of the duration of the TLR4 presence at the plasma membrane after binding of LPS can affect the duration of the MyD88-dependent signaling and reciprocally also the activation of the TRIF-dependent endosomal pathway. The TLR4-triggered signaling is also modulated by the mechanisms controlling the intracellular trafficking of the receptor. Intracellular trafficking of TLR4: an overview TLR4 is synthetized in the endoplasmic reticulum where gp96, a paralog of Hsp90, is involved in proper folding of the receptor, while PRAT4A participates in its maturation (glycosylation). These proteins control folding and maturation of all TLRs [175, 176]. The newly synthetized TLR4 is transported to the Golgi apparatus, where it associates with MD-2; potentially TLR4 can reach the cell surface without an MD-2 assistance and then bind secreted MD-2 [177, 178]. In unstimulated cells, TLR4 can be detected not only in the endoplasmic reticulum, Golgi apparatus and the plasma membrane but also in Rab11-positive ERC [92, 94, 95, 179]. Latz et al. showed that in HEK293 cells transfected with TLR4 and MD-2, the receptor cycled between the Golgi apparatus and the plasma membrane. This suggested that the TLR4/MD-2 complex is internalized and directed to the Golgi apparatus to be transported back to the plasma membrane [92]. Later studies indicated that LPS binding changes the intracellular trafficking of TLR4 and revealed the complexity of this process. Thus, LPS-activated TLR4 is internalized and thereby its amount at the cell surface decreases with the duration of the stimulation [17, 40, 141]. TLR4 internalized by murine immortalized BMDM stimulated with 1 μg/ml LPS is first (after ~ 10 min of stimulation) detected in submembrane Rab5-positive vesicles related to early endosomes (Fig. 2, I) [180]. In murine microglia (BV2 cells), the TLR4 translocation to the Rab5-positive vesicles seems to be slower than in murine macrophages since a colocalization of TLR4 and Rab5 was visible only after 2 h of stimulation with the same LPS concentration [156], suggesting that the dynamics of TLR4 endocytosis is cell type specific. In the Rab5-positive endosomes TLR4 interacts with TRAM and activates the TRIF-dependent signaling cascade leading to the production of type I IFN and expression of IFN-induced genes. Next (after ~ 30–60 min of stimulation of immortalized BMDM) TLR4 is found in the membrane of Rab7-positive endosomes identified as late endosomes, where it also colocalizes with TRAM (Fig. 2, II) [180]. The endosomes marked by Rab7a or Rab7b are multifunctional in the context of TLR4 activity as the TRIF-dependent 13 A. Ciesielska et al. signaling can be maintained in some of them while others are sites of TLR4 degradation that progresses in lysosomes [94, 181–184], as discussed in more detail in the section below. Ubiquitination is likely a signal targeting TLR4 for degradation and it increases significantly after stimulation of cells with LPS, but the exact type of this modification has not been established [41]. Ubiquitinated TLR4 is recognized by hepatocyte growth factor regulated tyrosine kinase substrate (Hrs), an early endosome-associated component of the ESCRT-0 complex which, together with other ESCRT complexes, causes clustering of the receptors and targets them to intraluminal vesicles in multivesicular bodies (MVB) and consequently to lysosomal degradation (Fig. 2, III and IV) [41]. Beside the Rab5- and Rab7-positive endo-lysosome compartment, in Raw264.7 cells, LPS-activated TLR4 (~ 10–20 min) is also found in Rab10-positive vesicles identified as a subtype of recycling endosomes, while prior to the stimulation TLR4 is located to the Golgi apparatus where also Rab10 can be detected (Fig. 2, V). Silencing of Rab10 decreased the amount of TLR4 in the plasma membrane and diminished the activation of both the MyD88- and the TRIF-dependent signaling pathway. It has been, therefore, proposed that Rab10 controls the replenishing of the plasma membrane pool of LPS-activated TLR4 from its reservoir in the Golgi apparatus [179]. Additionally, recent data have indicated that in J774 cells stimulated with LPS the plasma membrane pool of TLR4 can be replenished by de novo synthetized protein as well as by recycling of previously internalized receptor. The recycling route of TLR4 probably engages the ERC and ERC-related vesicles in which internalized TLR4 is transported back to the plasma membrane. This process depends on the plasma membrane-localized SNARE proteins syntaxin 11 and its partner SNAP-23 which can regulate fusion of the TLR4-bearing vesicles with the plasma membrane. A knock-down of syntaxin 11 or SNAP23 suppressed the LPS-induced transport of TLR4 to the cell surface and promoted its lysosomal degradation [185]. Recycling of TLR4 can be down-regulated by TRPM7, as suggested by Granucci’s analysis of the TLR4 reappearance on the surface of TRPM7-depleted and LPS-stimulated macrophages [186]. Finally, unusual Rab11-dependent trafficking of an intracellular pool of TLR4 bypassing its plasma membrane localization was detected during phagocytosis of E. coli by human monocytes (Fig. 2, VI) [95, 187]. Rab11-positive vesicles also carry TRAM to the phagosomes, while in LPS-stimulated cells they participate in transporting GOLD domain-containing proteins to TLR4/LPS-harboring endosomes, thereby affecting the duration of the TRIFdependent signaling [181, 182]. Thus, the Rab11-positive compartment plays an important role in the regulation of TLR4 and CD14 trafficking and its influence on LPS‑induced pro‑inflammatory signaling 1243 Fig. 2  Intracellular trafficking pathways of TLR4 and TRAM. In unstimulated cells, TLR4 is present in the plasma membrane, Golgi apparatus and the ERC, whereas its adaptor protein TRAM is located in the ensosomes, the plasma membrane and Golgi apparatus. (I) After activation by LPS, TLR4 is internalized and translocates to Rab5- and EEA1-positive early endosomes where it interacts with TRAM and TRIF and initiates the TRIF-dependent signaling. (II) The association of TRAM with TLR4 and the activation of the TRIF-dependent pathway is sustained during maturation of early endosomes to Rab7a- or Rab7b-positive late endosomes. In late endosomes TMED7 and TAG disrupt the TRIF—TRAM interaction and facilitate termination of the signaling. (III) In early endosomes, Hrs and ESCRT recognize ubiquitinated TLR4 and sort it for degradation in late endosomes/MVB and lysosomes. (IV) TLR4 degrada- tion and signaling can be regulated by proteins involved in the functioning of the endo-lysosomal compartment, like Lyst, GMFG, Rab7a and Rab7b, and Vps33B. (V) Upon stimulation with LPS, the intracellular TLR4 originally associated with the Golgi apparatus is transported via Rab10-positive endosomes to the plasma membrane. (VI) The intracellular pool of TLR4 derived from the ERC can trigger the TRIF-dependent signaling at the phagosome membrane. Both TLR4 and TRAM are transported to the phagosome membrane with a contribution from Rab11. (VII) Possible intracellular transport of TRAM in Rab11-positive vesicles to the early endosomes. It is unknown whether the well-established protein trafficking pathway from the early endosomes to the Golgi apparatus (dashed gray arrow) can be used by TLR4 the TRIF-dependent signaling pathway of TLR4 discussed in detail in the following section. The data presented above indicate that activation of the TRIF-dependent signaling cascade is coupled with the trafficking of TLR4 through endo-lysosomal compartments. Therefore, the process of transformation of early endosomes into late endosomes and lysosomes, which is correlated with changes of the protein and lipid 13 1244 composition of their membrane, acidification of the lumen and enrichment in hydrolytic enzymes, is crucial for both the signaling and the degradation of TLR4. Regulation of TLR4 signaling in endo‑lysosomal compartment Progressive acidification of the endosome interior provides an optimal environment for the dissociation of ligands from their receptors and for the activity of hydrolytic enzymes. In agreement, acidification of endosomes was found to induce splitting of TLR4/MD-2 dimers and dissociation of LPS from the receptor [188, 189], albeit the pH optimum for those processes has not been determined. In general, activation of endosomal proteases facilitates degradation of the internalized proteins, but for endosomal TLRs a limited proteolysis catalyzed by cathepsins and asparaginyl endopeptidase is necessary for their dimerization and signal transduction [152, 190]. Notably, TLR4 does not undergo such modification and binding of LPS overcomes the repulsion of two receptor ectodomains and forces TLR4 dimerization [69]. The acidic pH of endosomes could facilitate induction of the TRIF-dependent signaling by TLR4. Such hypothesis has been put forward by Ganglioff and co-workers based on their analysis of the crystallographic structure of TLR3, a receptor residing in endosomes and triggering the TRAM/TRIFdependent signaling pathway. By analogy with TLR3, the acidic environment of endosomes (pH 5.5) would induce changes in the position of TLR4 ectodomains which after internalization face the endosome lumen. Concomitant conformational changes of the transmembrane and cytosolic regions of the receptor together with specific features of the endosome membrane, such as its high curvature and the presence of distinct species of PIs, could facilitate recruitment of TRAM to TLR4 [191]. Two approaches have been used to examine the role of endosome acidification in LPS-induced signaling of TLR4. Treatment of Raw264.7 cells with chloroquine, which neutralized the pH of the endosome interior, enhanced the depletion of LPS-activated TLR4 from the cell surface [155]. In agreement, chloroquine was shown to induce intracellular accumulation of LPS-activated TLR4 instead of its degradation, thus the stability of the TLR4/MD-2 complex was also prolonged [41, 189]. In chloroquine-treated Raw264.7 cells, the production of the MyD88- and TRIF-dependent cytokines was decreased, which was interpreted as a result of impaired recycling of TLR4 from endosomes to the plasma membrane [155]. On the other hand, knock-down of a ATP6V0D2, a subunit of the V-ATPase responsible for acidification of endosomes, attenuated the TRIF-dependent signaling pathway in 13 A. Ciesielska et al. Raw264 cells as reflected by a decreased expression of Ifnb1 and upregulated expression of the MyD88-dependent Tnfa, Il6 and Il12p40 [152]. In this case, the enhancement of the MyD88-dependent signaling and the reduction of the TRIF-dependent one correlated with reduced endocytosis of TLR4 and its prolonged maintenance on the cell surface. At the basis of this effect was an impaired interaction of the V-ATPase complex with Arf6 caused by the silencing of ATP6V0D2 [152], indicating that the regulation of TLR4 signaling via V-ATPase goes beyond controlling the endosome acidification and also involves its influence on the Arf6 activity. Ample data indicate that several other proteins which determine the functionality of the endo-lysosomal compartment, such as Lyst, Vps33B, GMFG and Rab7b, affect the TLR4-triggered signaling and the receptor degradation (Table 1). Lyst is a lysosomal trafficking regulator contributing to endo-lysosomal biogenesis and also takes part in terminal maturation of secretory vesicles [192]. Structurally, Lyst belongs to BEACH domain-containing proteins which are large scaffolding proteins associated with cellular membranes due to the interaction of their PH domain with phospholipids, mainly PIs [193]. In murine BMDM and bone marrow-derived DC (BMDC) bearing a mutation in the Lyst gene leading to the production of a dysfunctional Lyst, neither the LPS-induced MyD88-dependent activation of MAP kinases and IκB nor the TLR4 endocytosis were affected; however, the TRIF-dependent IRF3 phosphorylation and its translocation to the nucleus were impaired. In agreement, that deleterious mutation of Lyst decreased the production of IFN-β, TNFα, and IL-12. Similar effects were observed in THP-1 cells of human origin. In vivo studies of pulmonary inflammation in mice showed that the Lyst dysfunction led to a lower production of TNF-α and IFN-β after administration of LPS, and protected the animals from endotoxininduced lethal shock. This suggests that the dysfunction of the endo-lysosomal compartment observed in cells expressing the mutated form of Lyst inhibited the TRIF-dependent signaling. One can only speculate whether this impairment resulted from a faster degradation of TLR4 or an inefficient formation of its endosomal signaling complex. Later studies on the phagocytosis of E. coli and LPS-coated beads by BMDC expressing the mutated Lyst indicated that the recruitment of Rab7 (also called Rab7a) to maturing phagosomes, but not their acidification, was affected [183]. Vacuolar protein sorting-associated protein (Vps) 33B, a homolog of yeast Vps33p, is another factor regulating TLR4 signaling and degradation. Mammalian Vps33B and closely related Vps33A are soluble proteins of the Sec1/ Munc18 family which, together with SNAREs, allow fusion of intracellular compartments with lysosomes. Vps33B interacts with the VIRAP protein and with Rab10, Rab11, and Rab25 [194, 195]. After silencing of Vps33B TLR4 and CD14 trafficking and its influence on LPS‑induced pro‑inflammatory signaling in murine macrophages, the phagosomal clearance of E. coli was impaired even though the rate of phagocytosis was not changed. Furthermore, the trafficking of LPS-activated TLR4 to Rab5- and further to Rab7-positive endosomes was also similar to that in wild-type cells. In both Vps33Bdepleted and control macrophages, TLR4 was also present in Rab11-positive endosomes. However, degradation of LPS-activated TLR4 was impaired after Vps33B depletion and the receptor accumulated in LPS-stimulated cells, which resulted in an overproduction of cytokines such as IL-6, TNF-α and IFN-β. Collectively, these data suggest that Vps33B has an important function in the maturation of phagosomes and also in a late step of endosome maturation, i.e., the fusion with lysosomes. Therefore, Vsp33B can be viewed as a negative regulator of the TLR4-induced proinflammatory response [180]. Glia maturation factor-γ (GMFG) is a newly discovered actin depolymerization factor which binds the Arp2/3 complex and inhibits nucleation of actin monomers and also promotes debranching of actin filaments, processes important for endosome trafficking. GMFG is expressed mainly in immune cells and microvascular endothelial cells, where it facilitates recycling of internalized β1-integrin [196]. GMFG also affects the internalization and trafficking of LPS-activated TLR4 and thereby the TLR4-induced proinflammatory responses. In THP-1 cells, GMFG associates with early and late endosome markers, including EEA1, Rab5, and Rab7. Depletion of GMFG in those cells and in primary human macrophages resulted in a delayed internalization of TLR4 and its abnormal accumulation in the plasma membrane and Rab5-positive vesicles. This retention of TLR4 in the plasma membrane and in early endosomes significantly enhanced the LPS-induced activation of NF-κB, MAP kinases and IRF3, and the production of TNF-α, IL-6 and IFN-β at both the mRNA and protein level. In accordance, GMFG overexpression decreased the production of pro-inflammatory cytokines. Interestingly, production of the anti-inflammatory IL-10 was slightly increased in these conditions [196]. Rab7b is expressed selectively in some cell types, e.g., in monocytes and monocyte-derived immature DC, and is involved in the transport of cargo to late endosomes and lysosomes [184, 197, 198]. Silencing of Rab7b in Raw264.7 cells increased the overall level of TLR4, including its cellsurface pool [184]. More-detailed studies on TLR4 localization in EEA1- and LAPM-1-positive compartments indicated that silencing of Rab7b caused accumulation of TLR4 in early endosomes, while overexpression of Rab7b led to an accelerated TLR4 translocation to late endosomes/lysosomes. Those data suggested that Rab7b is required for lysosomal degradation of TLR4. Indeed, overexpression of Rab7b in peritoneal macrophages and Raw264.7 cells decreased TLR4 protein level without affecting its mRNA 1245 level [184]. An increased expression of Rab7b also downregulated TLR4 surface level in human DC treated with a water-soluble fraction of the worm Trichuris suis [199]. In addition, studies on the cerebral ischemic stroke in the rat revealed an increased expression of Rab7b in the brain after the stroke. In turn, overexpression of Rab7b in rat brain reduced the TLR4 and NF-κB levels following the stroke and also reduced the production of pro-inflammatory cytokines in both signaling cascades of the receptor. Thus, the overexpression of Rab7b down-regulated the activation of MAP kinases, NF-κB and IRF3 as well as inhibited the production of IL-6 and IFN-β in the rat brain [200]. Also in other studies, overexpression of Rab7b reduced the TLR4-triggered cytokine release, including that induced by a water-soluble fraction of T. suis in human DC [199]. It should be emphasized, however, that the overexpression of Rab7b led to a down-regulation of NF-κB level in addition to that of TLR4, suggesting that also this effect contributed to the reduction of the TLR4-triggered pro-inflammatory responses [199, 200]. Rab7a and Rab7b exhibit 65% sequence similarity [197] and have partially overlapping cellular localizations and functions in maturation of endosomes, yet the two proteins regulate TLR4 signaling differently. Rab7a is required for the activation of the TRIF-dependent signaling cascade in LPS-stimulated BMDMs and does not interfere with the MyD88-dependent one. Also during phagocytosis of LPScoated beads, Rab7a-positive phagosomes recruited TBK1, the kinase responsible for phosphorylation of IRF3 [183]. Recent studies have indicated that both Rab7a and Rab7b can be involved in retrograde transport of cargo from late endosomes to the trans-Golgi network and called into question the involvement of Rab7b in the transport of selected proteins from early to late endosomes [198, 201]. The retrograde transport is mediated by a multiprotein complex called retromer which retrieves membrane-bound receptors, e.g., cation-independent mannose-6-phosphate receptor, from endosomes and transports them to the trans-Golgi network and to the plasma membrane. The core of the retromer is formed by the heterotrimer of Vps35, 29 and 26. Silencing of Vps35 in microglia increased their inflammatory response to LPS, which was linked with an impaired trafficking of Trem2, an immunomodulatory microglial surface receptor [202]. Taking into account that Rab7a, Rab7b, and Vps35 alter the LPS-induced inflammatory responses it seems plausible that retrograde transport can be important for the recycling/retrieval of TLR4, but further studies are required to confirm this hypothesis. Maturation of endosomes is linked with changes of the lipid composition of their membrane. Our studies also indicated that such changes are important for the regulation of the intensity of TLR4-dependent proinflammatory responses. We found that accumulation of 13 1246 lysobisphosphatidic acid, a phospholipid enriched in late endosomes/lysosomes, strongly inhibited the TRIF-dependent signaling cascade and decreased LPS-dependent CCL5/ RANTES production [203]. Taken together, the data discussed above indicate that disturbances in the endocytosis of LPS-activated TLR4 and also dysfunctions of the endosomal/lysosomal machinery ultimately affect TLR4-triggered pro-inflammatory responses. A proper maturation of endosomes is necessary for the TRIF-dependent signaling to occur and can also affect the MyD88-dependent one, while slowing down of the TLR4 degradation increases the pro-inflammatory response. Whether a retrograde transport of TLR4 from Rab7-positvie endosomes via the Golgi to the plasma membrane, which could sustain activation of the receptor by extracellular LPS, is a physiological phenomenon remains an intriguing open question. Rab11‑positive recycling endosomes in LPS‑induced trafficking of TLR4 and TRAM Early endosomes identified by Rab5 presence provide a sorting environment for diverse internalized membrane proteins. Some of them recycle back to the trans-Golgi network or to the plasma membrane, others are directed to lysosomes for degradation. In general, the routes of protein recycling to the plasma membrane are classified as “fast” and “slow”. Fast recycling involves Rab4- or Rab35-marked vesicles which bud off of the early endosomes and next fuse with the plasma membrane [204]. On the other hand, proteins retrieved by the slow recycling route are delivered from early endosomes to the Rab11-positive ERC closely opposing the Golgi apparatus, and then are transported back to the plasma membrane by Rab11- and EHD1- or Arf6-positive recycling endosomes [153, 204, 205]. Rab11-poisitive vesicles participate in the trafficking of the archetypical recycling protein, the transferrin receptor [206]. Rab11-positive compartments are also crucial for the TLR4 trafficking and activation during phagocytosis of Gram-negative bacteria. It was found that Rab11-bearing ERC is a source of TLR4 that is transported in a Rab11dependent manner toward forming phagosomes (Fig. 2, VI). This intracellular pool of TLR4 omits the plasma membrane and triggers the TRIF-dependent signaling at the phagosome membrane. TRAM, required at the onset of this signaling pathway, is also transported to the phagosome membrane with a contribution from Rab11. Local accumulation of IRF3 and production of IFN-β follow [95]. Further studies have revealed that FIP-2, a Rab11 effector protein, is required for the TRAM recruitment to the forming phagosomes. Rab11, FIP-2, TRAM, and TRIF assembly into a 13 A. Ciesielska et al. complex which is recruited to the TIR domain of activated TLR4. FIP-2 guides the TRAM accumulation at the forming phagosomes to activate Rac1 and Cdc42, thereby governing actin filament rearrangement and the uptake of the bacterium, and also to induce the TRAM/TRIF-dependent signaling of TLR4 [187]. Rab11-positive recycling endosomes can also be involved in the intracellular trafficking of TLR4 during stimulation of cells with LPS. Indeed, a FRAP analysis of TLR4 mobility in LPS-stimulated cells revealed its high dynamics suggesting that TLR4 enters and exits the ERC [164]. Moreover, silencing of Rab11 or FIP-2 inhibited the TRIF-dependent signaling of TLR4 triggered by LPS, resembling the effects seen during phagocytosis [95, 187]. At present there are no other data showing that during stimulation of cells with LPS Rab11-positive vesicles can transport TLR4 from its intracellular reservoir directly to endosomes. More likely is a contribution of Rab11 to the transport of TRAM toward the endosomes which acquired LPS-TLR4 from the plasma membrane following induction of the MyD88-dependent signaling cascade. Rab11 can determine the localization of TRAM in unstimulated cells. Overexpression of a Rab11 in HEK293 cells expressing also TLR4, MD-2, CD14, and TRAM resulted in accumulation of TRAM in the ERC, which was concomitant with its depletion in the Golgi apparatus [164]. As mentioned above, the pool of TRAM involved in the endosomal TLR4 signaling does not traffic to endosomes from the plasma membrane [40, 182]; therefore, its possible sources include the Golgi apparatus and the Rab11-positive ERC (Fig. 2, VII). TRAM can be accumulated in the ERC with the help of Arf6-dependent transport from the vicinity of the plasma membrane [153]. Upon a subsequent stimulation of cells with LPS, the TRAMRab11-FIP-2-TRIF complex can be transported from the ERC to LPS/TLR4-harboring endosomes where the TRIFdependent signaling is triggered [95, 164]. This Rab11dependent trafficking of TRAM could facilitate activation of the endosomal signaling pathway of TLR4. Expression of a dominant negative or constitutively active Arf6 variant interfered with the endosomal TLR4 signaling indicating its dependence on the Arf6-mediated intracellular TRAM trafficking [153]. Rab11 is also involved in the delivery to endosomes of two other proteins—transmembrane emp24 domain-containing protein (TMED) 7 and TRAM adaptor with GOLD domain (TAG), both involved in the TRIF-dependent signaling of TLR4. TMED7 and TAG cooperate to promote termination of TLR4 signaling. Silencing of TAG or TMED7 resulted in upregulation of the TRIF-dependent signaling reflected by an increase of the ISRE reporter gene expression and enhanced production of CCL5/RANTES. Moreover, in cells with a reduced level of TAG or TMED7 degradation of TLR4 was inhibited [181, 182]. At the basis of TLR4 and CD14 trafficking and its influence on LPS‑induced pro‑inflammatory signaling the regulatory function of TMED7 and TAG in TLR4-triggered signaling is their negative influence on the stability of the TRAM/TRIF complex. These two proteins contain a GOLD domain allowing their homotypic interaction [181, 182]. TAG, expressed exclusively in humans, is a splicing variant of TRAM and also carries the TIR domain [181]. Upon binding to the TIR domain of TRAM, TAG displaces TRIF from the TRAM/TRIF complex. In unstimulated cells, TAG localizes mainly to the endoplasmic reticulum while TMED7 is present in the Golgi apparatus. After LPS stimulation, the two proteins traffic to endosomes. The colocalization of TMED7 with TLR4 and TRAM begins in EEA1positve early endosomes and culminates in Rab7-positive late endosomes where TMED7 facilitates disruption of the TRAM/TRIF complex by TAG, which eventually leads to degradation of TLR4 [181, 182]. The above data indicate that Rab11-positive compartments are involved in the LPS-induced trafficking of TRAM and also of other proteins regulating the LPS-induced TRAM/TRIF-dependent signaling. In the following sections, we discuss how the TLR4-triggered signaling can be affected by cellular trafficking of CD14 and internalization of LPS. Cellular trafficking of CD14 and its relation to the trafficking of TLR4 The participation of CD14 in the activation of TLR4 by LPS and in the endocytosis of LPS-activated TLR4 followed by the TRIF-dependent pro-inflammatory signaling is well established, as discussed above. Notably, CD14 is also involved in the internalization of high doses of LPS which engages scavenger receptors and leads to LPS detoxification by J774 cells [160], as described in the following section. However, it has to be emphasized that apart from LPS, CD14 binds a broad spectrum of other molecules, including PAMPs, like peptidoglycan, lipoteichoic acid, CpG DNA, and phospholipids [51, 207], and also DAMPs such as β-amyloid [208]. Thus, CD14 can affect the activity of several PRRs. The binding of phospholipids by CD14 is interesting in the context of the LPS-induced pro-inflammatory signaling of TLR4. Among the phospholipids bound by CD14 are 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero3-phosphorylcholine, two forms of oxidized 1-palmitoyl2-arachidonyl-sn-glycero-3- phosphorylcholine (oxPAPC) released from dying cells at the site of tissue injury. The oxPAPC-induced internalization of CD14 depletes its cell surface pool and as a consequence makes these cells less sensitive to LPS. However, in LPS-primed murine macrophages and DC, the CD14-dependent delivery of oxPAPC into the cells leads to the activation of caspase-11 and 1247 caspase-1 followed by IL-1β release. Notably, oxPAPC did not induce pyroptosis of such cells leaving them hyperactive to produce IL-1β without the lethal outcome of an inflammatory response and sepsis in mice [144, 209]. These data support the TAXI name given to CD14 as it is able to capture various cargo in addition to TLR4/MD-2/LPS and deliver them to signaling-competent locations [17]. Only scarce data allow speculation on the pathway(s) involved in the internalization of CD14 carrying LPS and other ligands, like oxPAPC, and also in the constitutivie endocytosis of CD14 in resting murine macrophages [17]. The latter is of importance since a down-regulation the cell surace level of CD14 and TLR4 in resting cells should prevent excessive LPS-induced pro-inflammatory signaling of TLR4 [161, 210]. This mechanism can also contribute to the antagonistic activity of R. spheroides LPS toward human and murine TLR4. Studies performed on murine BMDM showed that this LPS species induced CD14 endocytosis preventing subsequent binding of the pro-inflammatory E. coli LPS to CD14 and consequently to TLR4. The ability of R. spheroides LPS to induce CD14 endocytosis was determined by diphosphorylation of its lipid A [17]. The LPS-induced endocytosis of CD14 is independent of the signaling activity of TLR4, but it was reduced by a Syk inhibitor or a knocdown of PLCγ2 [33]. This suggests that, upon LPS binding, CD14 and TLR4 are internalized by the same route discussed above for TLR4 endocytosis. Since this LPS-induced uptake of CD14/TLR4/MD-2 does not require the signaling activity of TLR4, it has been proposed to be controled by CD14 itself [17]. However, it is still unknown whether the endocytic pathway triggered by LPS is the same as that involved in the constitutive internalization of CD14 in unstimulated cells or in the CD14-mediated uptake of other ligands. Studies on the endocytosis of other GPI-anchored proteins (GPI-APs) may shed light on the LPS-independent internalization of CD14. The majority of GPI-APs are internalized via the dynamin- and clathrin-independent carrier (CLIC) endocytic pathway which also contributes markedly to fluid phase uptake. The internalization of CD14 detected in resting J774 cells displayed properties of a CLIC-mediated uptake; however, the apparent similarities between the CD14 and TLR4 uptake found in those inhibitor-based studies do not allow unequivocal identification of the endocytic pathway involved [161]. The CLIC-mediated endocytosis is sensitive to actin depolymerization and requires activation of the Arf1 and Cdc42 GTPases [211]. Cdc42 interacts with PS in the inner leaflet of the plasma membrane. Depletion of CDC50, the α-subunit of the flippase complex, and the following inhibition of PS transport from the outer to the inner monolayer of the plasma membrane increased the surface level of CD14 in unstimulated THP-1 macrophages [212]. These data suggest that CD14 can be internalized by 13 1248 CLIC-mediated endocytosis involving Cdc42 and PS. An attempt has been undertaken to assess whether the CLICmediated endocytosis is also involved in the internalization of CD14 upon LPS binding. It was found that, depletion of galectin-3, a protein triggering CLIC-mediated endocytosis via clustering of cell-surface glycosylated proteins with glycosphingolipids, did not affect the LPS-induced internalization of either CD14 or TLR4 [17, 213]. Those results spoke against a role of the galectin-3-facilitated endocytosis of CD14 in LPS-stimulated cells; however, a hypothetical involvement of galectin-3-independent CLIC-mediated pathway(s) cannot be ruled out. Internalized GPI-APs accumulate in early endosomes which therfore earn the name of GPI-AP-enriched early endosomal compartmet (GEEC). The GEEC undergoes fast but only modest acidification to pH ~ 6, which is above the average pH of other early endosomes. The acidic pH of GEEC can facilitate the release of ligands from the internalized GPI-APs [211, 214]. Inhibition of V-ATPase reduced the CLIC/GEEC endocytosis of dextran, a fluid phase marker [211]. As mentioned earlier, a knock-down of the ATP6V0D2 subunit of V-ATPase also impaired the LPS-induced endocytosis of TLR4 [152]. It remains to be established whether in those conditions, the inhibition of the internalization of TLR4 was a consequence of a disturbed uptake of CD14. At present, there are no data indicating that upon LPS binding CD14 can recycle to the plasma membrane either via the fast Rab4/Rab35-dependent pathway, the slow one dependent on Rab11, or via Rab10-enriched recycling endosomes, as it was shown for TLR4 [179]. On the contrary, it has been established that at least a fraction of the internalized CD14 is degraded in lysosomes [17]. Even in cells not stimulated with LPS CD14 was detected in perinuclear structures and in MVB-like vesicles [163]. Stimulation of cells with LPS increased the degradation of CD14 in lysosomes while chloroqine treatment inhibited this process [17]. Notably, CD14 internalized by human monocytes during phagocytosis of E. coli or monosodium urate crystals (a sterile phagocytic stimulus) is cleaved in phagolysosomes by elastase and relased as presepsin [54] (see chapter “Signaling pathways triggered by TLR4”). It is an open question whether disturbances in the endolysosome functioning caused, e.g., by mutation/knock-down/ inhibition of endosomal proteins listed in Table 1 can affect the cellular level and localization of CD14 and thereby the LPS-induced TLR4 activation. This possibility is usually neglected in analyses of the impact of endo-lysosomal proteins on TLR4 trafficking and signaling. 13 A. Ciesielska et al. LPS uptake and detoxification Clearance of LPS from circulation and its detoxification help resolve the inflammation induced by bacterial infection. These processes are also important for the removal of the gut microbiota-derived LPS which got access to the bloodstream [215]. There are several mechanisms of LPS inactivation in the human body acting both extracellularly and intracellularly. The latter follows LPS uptake by hepatocytes which remove LPS into the bile, and by immune cells capable of detoxifying the endotoxin enzymatically [216]. The extracellular inactivation of LPS is executed by several lipid A-neutralizing proteins which circulate in the bloodstream, like bactericidal permeability-increasing protein, lactoferrin, lysozyme, collectins, and also anti-LPS antibodies [217]. Also in the blood LPS can be bound by lipoproteins, such as chylomicrons and high, low, and very low-density lipoproteins (HDL, LDL, VLDL) to be next transported to the liver [218, 219]. The binding of LPS to the lipoproteins is catalyzed by phospholipid transfer protein, cholesteryl ester transfer protein, LBP, and sCD14. Liver is the main source of sCD14, also LBP is produced predominantly by the liver and in addition by epithelial cells of lungs and the gastrointestinal track [220]. LPB and sCD14 are both acute phase proteins upregulated significantly during sepsis [45, 221]. High concentrations of LBP attenuate TLR4 activation by inhibiting the transfer of LPS from CD14 to MD-2 while sCD14 can accept LPS form membrane-bound CD14 and accelerate its transfer to HDL and subsequent detoxification in the liver [49, 207, 215, 222, 223]. Liver also plays an essential direct role in the clearance of LPS from circulation and its excretion into the bile. In mice injected in the tail vein with 5 μg LPS, 90% of the endotoxin accumulated in the liver within 60 min [224]. The mechanism of the LPS detoxification in the liver is not fully clear but it is known to engage various types of hepatic cells, including liver sinusoidal endothelial cells, hepatocytes, and hepatic macrophages—the Kupffer cells. All these cells can internalize LPS, but mainly the macrophages inactivate the internalized endotoxin enzymatically [224–227]. If not internalized by hepatocytes directly, LPS is transferred to them from the other cells and is next secreted into the bile [224]. It is worth emphasizing that the mechanism of LPS endocytosis preceding its intracellular detoxification depends on its formulation which includes aggregates, OMV, lipoprotein-bound LPS, and LPS monomers. OMV and LPSenriched liposomes are likely internalized in a clathrindependent and CD14-independent way by macrophage. The binding of LPS to lipoproteins affects the way it is detoxified in the liver. The endocytosis of HDL-LPS by Kupffer cells was not as effective as that of free LPS [224], indicating that the HDL-LPS complexes are rather internalized by cells TLR4 and CD14 trafficking and its influence on LPS‑induced pro‑inflammatory signaling highly expressing scavenger receptors, such as liver sinusoidal endothelial cells and hepatocytes [227]. The role of the scavenger receptors, e.g., CD36, CLA-1/SRB1, CLA-2 and SR-A of immune cells, in LPS-induced processes is more intricate and includes the CD14-dependent uptake of high doses of LPS but also the activation of immune responses [160]. It has been found recently that LPS can also be internalized in complex with secretoglobin 3A2 and HMGB1. The first protein is abundantly expressed in airway epithelial cells and is internalized via the SDC1 receptor. The endocytosis of HMGB1, in turn, is mediated by the RAGE receptor expressed in epithelial cells and macrophages. Both proteins facilitate leakage of LPS from endosomes to the cytosol where it is bound by pro-caspase-11 and activates the NLRP3 inflammasome [113, 228]. TLR4 makes a small contribution to the internalization of LPS by immune cells [229]. In agreement, blocking of TLR4 with a neutralizing anti-TLR4 antibody did not affect the internalization of a relatively high dose of LPS (1000 ng/ ml) [160]. Although TLR4 did not directly participate in the uptake of high amounts of LPS by macrophages, it could indirectly reinforce its clearance from plasma by hepatocytes, as a knock-down of hepatocyte TLR4 inhibited this process [26]. It was also shown that TLR4, CD14, and MD-2 participate in the clustering of CD11b/CD18a integrins involved in LPS endocytosis in hepatocytes [230]. Moreover, TLR4 can regulate deacylation of LPS by immune cells. In TLR4-deficient mice, significantly less LPS was deacylated at the site of infection than in wild-type littermates [231]. In accordance, LPS and commensal Gram-negative bacteria increased the expression of acyloxyacyl hydrolase (AOHA) in macrophages and DC suggesting that activation of TLR4 can activate production of this enzyme [232, 233]. AOHA is a highly conserved hydrolase which recognizes LPS and removes secondary fatty acids at positions 3′, 2′ and/or 2 of lipid A, converting a hexa-acyl (also hepta- or penta-acyl) structure into one that has only four acyl chains. The resulting tetra-acyl LPS molecule binds to MD-2 but does not initiate TLR4 signaling and is also inert to caspase-based detection systems [234]. Similarly, weaker inflammatory responses are induced by LPS dephosphorylated by host phosphatases, like the intestinal alkaline phosphatase which participates in detoxification of LPS in the gut lumen [235]. Deacylation and dephosphorylation of LPS deprived it of a pro-inflammatory activity especially in human cells [59, 61], due to a weaker or no activation of human TLR4 by these LPS species, as discussed above. Notably, the vast majority of in vivo studies on LPS detoxification have been carried out on mice the TLR4 of which can be activated by a broader range of LPS species (see above), but which are much less sensitive to LPS-induced septic shock than humans [236]. 1249 Intestinal alkaline phosphatase is produced in the duodenum, whereas high expression of AOAH was found in the liver, mainly in Kupffer cells with a minor contribution of DC [235, 237]. AOHA expression was also detected in macrophages and DC from the large intestine and in lung macrophages and neutrophils [232]. When LPS was applied subcutaneously, mimicking its release from bacteria in a tissue other than blood, up to 70% of the LPS was deacylated by AOHA before it left the site of injection. This in situ detoxification of LPS is carried out by immune cells, including neutrophils, DC, and macrophages [231]. Deacylation of LPS is a slow process taking several days. Therefore, during infection, the activity of AOHA has little impact on the early response to LPS and cytokine production, but it is necessary to terminate the inflammation and affects the activation of B lymphocytes [231, 237]. Many aspects of AOHA activity are unresolved, the most intriguing one being whether the main side of its action are organelles of immune cells or the extracellular space [231]. In light of the data on the role of metabolic endotoxemia in the development of metabolic diseases, and a growing number of fatal sepsis cases worldwide, the mechanisms of endotoxin disposal by immune cells and overall by the human body deserve close attention. Contribution of abnormal trafficking of TLR4 and CD14 to pathogenesis of certain diseases The multiple pathways of TLR4 trafficking in diverse cells require tight regulation to ensure an optimal magnitude and duration of pro-inflammatory responses induced by LPS and DAMPs. For this reason, several human hereditary and neurodegenerative diseases caused by disturbances in the functioning of the endo-lysosmal compartment are linked with TLR4 hyperactivation and inflammation contributing substantially to their pathogenesis. This fatal relationship is exemplified by lysosomal storage disorders (LSD) caused by hereditary dysfunctions of lysosomes which are accompanied by an abnormal activation of TLR4 and production of pro-inflammatory cytokines [238, 239]. These responses can be triggered by an extracellular accumulation of undegraded substances which act as DAMPs and also by excessive TLR4 stability and persistent signaling caused by an impaired lysosomal proteolysis in cells of LSD patients. In particular, TLR4 has been shown to be involved in the development of mucopolysaccharidosis (MPS), a heterogenous group of LSD resulting from deficiencies in lysosomal enzymes degrading glycosaminoglycans (GAGs). Various MPS subtypes affect different tissues and organs, including central nervous system, bones, muscles, and the connective tissue [238]. Under physiological 13 1250 conditions, hyaluronian of the GAG family is transported to the extracellular matrix while other GAGs, like heparan sulphate, are exposed on the cell surface and remain bound to the plasma membrane via a proteoglycan core. Thereby heparan sulphate of epithelial cells affects the adhesion of immune cells during inflammation. Several GAGs also induce production of pro-inflammatory chemokines and cytokines. Studies conducted on animal models of MPS VI and/or VII showed an increased level of IL-1β and TNF-α and also an upregulated expression of several other inflammatory markers in synovial fluid, fibroblast-like synoviocytes and serum. Those changes were linked with TLR4 activity as a knock-down of TLR4 in MPS VII animals normalized the serum level of TNF-α and phosphorylation of chondrocyte STAT1 and also alleviated some of the pathological effects of the disease in bones and joints [240, 241]. A similar line of data indicated a contribution of TLR4induced pro-inflammatory signaling of microglia to the onset of neurodegenerative MPS III [242]. The pro-inflammatory signaling of TLR4 is considered to be triggered by GAG— DAMPs which accumulate in the extracellular space. These include hyaluronian which stimulates TLR4 in cooperation with CD44, and also heparan sulphate and its derivatives resulting from an incomplete degradation of this GAG in lysosomes and subsequent exocytosis of its products. However, an increased level of TLR4 has also been detected in fibroblast-like synoviocytes of MPS VI animals. In addition, these cells have increased mRNA levels of LBP, CD14, and MyD88. The upregulation of TLR4 level was caused by its accumulation in dysfunctional lysosomes with an unchanged expression of the Tlr4 gene [240, 241]. Collectively, these results indicate that an impaired lysosomal degradation of TLR4 contributes to the inflammation in MPS. An increased activity of TLR4 is also linked with the pathogenesis of another LSD, Niemann–Pick’s type C (NPC) disease leading to progressive neurological dysfunctions in children. NPC is caused by mutations in the NPC1 or NPC2 genes causing malfunctioning of the encoded proteins, which results in an aberrant endosomal membrane flow and accumulation of cholesterol and other lipids in endo-lysosomes. TLR4 level is increased in fibroblasts and glial cells of NPC patients and in Npc1 knock-out mice, respectively, as a result of its impaired degradation. This leads to the accumulation of TLR4 in endosomes and upregulation of its signaling pathways enhancing the production of pro-inflammatory cytokines, such as IL-6, IL-8, and IFN-β. The increased inflammatory response, particularly the glial production of IL-6, can contribute to the pathogenesis of NPC via both the autocrine and paracrine effects of this cytokine [243]. A participation of the TLR4 receptor has also been noted in the development of cystic fibrosis (CF)—an autosomal recessive disorder caused by mutations in the CFTR gene 13 A. Ciesielska et al. encoding cystic fibrosis transmembrane conductance regulator, a chloride channel expressed in airway epithelial cells and, to a lower extent, also in other cells. The disease is manifested chiefly by a dysfunction of the airway epithelium, which leads to a dehydrated and acidic airway surface liquid favoring chronic bacterial infections [244, 245]. A characteristic feature of cystic fibrosis is an excessive inflammatory response leading to lung damage [244, 245]. Data concerning the role of bronchial epithelial cells in the inflammatory response in cystic fibrosis are inconsistent. Early studies reported a decreased or unchanged expression of TLR4 in those cells in comparison with the cells of healthy donors [244, 246]. In contrast, a recent study on immortalized bronchial epithelial cell derived from a CF patient showed an increased level of TLR4 on the cell surface and its impaired transport to lysosomes likely contributing to an enhanced inflammatory response to LPS [247]. However, the hyperinflammatory response in CF lungs is mainly attributed to macrophages. Accordingly, macrophages from CFTR knockout mice had an elevated level of TLR4 compared with the wild type, with no differences in the amount of TLR4 mRNA [248]. The deletion of the Cftr gene caused accumulation of TLR4 in early endosomes in macrophages and compromised its degradation, thereby leading to an increased activation of NF-κB, MAPK, and IRF3. The retention of TLR4 in early endosomes could ensue from an aberrant maturation of endosomes in CFTR-depleted LPS-stimulated cells, insufficient endosomal acidification, or perturbations in cholesterol trafficking caused by the CFTR deficiency [248]. These mechanisms can also cause the accumulation of TLR4 and an excessive pro-inflammatory response to LPS of human macrophages ultimately leading to the lung disease of cystic fibrosis patients [248]. The arthrogryposis–renal dysfunction–cholestasis syndrome is a rare fatal disease caused by autosomal recessive mutations in the VPS33B gene. Characteristic symptoms of this disease comprise arthrogryposis, renal tubular acidosis, and neonatal cholestatic jaundice. The patients also suffer from persistent infections and sepsis [180, 249]. As it was described above, dysfunction of VPS33B leads to an impaired degradation of TLR4 and enhanced pro-inflammatory response of macrophages to LPS via the MyD88and TRIF-dependent pathways. It has been proposed that such exaggerated TLR signaling causes so-called immune paralysis whereupon the immune system loses the ability to respond to secondary infection [180]. Studies on Chédiak–Higashi syndrome (CHS) have expanded the list of diseases linked with disturbances in TLR4 trafficking and signaling. CHS is a rare autosomal recessive disease characterized, among others, by partial albinism, neurological problems, and immunodeficiency. CHS patients suffer from persistent and recurrent infections of the skin, respiratory tract, and mucous membranes. TLR4 and CD14 trafficking and its influence on LPS‑induced pro‑inflammatory signaling Human CHS and its corresponding mouse disorder beige are caused by mutations of the LYST gene [192]. Recent studies indicate that Lyst controls endosomal TRIF-dependent signaling of TLR4, as described above. In mice, a loss of a functional Lyst impaired inflammatory responses against Gram-negative bacteria and enhanced bacterial dissemination in tissues but also lowered the susceptibility to LPSinduced septic shock as a result of a reduced production of pro-inflammatory cytokines [183]. TLR4 also participates in the development/progression of neurodegenerative disorders associated with Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis, and amyotrophic lateral sclerosis (ALS). Due to its engagement in neurodegeneration, TLR4 is considered a therapeutic target in the treatment of these diseases [250]. At the early stages of the development of neurodegenerative diseases, activation of TLR4 may be beneficial for the clearance of β-amyloid (AD) and α-synuclein (PD) and can prevent accumulation of their aggregates characteristic for these diseases. However, a prolonged overactivation of microglia may lead to the release of neurotoxic products and damage neurons [251, 252]. Such increased activity of microglia can result from their abnormally high content of TLR4. An increased expression of TLR4 was observed in brains of a murine AD model (overexpressing amyloid precursor protein) and in AD patients in areas surrounding plaques [251, 253]. Moreover, β-amyloid increases expression of TLR4 in cortical neurons, which further activates JNK and caspase-3 and contributes to neuron death [254]. An elevated expression of TLR4 was also detected in ALS and PD patients [255]. In addition to TLR4, also CD14 is important for the activation of inflammatory responses and phagocytosis upon exposure of microglia to β-amyloid [251, 252]. In agreement, an elevated level of CD14 was detected in brains from murine models of AD, PD, and ALS, and from AD patients [256–258]. However, the outcome of a CD14 engagement in AD can be both beneficial and detrimental [259]. The elevation of the amounts of TLR4 and CD14 in microglia and exaggerated production of pro-inflammatory mediators can result from disturbances in endo-lysosomal functioning in analogy to the LSD described above. This assumption is supported by the fact that among the features of AD, PD and ALS are defects of endo-lysosomes caused by mutations of genes encoding proteins involved directly or indirectly in their functioning [260, 261]. Recent data link TLR4 activation with the inflammatory process induced by cerebral stroke and leading to ischemic brain damage. TLR4 was upregulated after ischemia-induced stroke, and mice deficient in TLR4 exhibited less-severe cerebral infarction and neurological deficit compared with wild-type counterparts [200]. TLR4 is also associated with the development of allergies and some autoimmune diseases, like Crohn’s disease, asthma, and type 1 diabetes [262–264]. 1251 Further studies are required to establish the contribution of disturbances in TLR4 trafficking to these processes. In summary, the involvement of TLR4-triggered signaling in the development and progression of multiple diseases is often linked with its disturbed trafficking through the endo-lysosomal compartment, as demonstrated for LSD and strongly suggested for neurodegenerative diseases. It remains an open question whether defects in TLR4 trafficking contribute also to the severity of acute and chronic inflammation caused, respectively, by infection and metabolic endotoxemia, the latter known to participate in the development of type 2 diabetes. A comprehensive knowledge of the TLR4 trafficking pathways and the role of CD14 in these processes is key to understanding mechanisms regulating TLR4-induced inflammatory response in both infectious and non-infectious diseases. This in turn should facilitate the design of new therapeutic strategies for treatment of these diseases. Acknowledgements This work was supported by grant number 2016/23/D/NZ3/02212 from the National Science Centre, Poland to A. Ciesielska. We thank Dr. Jan Fronk from the Faculty of Biology, University of Warsaw for helpful comments and critical discussion. Author contributions A.C. and M.M.: original draft preparation and editing, K.K.: review and editing. Compliance with ethical standards Conflict of interest The authors declare no conflict of interest. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. 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    www.bioinformation.net Volume 15(6) Research Article Identification of novel inhibitor targeting Fyn kinase using molecular docking analysis Dharani Nedunchezhian1, Kulanthaivel Langeswaran2* & Sundar Santhoshkumar3 1Department of Biotechnology and Bioinformatics, Bishop Heber College, Bharathidasan University, Tiruchirapalli, Tamil Nadu, India; Genetics & Molecular Biology Laboratory, Department of Bioinformatics, Science Campus, Alagappa University, Karaikudi, Tamil Nadu, India; 3Department of Computer Science, Science Campus, Alagappa University, Karaikudi, Tamil Nadu, India; Dr. V. K. Langeswaran - Email: dr.langeswaran@gmail.com; *Corresponding author 2Cancer Received May 21, 2019; Accepted June 3, 2019; Published June 15, 2019 DOI: 10.6026/97320630015419 Abstract: Identification of tyrosine Fyn kinase inhibitor is recognized as an effective and feasible therapeutic measure in reducing consequences of memory loss disorder Alzheimer's. The present investigation has been attempted with an objective to find out a novel potent inhibitor with similar homological structure to Fyn kinase using structure based in silico screening measure. Such derived structure was compared with natural data base pool and were systematically analyzed. Ligand based interaction was also tested and evaluated. We applied a molecular dynamic simulation technique to validate the stability of the identified complexes and to understand the ligand binding mechanism. Results provide information on the characteristics of novel and potent inhibitor for tyrosinase Fyn kinase protein so as to develop an innovative strategy to treat AD. Keywords: Tyrosine fyn kinase, ligand binding, dynamic simulation Background: Tyrosine Fyn Kinase is a determined protein factor involved in the activity of cell signaling pathway. Unusual occurrence in the rate of activity of Fyn Kinase in signaling, render toxic substances, resulting chronic disorder such as Alzheimer's [1]. This played a crucial role in phosphorylation and anti-phosphorylation process and confers its intra molecular affinity [2], Cell adhesion, synaptic activity and signaling [3]. Several earlier reports were demonstrated and evident that Tyrosine Fyn was recognized as driven force causing AD by interacting Amyloid B and Tau, conferring a novel therapeutic strategy to address and minimize the consequences of AD. Regulation of Fyn activity was obviously studied in several types of signaling pathways [4]. Shirazi et al. [5] have reported that Fyn Protein showed immune reactivity while unusual phosphorylated Tau protein. However, several studies were focused on inter linkage of Tyrosine Fyn Kinase protein with AD. Participation of Fyn Kinase Protein in inducing and act as causative agent for chronic disorder. Therefore, it received more attention on its ability in signaling pathways. Advancement of docking based virtual screening technique in accordance with affinity of protein and ligand conferring an ideal way for screening the lead novel compounds in the field of molecular docking [6]. Identification of macromolecules and validity was the remarkable factor in targeting the drugs, however, it is still unclear and debatable [7]. Therefore, in the present investigation, a trial has been taken up (i) to predict the novel potent inhibitor targeting Fyn Kinase and (ii) to identify the ligand by comparing natural database and (iii) to explore protein ligand complex stability and its behavior.   ISSN 0973-2063 (online) 0973-8894 (print)   Bioinformation 15(6): 419-424 (2019)   419 ©Biomedical Informatics (2019)        Table 1: Virtual screening of ZINC-NPD, NPB and NCI results S. No Natural I.DS GE VDW COUL GEM compounds ZINC-NPD 1 144027 -16.572 -68.639 -37.652 -30.987 -121.633 2 120095 -15.884 -82.871 -46.541 -36.330 -113.640 3 128794 -15.848 -90.119 -58.883 -31.236 -130.885 4 2582 -15.812 -79.383 -52.157 -27.227 -105.107 5 118082 -15.799 -82.457 -55.718 -26.739 -99.998 NPB 1 2455 -9.946 -63.013 -51.890 -11.123 -92.357 2 7898 -9.838 -64.901 -41.363 -23.538 -81.703 3 2407 -9.595 -58.628 -47.704 -10.923 -81.459 4 4794 -9.096 -42.552 -39.816 -2.737 -65.886 5 2786 -8.961 -53.323 42.002 -11.321 -72.928 NCI 1 9129 -10.713 -46.113 -35.774 -10.339 -68.734 2 6152 -10.199 -35.153 -19.122 -16.031 -48.802 3 4036 -10.026 -39.686 -23.145 -16.541 -52.388 4 9037 -9.874 -38.099 -25.992 -12.107 -65.595 5 2608 -9.845 -35.570 -23.836 -11.734 -61.072 (ZINC-NPD, NPB, NCI) Natural compound database; DS-Docking energy; GE- Glide energy; GEVDE-VDE; GECOUL-COUL; GEMODE-GEM. Table 2: ADME results of the top compounds S. No COMPOUND MW DONAR HB 01 ZINC_NPD144027 396.463 1 02 NPB: 2455 240.258 3 03 NCI: 9129 478.636 1 ACCEPT HB 9 2.25 10.5 % HOA 88.588 86.594 85.564 Q plogPo/W -5.76. 2.038 2.919 Materials and Methods: Using Software packages such as CPU platforms performed aforementioned tasks. Stipulations were CPU-Supermicro 32Gb Nvidia Quadro K620 with 2GB included, LLC, 2019 Desmond. Fyn protein complex code (PDB ID-2DQ7) with a resolution of 2.8 Armstrong was used for virtual screening and molecular docking. They contain heavy atoms, covering its cofactors. However, to avoid left out information on connectivity, protein structure is imported from PDB into Maestro in the protein preparation wizard with hydrogen polar. Optimization of Hydrogen bond was employed and rely on clusters of hydrogen-bonded species were selectively optimized. Minimizing the structure has been performed to refine the structure, with a restrained minimization. The RMSD value of the atom was minimized. The partial atomic charges were also computed and analyzed. QplogHERG -5.708 -4.643 -5.399 QPP Caco 0.235 463.328 209.104 QPPMDCK 0.059 215.385 128.805 in the workspace. Van der Waals (vdW) radius scaling of 14 for closely contacts with the ligand and the receptor (a protein molecule) is used. Scaling factor default value is 0.30 and the partial charge cut off default value is 0.25. Scaling of van der Waals radii was performed on the non-polar atoms. Scale factor default value is 1.0 as the contact by small gaps in the solvent-exposed regions and also determines where in the scoring grids are located. Energetic program was used to analyses spectrum to predict the modes of the protein ligand complexes. Workflow filtering method was used. It was also evaluated the acceptability of analogues based on Lipinski’s rule of 5 which was essential to ensure the drug-like properties of the molecules were assessed. GSAP complexed with compounds from Zinc-NPD, NPB, Nci-NPD database was employed by using the Desmond modular. In this present study, totally three from Zinc-NPD, NPB, Nci-NPD database (144027, 2455, 9219) were chosen, theses three are the top leads from each database identified based on the docking score. All these three complexes were simulated for 10 analyzed deviations (RMSF) and considered as backbone of the protein. The RMSF was assessed to estimate fluctuation. The generated structure of a prepared protein showed in an order such as receptor, site, constraints and rotatable groups. Receptor is a workspace and the ligandwere identified as a molecule. The green color marker should be shown after the ligand molecule is picked   ISSN 0973-2063 (online) 0973-8894 (print)   Bioinformation 15(6): 419-424 (2019)   420 ©Biomedical Informatics (2019)        Figure 1: Interaction profile and docking pose of the best hit compounds obtained from screening for FYN 2DQ7 with Zinc NPD(a, b), NPB(c, d), NCI-NPD(e, f) databases.   ISSN 0973-2063 (online) 0973-8894 (print)   Bioinformation 15(6): 419-424 (2019)   421 ©Biomedical Informatics (2019)        Figure 2: Molecular dynamics simulation results of the best hit compounds from three databases Result and Discussion: In the present study high throughput virtual screening (HTPS) method was used to identify potential molecule inhibitors using compounds obtained from pharmacophore against active FYN kinase protein of the ligands were further docked flexibly by XP which generates compounds were tabulated in Table 1 and shown in Figure 1. Drug likeness was tested and followed by Quikprop method of Schrodinger ADME properties of identified inhibitors were recorded systematically and tabulated (Table 1) based on Lipiski’s rule of 5 [8]. Results of the docking indicated that efficient inhibitors identified among three database, chief lead compound was found to be G Plog Po and was promising alternate. Final results of screened compounds were selected data base as an input to find matches to hypothesis to screen the best ligands molecules. In earlier studies, virtual screening molecules against several proteins related to human disorder were reported. Poli et al. [9] have demonstrated that few efficient Fyn inhibitors have identified and proved to be blocked the activities of Fyn kinase.In our study, three different Natural compounds databases were chosen namely ZINC NPD (3,76,884 Ligand molecules), NPB (20,783 Ligand molecules), NCI(56,000 Ligand molecules) were taken for screening process. Based on the docking score top three compounds were selected from each database.   ISSN 0973-2063 (online) 0973-8894 (print)   Bioinformation 15(6): 419-424 (2019)   422 ©Biomedical Informatics (2019)        Analysis on ADME of inhibitors: ADME properties evaluation of identified chief compound, A Plogpo scored highest range (Table 2). It was observed that identified compounds with a group of hydrogen bonding donor and acceptor pair as found This present study results were matching the findings of Zhu [10]. The biological membrane access ranges were found to be range from 293.11 to 2288.96 and the range of QPPMDCK was 96.8 to 100 [11]. Thomas et al. [12] have established that improvement in ADME properties of identified compounds are attributed and translate to the pharmacokinetic properties. Findings were correlated with earlier study results.In the present investigation two ligands from the ZINC-NPD database (95914724), NPB database (2455) and NCI-NPD database (6152) were chosen. 95914724, 2455, 6152 were the top lead identified based on docking score. All two complexes were calculated and analyzed RMED value in order to evaluate deviation in the structure during simulation (RMSD). The stability of protein-ligand (2DQ7-144027, 2DQ7- 2455, 2DQ7-9219) complexes is shown. Figure 2 exhibited that the deviations and fluctuations were not observed much and hence the results were agreeable. These results inferred that the compounds have slight fluctuations, which are observed, only in the loop region of the target protein. Zinc complexes in lead molecules may contribute and involved in coordination if neurons characterized by fast ligand exchange [13]. In the present study, it was evident that interaction of compound with zinc was attributed to the selected ligand and it was agreement with our findings [14]. The results of the RMSD plot reported that high scored lead compound could be satisfactorily explored in the present simulation experiment. simulation have proved and supported our claims to be the useful tool and the best ligand to block the Fyn kinase. This study has confirmed that these compounds can be a potent inhibitor to block the interactions between the targets and inhibit the progression of Alzheimer’s disease. Acknowledgement: The article has been written with the financial support of RUSA – phase 2.0 grant sanctioned vide Letter No.F.24-51/2014-U policy (TNMulti – Gen), Dept of Edn. Govt of India Dt, 09.10.2018. Conflict of Interest The authors declared that there are no conflicts of interest. References [1] Poli G et al. J Chem Inf Model. 2013 53:2538. [PMID: 24001328] [2] Krämer-Albers EM et al. Cell Mol Life Sci. 2011 68: 2003 [PMID: 21207100] [3] Resh MD. Int J Biochem Cell Biol. 1998 30:1159.[PMID: 9839441] [4] Hu JL et al. Mol Brain. 2010 3:20. [PMID: 20569495] [5] Shirazi SK et al. Neuroreport. 1993 4:435. [PMID: 8388744] [6] Nguyen TH et al. J Biol Chem. 2002 277:24274. [PMID: 11983687] [7] Moellering RE et al. Nature 2009 462:182. [PMID: 19907488] [8] Lipinski CA et al. Adv Drug Deliv Rev. 2001 46:3. [PMID: 11259830] [9] Goldsmith JF et al. Biochem Biophys Res Commun. 2002298:501. [PMID: 12408980] [10] Zhu L et al. Mol Divers. 2018 22:979. [PMID: 30083853] [11] Thomas SM et al. Annu Rev Cell Dev Biol. 199713:513. [PMID: 9442882] [12] Thomas R et al. Pediatrics. 2015 135: e994. [PMID: 25733754] [13] Elena Atrián-Blasco et al. Dalton Trans. 2017 46: 12750. [PMID: 28424538] [14] Monika et al. Bioinformation 2013 9: 583. [PMID: 23888100] Conclusion: We are interested identifying the inhibitors targeting the Fyn kinase for the treatment of Alzheimer’s disease. It was concluded that Fyn kinase inhibitors are, therefore promising novel molecules and in disrupting a neurodegenerative disorder. Computational studies such as receptor based screening and molecular dynamics Edited by P. Kangueane Citation: Nedunchezhian et al. Bioinformation 15(6): 419-424 (2019) License statement: This is an Open Access article which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. This is distributed under the terms of the Creative Commons Attribution License   ISSN 0973-2063 (online) 0973-8894 (print)   Bioinformation 15(6): 419-424 (2019)   423 ©Biomedical Informatics (2019)        Journal   ISSN 0973-2063 (online) 0973-8894 (print)   Bioinformation 15(6): 419-424 (2019)   424 ©Biomedical Informatics (2019)   

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Aromatic Hydrocarbons as Molecular Propellants for Electric Propulsion Thrusters André Nyberg Borrfors KTH Royal Institute of Technology Dan J. Harding KTH Royal Institute of Technology Jonas Weissenreider KTH Royal Institute of Technology Simone Ciaralli OHB Sweden AB Ashley Hallock OHB Sweden AB Tore Brinck (  tore@kth.se ) KTH Royal Institute of Technology Research Article Keywords: Posted Date: March 16th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2666479/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Aromatic Hydrocarbons as Molecular Propellants for Electric Propulsion Thrusters André Nyberg Borrfors1, Dan J. Harding2, Jonas Weissenreider3, Simone Ciaralli,4 Ashley Hallock,4 and Tore Brinck1* 1Department of Chemistry, KTH Royal Institute of Technology, SE 100 44 Stockholm, Sweden 2Department of Chemical Engineering, KTH Royal Institute of Technology, SE 100 44 Stockholm, Sweden 3KTH, Materials and Nano Physics, Hannes Alfvens väg 12, 11419 Stockholm, Sweden 4OHB Sweden AB, Viderögatan 6, 164 29 Kista, Sweden *tore@kth.se ORCIDs: DJH: 0000-0002-8622-9046, JW: 0000-0003-1631-4293, AH: 0000-0001-6801-5186, TB: 0000-0003-2673-075X ABSTRACT The aromatic hydrocarbons (AHs) fluorobenzene, naphthalene, and 1-fluoronaphthalene are introduced as promising alternatives to xenon as propellant for in-space electric propulsion (EP). These storable molecules have similar mass, lower cost, and lower ionization energies compared to xenon, as well as the critical advantage of low post-ionization fragmentation compared to other molecular propellant candidates. The ionization characteristics of AHs are compared with those of xenon and the diamondoid adamantane, previously evaluated as a molecular propellant for EP. Quantum chemical calculations and BEB theory together with 25 eV electron ionization mass spectrometry (EI-MS) measurements have been used to predict the fragmentation of the AHs and adamantane when ionized in a plasma with an electron temperature of 7 eV (a typical electron temperature in EP plasmas). A high fraction (81 - 86 %) of the detected AH ions originate from intact molecules, compared to 34 % for adamantane. indicating extraordinarily low fragmentation for the selected AHs. The ionization potential of the AHs is similar to that of adamantane but lower compared to xenon (8.14-9.2 eV for the AHs, 9.25 for adamantane and 12.13 eV for xenon). BEB calculations have also been used to predict total ionization cross sections. The calculated ionization cross section of the AHs is comparable to that of adamantane but 3-5 times higher than that of xenon, which together with the low ionization potential can contribute to more efficient ionization. The AHs may have the potential to perform better than xenon, despite the absence of fragmentation in xenon. Acknowledgments Special thanks to Dr. Abdusalam Uheida of the KTH Functional Materials Division for allowing us access to his lab and equipment, saving us precious time during the early measurements. Technical assistance by Lea Hohmann at KTH Dep. of Chemical Engineering is gratefully acknowledged. Page 1 of 23 DECLARATIONS Funding This work was initiated by OHB Sweden through the NFRP 4 program and is supported by the Swedish National Space Agency. Additional funding to DJH was provided by the Swedish Foundation for Strategic Research (SSF) (ITM17-0236). Additional funding to JW was provided by the Swedish Research Council (VR) (2021-87883). Conflicts of interest The authors have no competing interests to declare that are relevant to the content of this article. Data availability Computational and experimental data are available from the corresponding author upon reasonable request. Code availability Not applicable. Authors' contributions André Nyberg Borrfors (ANB), Simone Ciaralli, Ashley Hallock and Tore Brinck (TB) contributed to the study conception and general design. ANB, Daniel J. Harding and Jonas Weissenrieder contributed to the experimental design and data acquisition. ANB and TB devised the theoretical procedure. ANB performed all computations and analysis of experimental data. ANB wrote the first draft. All authors commented on previous versions of the manuscript, read and approved the final manuscript. Page 2 of 23 1. INTRODUCTION Electric propulsion devices transfer momentum to a spacecraft by ionizing a propellant and electrically accelerating the resulting plasma to exhaust velocities an order of magnitude greater than what can be achieved with conventional chemical thrusters, allowing delivery of a higher delta-v compared to chemical propulsion for the same propellant mass. With the commercialization of space and the steadily increasing demand for affordable satellites, the market for more efficient and cost-effective electric propulsion thrusters has grown. Xenon has historically been the most common propellant for electric propulsion subsystems, but has begun to be replaced with more affordable and more readily available alternatives such as krypton. A storable alternative to xenon offers advantages on subsystem level by eliminating the need for high-pressure propellant storage. In recent years the use of storable atomic propellants like bismuth and mercury for high thrust applications, and zinc and magnesium for high specific impulse applications has been investigated [1–4]. Some molecular propellants have been investigated, such as water (where the thruster operates on hydrogen and oxygen gas created by electrolysis) [5,6], iodine [7–13], and adamantane [14–16], but were all found to have prohibitive disadvantages such as significantly lower performance compared to xenon. A paper on the first in-space demonstration of (low-power) thruster operation on iodine vapor was recently published in Nature [13]. Iodine offers a high storage density and vapor pressure combined with low ionization energy; however its reactivity introduces major challenges to development and implementation, especially for thrusters operating above 100 kW. The goal of this work is to identify and evaluate potential storable alternative EP propellants that have a mass and ionization efficiency comparable to that of xenon. 1.1 Ionization Properties of Molecular Propellants The hydrocarbon molecule adamantane has previously been explored as a propellant for use in electric propulsion thrusters [14–16]. Adamantane is the smallest diamondoid and has a mass similar to the xenon atom. Its ionization cross section is five times larger than that of xenon and its ionization energy (IE) is 9.25 eV compared to 12.13 eV for xenon [17]. It can serve as a scaffold for further molecular design and it is easily modified with a wide range of possible substituents; however experiments involving thruster operation on adamantane and a few of its substituted derivatives show fragmentation levels too high for these molecules to be effectively used as propellants for electric propulsion [14,15]. In this study we propose aromatic hydrocarbons (AH) for use as molecular propellants as an alternative to xenon. In particular, we highlight the advantages of the hydro- and fluorocarbons fluorobenzene, naphthalene, and 1-fluoronaphthalene (Fig. 1). The availability of AHs (especially the smaller variants) is reflected in their low prices as is evident from Table. 1 of Appendix A. While these are estimates and based on what was available from resellers at the time of asking, the AHs are generally around 10-100 times less expensive than xenon and much easier to procure. Page 3 of 23 Fig. 1 Molar mass and (adiabatic) ionization energies of adamantane and the AHs considered for use as molecular propellants in electric plasma thrusters These candidates were selected from a set of 13 different AHs (see Table. 1) by screening for desirable properties like high vapor pressure, appropriate phase change temperatures, and low fragmentation during ionization as measured using standard electron ionization mass spectrometry. An inherent energy sink associated with ionizing molecules is the occurrence of fragmentation wherein some molecular ions break apart into multiple fragments where only one fragment remains charged. The additional energy of having to ionize any resulting neutral fragments can quickly compound because fragments are typically less stable than the original molecule and can dissociate even further. Therefore, molecular propellants that are stable both before and after ionization could offer a higher ionization efficiency compared to molecules with higher rates of fragmentation. Compared to other molecules, AHs are unique in their stability towards ionization-induced dissociation because of their unique electronic and nuclear structures. In an AH, the electronic structure consists of a combination of - and -orbitals, which provides additional stabilization upon ionization compared to saturated hydrocarbons such as adamantane. The -orbitals can be divided into lower-energy core orbitals and bonding valence orbitals. The latter make up a skeleton of single bonds between all atoms in the molecule. The -orbitals are generally higher in energy and more delocalized in character, and together they contribute a partial double bond character to the bonds between all atoms of the ring system. Because of their higher energies, ionization typically leads to expulsion of an electron from a -orbital, and this only partially reduces bond orders between the ring atoms as the -orbital single-bond skeleton remains intact. In the more unlikely event of ionization from a -orbital, individual -bonds may be weakened, but due to the extra bond-stabilization from the -orbitals this is not likely to lead to bond rupture. In a saturated hydrocarbon, such as adamantane, ionization occurs predominantly from the valence sigma-orbitals, and this will inevitably weaken the -bond skeleton, and it is highly likely to lead to bond rupture and fragmentation. The higher stabilities upon ionization of AHs compared to saturated hydrocarbons can be verified by comparing their electron ionization mass spectra, which show significantly higher fragmentation for the saturated hydrocarbons. AHs also possess remarkable resistance towards thermal degradation [18–21], with gas phase benzene and naphthalene starting to decompose in the range of 600-650 °C [22]. Heavier AHs than those in Fig. 1 consisting of an increasing number of fused carbon rings can also serve as viable propellant candidates and have even higher decomposition temperature but their vapor pressure is lower than that of their smaller counterparts. This can lead to higher required heater power to maintain the propellant as a gas in the propellant feed system and potential deposition of propellant from the thruster plume on spacecraft surfaces. Vapor pressure curves for many AHs can be found in Appendix A, Fig. 6. Page 4 of 23 The properties of AHs are readily modified by chemical functionalization, i.e. one or more of the hydrogens can be substituted for another atom or functional group. The substitution of a hydrogen for a heavier element typically reduces the resistance towards ionization-induced fragmentation. The only obvious exception is fluorine substitution, which generally has a minor effect on the fragmentation as long as the number of substitutions is limited. This anomaly can be traced to the relatively low weight of the fluorine in combination with the relatively high strength of the C-F bond. Substituting one of the hydrogens in naphthalene with a single fluorine atom increases the molecular mass by 14 % from 128.2 to 146.2 amu and the aggregation state changes from solid to liquid at ambient temperature. Depending on the position of the fluorine two different naphthalene isomers can be constructed, namely 1-fluoronaphthalene and 2fluoronaphthalene. These have slightly different physical properties, e.g. melting point, but their ionization behaviors and mass spectra are similar. Considering the similarity of the two isomers, only 1-fluoronaphthalene is considered in this work together with the other AHs. The ionization cross sections of molecules are generally larger than for atoms and therefore a shorter plasma ionizing region is sufficient to reach the required degree of ionization for a molecular propellant. The most important ionization process taking place in typical electric propulsion thrusters is (e,2e) electron ionization, where a single incident electron with kinetic energy T collides with a target and results in the expulsion of a bound electron. This creates a positively charged radical cation, which can be accelerated by an electric field to produce thrust. If T is lower than the IE of the target atom or molecule, no ionization can occur from a single collision. If multiple collisions between a target and electrons (or other molecules when the pressure is high) are allowed, as in an ionizing plasma, less energetic collisions can still accumulate enough energy in the target to ultimately lead to ionization. In this article, we will describe the experimental and analytical method of investigation of the ionization behavior of the molecules in Fig. 1. Results and discussion of ionization cross section calculations for the AHs and adamantane are followed by results and discussion of measurements of the fragmentation of the AHs and adamantane, including a comparison of the estimated ionization efficiency of the AHs and that of xenon. The predicted performance of a thruster operating on AHs is then discussed, along with a discussion of accommodation, safety and environmental aspects. The article concludes with a summary of findings regarding the suitability of the AHs as an alternative EP thruster propellant. 2. METHODOLOGY 2.1 Method of Calculation of Ionization Cross Section All quantum chemical calculations were performed using the Gaussian 16 software suite [23]. Relaxed molecular geometries were obtained through structure optimization using the hybrid M06-2X functional and the 6-311g(d,p) basis set. Vertical ionization energies (IE) used for producing BEB cross sections (see Appendix B, Eq. 3 and [24,25]) were obtained using electron propagator theory (EPT) based on Hartree-Fock (HF) wavefunctions and the 6-311+g(2df,2p) basis set. In the cases where relativistic effects had to be accounted for, such as for BEB calculations involving xenon and iodine, the Def2-TZVPP basis set with effective corepseudopotentials was used. BEB theory is most sensitive to the accuracy of the IE (B in Eq. 3) associated with the highest energy valence orbitals. Therefore, when used to calculate t=T/B for valence orbitals, the value of B was taken to be the more accurate IE obtained by applying the P3 propagator in EPT. As EPT often struggles to converge when the IE is too high, only values of B<20 eV were obtained using P3. For the remaining orbitals, and in the rare instances when P3 failed to converge while below the threshold, the IE was, by applying Koopmans theorem, taken to be Page 5 of 23 the negative of the orbital binding energy obtained at the HF level of theory. HF was also used in producing the value of the orbital kinetic energy U, and by extension its reduced form u=U/B. 2.2 Method of Measurement of Ionization-induced Fragmentation Mass spectra were collected using a Pfeiffer QMA 400 Quadrupole mass spectrometer (MS) equipped with a Faraday cup detector and mounted on a small vacuum chamber capable of reaching 1 × 10−8 mbar base pressure. Software for controlling the MS and collecting spectroscopic data was QUADERA (build 4.62.004). The chamber pressure was measured using a Balzers IKR-020 Penning Vacuum Gauge placed out of the line of sight of the MS ionizer. The Penning pressure detector is a cold cathode ionization-type vacuum gauge that uses a high electron current with a non-discrete and complex energy distribution to ionize gases within the chamber. The reported pressure is linearly dependent on the resulting ion current, which, in turn, depends on the appropriate cross sections of the gases. For simple and common gases (e.g., H2, N2, Ar) at low pressures, the correction factor for determining the real chamber pressure is known, but this is not the case for the molecular analytes covered in this study. Initially, spectra were collected using a residual gas analyzer MS of the type RGA200 from Stanford Research Systems and showed a strong bias towards low mass fragments. Later measurements (Fig. 4-5 and EIMS in Fig. 7) instead used the Pfeiffer QMA 400 instrument, where effort was made to ensure that no detection bias was present. Samples were let into the MS with a variable leak valve, allowing control of the pressure. Before each MS measurement, volatile atmospheric gases introduced during sample loading were slowly evacuated by a turbopump until only a trace amount of water could be detected. To protect the thermionic filament from overheating when running the MS at low electron energies an emission current of 0.1 A was used for all measurements (one tenth of the standard setting). To compensate for the resulting loss in signal intensity the chamber pressure was allowed to reach up to 5 × 10−6 mbar during measurements, as reported by the Penning gauge. To reduce baseline noise the collection of each spectrum was cycled four times and averaged. Measurements at different electron energies were performed in quick succession to ensure the chamber pressure did not drift appreciably. Analytes were purchased from SigmaAldrich (≥99 % purity) and used as received. 3. RESULTS AND DISCUSSION 3.1 Ionization Cross Section Calculation Results and Discussion The ionization cross section (analogous to the ionization probability) and the ionization energy (IE) are important properties of a propellant to maximize the performance and energy efficiency of a plasma thruster [26]. The IE of AHs can easily be found in the literature and are collected in Table. 1 of Appendix A, but experimental cross sections are only available for some atoms and for the simplest AHs, and we therefore refer to ab initio quantum chemical calculations instead. In Fig. 2 the total ionization cross section curves from (e,2e) ionization as calculated using Binary Encounter Bethe (BEB) theory are shown for a selection of relevant atomic and molecular species. Through experiment the total cross sections of the noble gases argon and xenon have been determined to 1.23-3.52 and 2.62-7.31 Å2, respectively, when ionized by 70-75 eV electrons [27,28]. Through BEB calculations we obtain 2.06 and 4.17 Å2, respectively, which are within the range of the experimental values. More importantly, BEB has been shown to accurately predict cross sections of many low mass molecules, including a range of different hydro-and fluorocarbons [29–31]. BEB cross sections of aromatic ring systems similar to small AHs are also known to be in excellent agreement with experimental values [32]. Page 6 of 23 Fig. 2 Total ionization cross sections calculated using BEB theory for the molecules in Fig. 1, argon, xenon, and atomic/molecular iodine. T is the kinetic energy of the incident electron impinging on the atom/molecule A clear separation in the cross sections between the included atoms and molecules can be observed in Fig. 2, where the former all have a cross section maximum in the range of 2 to 5 Å2. The maxima of the latter lie above 8 Å2 and rapidly ascend in order of increasing number of atoms of each respective molecule, starting with molecular iodine. For instance, adamantane and the naphthalenes have over 5 times the cross section of xenon, which would lead them being ionized after a much shorter time spent in the plasma. The cross-section curves of both atomic and molecular iodine are included in Fig. 2, as a large fraction of molecular iodine dissociates upon being ionized and the resulting uncharged iodine atom has to be ionized to produce thrust. The length L of the effective ionizing region of the plasma in a plasma thruster is proportional to the ionization cross section of a given propellant averaged over a Maxwellian velocity distribution [26] by 𝐿 ∝ 〈𝜎𝑣𝑒 〉−1 Where 〈𝜎𝑣𝑒 〉 ≈ 𝜎(𝑇𝑒 )𝑣̅ is the reaction rate coefficient that can be approximated by the product of the cross section averaged over a Maxwellian distribution of electrons with temperature 𝑇𝑒 and the average speed 𝑣̅ of those electrons. Increasing L will increase the degree of ionization of the propellant fed to the thruster by allowing for more collisions with the plasma electrons and more energy transfer into the propellant. However, increasing energy deposited into the propellant leads to decreasing energy efficiency of the thruster, and in the case of a molecular plasma promotes molecular dissociation processes. 3.2 Fragmentation Measurement Results and Discussion Incident electrons of kinetic energy T can cause ionization of the target atom or molecule through the (e,2e) mechanism, given that T exceeds their respective vertical IE. Energy deposition resulting from successful (e,2e) collisions have been calculated based on BEB theory, and the Page 7 of 23 gaussian-smoothed probability distributions (Eq. 4-7, Appendix B) are displayed in Fig. 3 for adamantane and the selected AHs. By increasing T, more strongly bound electrons in the molecules become accessible and can participate in (e,2e), resulting in the noticeable decrease in relative intensity of the lowest energy peak for each molecule. This decrease is less pronounced for adamantane in Fig. 3 (A) compared with the AHs (B-D) and should thus contribute to less variation in the degree of dissociation as T increases. We will see later, however, that the proclivity of adamantane to undergo ionization induced dissociation is already very high at T = 25 eV, and that the fragment abundance measured using standard 70 eV mass spectrometry is highly indicative of the stability (and lack thereof) at lower T. Fig. 3 Computed probability distributions of the energy deposition caused by single collision (e,2e) ionization for the molecules (A) adamantane, (B) fluorobenzene, (C) naphthalene, (D) 1fluoronaphthalene. The figure illustrates four (e,2e) scenarios: three where the incident electron kinetic energies T range from 25 to 70 eV, and one where T is dictated by a Maxwell-Boltzmann distribution of electron energies corresponding to a plasma temperature Te of 7 eV In electrostatic EP thrusters ions created through collisions with energetic electrons are accelerated to high velocities to produce thrust. Due to the significant difference in mass between the electrons and the larger particles within the anode chamber, a heterogeneous plasma can be sustained where the ionized species are expelled before thermal equilibrium with the electrons can be reached. The kinetic energy of the electrons in the ionizing plasma is here assumed to be well approximated by a Maxwell-Boltzmann distribution with a plasma temperature Te=7 eV (see Appendix B, Eq. 6). This Te was selected to obtain results comparable with those reported by Dietz et al in their study on plasma ionization of adamantane, xenon, and molecular iodine [15]. The calculated energy deposition caused by a Maxwellian plasma with an electron temperature Te = 7 eV is also included for the molecules in Fig. 3 (purple curves). These curves are very similar, but slightly lower, to the (blue) curves resulting from (e,2e) with T = 25 eV. With the assumption of a low number of electron collisions taking place within the 7 eV plasma, the 25 eV (e,2e) measurement is well suited for estimating the adamantane fragmentation within such a plasma. This was verified in the current study where the 25 eV mass spectrum of adamantane agreed well with measurements made in the 7 eV plasma thruster by Dietz et al (vide infra). Furthermore, the difference between energy deposition from low energy (e,2e) and plasma ionization for the selected AHs is more pronounced, indicating that electron energies even lower than T = 25 eV could be appropriate when simulating their behavior in a 7 eV plasma. The electron ionization mass spectra (EIMS) of adamantane and the AHs can be seen in Fig. 4. The intensities observed in the measurements cannot be used to compare absolute ionization cross Page 8 of 23 sections between the different analytes, as we had no reliable way of measuring the absolute pressure (see methods). However, the relative change in ion current as a function of T for each analyte is easily determined and can be found in the right column of the tables in Fig. 4, as well as in Appendix A, Table 2. In Table 2 we also list the relative cross sections calculated using the BEB method. Immediately evident from the spectra is the much lower fragmentation of the AHs when compared with adamantane, and the similarly sized fragmentation peaks of the naphthalenes. Fig. 4 Mass spectra from the molecules (A) adamantane, (B) fluorobenzene, (C) naphthalene, (D) 1-fluoronaphthalene, collected at different incident electron energies T. The spectra collected at 40 and 25 eV have been horizontally offset by 4 and 8 m/z, respectively. For each molecule is included a table with the relative ion current of M+ with respect to the total ion current 𝐼𝑀+ /𝐼𝑡𝑜𝑡 (left), and the relative total ion current with respect to the same total ion current measured at 70 𝑇 𝐼𝑡𝑜𝑡 /𝐼𝑡𝑜𝑡 (right) As mentioned earlier, adamantane has been shown to suffer from a high degree of fragmentation under conditions commonly found in EP thruster discharge chambers [15,16]. Per Fig. 2 and Table 2 (Appendix A), the BEB cross section of adamantane decreases by 12.3 and 43.1 % when electron energy is reduced from 70 eV to 40 and 25 eV, respectively. This is reasonably well reflected by our measurements in Fig. 4 (A) by a 14.6 and 51.1 % reduction, respectively. However, as can be seen in the low ratios in the left column of the table in (A), a very large part of the adamantane ion current stems from fragments of molecular dissociations. Even when using the low electron energy T = 25 eV only 33.9 % of the signal is due to the adamantane molecular ion (M+), a figure which rises to 41.3 % (Table. 2) if none of the neutral hydrogen loss from the parent molecule is counted towards fragmentation (i.e. M+ = [M-HX]+ where X≥0). Each signal in Fig. 4 caused by the detection of an ionic fragment is associated with at least one additional neutral fragment which does not appear in the measured spectra. In a plasma thruster all such Page 9 of 23 neutrals would have to be ionized before undergoing electric or electromagnetic acceleration, and for adamantane this would result in a very low energy efficiency because of the excessive fragmentation. The mass spectra measurements performed by Dietz et al on the exhaust plume of a modified RIT thruster operating on adamantane (see Figure 9 in [15],) show a very high abundance of fragments. The authors varied the mass flow of adamantane to the thruster as well as the power of the radio frequency generator (RFG) to estimate the effect of plasma temperature and electron-neutral collision frequency on the molecular plasma composition. At the lowest RFG power, and with plasma temperature Te estimated to be 7 ± 1 eV, the relative abundance of [MHX]+ ions exiting the RIT was roughly 36 % of the total ion current. This high fragmentation agrees well with the value of 41.3 % we obtained for T = 25 eV in the present work. The behavior of the AHs in Fig. 4 under ionizing conditions is distinctly different from adamantane, whereas the differences between the individual AHs are rather small. We will therefore discuss the naphthalene spectra in more detail and compare them to the adamantane spectra. The mass spectra of naphthalene in Fig. 4 (C) display much less intense fragment ion signals in relation to M+ for all T. Notably, naphthalene remains intact to a larger degree even at T=70 eV (IM+=47.8 %) compared with adamantane at T=25 eV (IM+=33.9 %). At 25 eV the percentage of intact naphthalene cations is 85.3 % of the total ion current (92.5 % for [M-HX]+), while the remaining 14.7 % (7.5 %) is due to detected fragment ions. The largest difference in fragmentation behavior is observed when T is increased from 25 to 40 eV, which is consistent with the corresponding curves in Fig. 3 where the yellow and red lines (40 and 70 eV) overlap to a large extent. This highlights the importance of optimizing the ionization conditions of the plasma thruster to prevent excess energy being deposited into the propellant, as all fragmentation constitutes a loss of thruster efficiency. To increase legibility, the 25 eV mass spectrum of naphthalene was converted to a histogram bar graph by integrating the signals about each integer m/z. The result can be seen in Fig. 5 where the region 20-105 m/z has been scaled up and labels for the most abundant fragments have been included. Fig. 4 Histogram mass spectrum of naphthalene obtained at T=25 eV incident electron energy, where the region 20-105 m/z has been scaled up From Fig. 5 it is clear that for single collisions with low energy electrons, naphthalene is likely only to dissociate into a few select fragments. The rightmost peaks (m/z=101,102 amu) in the + , is the fragment associated with loss of acetylene, C2 H2 , whose scaled-up figure, denoted C8 H5−6 signal is detected as the leftmost peak at m/z=26 amu. Together, these constitute the full naphthalene molecule or in the case of any associated hydrogen loss, some version of [M-Hx]+. + The remaining peak, C6 H4−6 , is associated with a fragment that appears with low intensity at m/z=50 amu. Page 10 of 23 The situation becomes significantly more complex when a plasma is used for ionization. By colliding with energetic electrons, the energy of a molecule increases. If the pressure is high, this energy is rapidly distributed through molecule-molecule collisions, resulting in a rise in temperature. At higher temperatures, more dissociation pathways become available and, consequently, more unique fragments are created during ionization and can be detected from mass spectrometry. We performed high temperature gas chromatography mass spectrometry (GC-MS) at 250 °C and T = 70 eV electrons on the analytes in Fig. 4 (except fluorobenzene) to evaluate how their respective fragment abundances change when heated. The superimposed spectra from GCMS with those from relevant measurements in Fig. 4 can be found in Appendix A, Fig. 7. In the case of adamantane, GC-MS resulted in a noticeable increase in fragment intensities while the effect was more subdued for the naphthalenes, almost to the point of being indistinguishable when compared with the cold EIMS experiment. Similar effects were reported by Eren et al [33] who noted that “… for small molecules (below 20 atoms) such as benzene (C6H6), no or very little vibrational cooling effect is observed in their Cold EI mass spectra, while compounds with 20-40 atoms show noticeable enhancement of their molecular ion abundance.” In a study by Alliati et al [34] where they measured ionization of naphthalene using a plasma sourced from a high-frequency RFG, the types of fragments detected was almost identical to those we found when using T = 40 and 70 eV electrons in (e,2e) of naphthalene. The relative intensities of fragment peaks following plasma ionization were, however, found to be much lower and on par with our 25 eV measurements. The authors did not specify an electron temperature associated with the plasma electrons, or even if they could be considered Maxwellian and have a well-defined temperature, and therefore it is difficult to draw conclusions beyond these general observations in relation to the results obtained in this work. Nevertheless, their results are very encouraging and support our observations of naphthalene’s stability under relevant conditions. Expanding the discussion to include the rest of the AHs in Fig. 4, the relative ion current at T = 25 eV for naphthalene appears slightly larger than for 1-fluoronaphthalene in accordance with the BEB cross sections in Fig. 2. Their fragmentation abundances are nearly identical at all T, confirming that the addition of a fluorine substituent has negligible effect on the molecular stability and that fluorine substitution is a useful tool which allows for expanding on the AHs capabilities as propellants. Fluorobenzene is slightly more prone to dissociate at T = 25 eV compared to the heavier naphthalenes, but the difference is minor and should not exclude fluorobenzene from being considered a viable propellant candidate. Treating [M-HX]+ ions as intact molecules despite loss of neutral hydrogen (as was done in the paper by Dietz et al [15] when analyzing adamantane) increases the intact-to-total-ion ratio of the T = 25 eV spectra from that of the right columns in Fig. 4 to 0.844, 0.925, and 0.897 for fluorobenzene, naphthalene, and 1-fluoronaphthalene, respectively. Disregarding hydrogen fragmentation is not entirely unwarranted, as both molecular and atomic hydrogen obtain considerable velocity when having undergone dissociation, due to their low mass. The ionization cross section of molecular hydrogen is around 0.5 Å2 when T=25 eV [35] and its 1st IE is over 15.4 eV, making it much less likely to be ionized by a plasma thruster designed and optimized for much heavier molecules with large cross sections and low IE. Atomic hydrogen even less so. A more likely outcome is that the dissociated neutral hydrogen will, to a large degree, exit the thruster without being ionized. However, the hydrogen could also participate in neutral-neutral and neutral-ionic collisions with other molecules. Both processes, which are inherent when dealing with molecular plasmas, contribute to increasing the ionization inefficiency of the thruster by promoting further dissociation of the propellant. Page 11 of 23 4. Performance, Accommodation, and Impact of AHs as EP Propellants 4.1 Performance Predictions for AHs Fluorobenzene has a relatively low molecular mass of 96.1 amu, high vapor pressure and low melting point, which could lead to low heater power requirements and higher specific impulse. It could be an attractive candidate for, e.g., power- and volume-constrained satellites, or long-range missions with low available power and high delta-v requirements. The naphthalenes are heavier and therefore better suited for high thrust applications. The larger ionization cross section and lower IE of the AHs compared to that of xenon could result in a comparable or even higher ionization efficiency compared to that of xenon despite ionization-induced fragmentation of the AHs. To put it in perspective, the difference in IE between naphthalene and xenon is 4 eV which translates to about 386 kJ mol-1 or 92.2 kcal mol-1 (1 mol is approximately 0.13 kg in this case). With an assumed propellant feed rate of 10 mg s-1 to the thruster (and disregarding any energy costs associated with fragmentation), the 4 eV difference would equate to a 30-W reduction in power required for total ionization. Furthermore, the mass of naphthalene (128.2 amu) is very close to that of xenon (131.3 amu), making it an ideal benchmark propellant for future testing. 4.2 Propellant Storage and Delivery In Table. 1 of Appendix A the boiling point and density of several AHs are listed. The density of the AHs in Fig. 1 ranges between 1020-1140 kg m-3 and the boiling point is in all cases high enough to ensure that they all remain non-gaseous at room temperature and under atmospheric pressure (i.e., at NTP). A propellant stored onboard a satellite can be kept close to NTP, and as the melting point of 1-fluoronaphthalene and fluorobenzene are negative on the Celsius scale, they will remain liquid. Naphthalene is solid at NTP, but the vapor pressure is sufficient to facilitate the transfer rate required to feed a mN-class thruster and the melting point is sufficiently high to avoid a solid-liquid phase transition. Vapor pressure data for the fluoronaphthalenes was not available, but the fluoronaphthalenes are assumed to have similar vapor pressure curves as naphthalene, since benzene and the fluorobenzenes have similar vapor pressure curves as shown in Fig. 6. While the AHs cannot be compressed to the density of pressurized gaseous propellants like xenon at 186 bar, or the density of heavier atomic propellants like iodine or mercury, they do avoid the added mass and safety considerations of the high-pressure propellant storage equipment required by the former and many material incompatibilities of the latter. 4.3 Accommodation in electrostatic EP thrusters Positively charged propellant electrostatically expelled from the thruster must be neutralized to prevent the accumulation of negative charge in the spacecraft. This is achieved by electron emission, which in addition to neutralizing the exhaust, also feeds electrons for the ionizing plasma. For high power thrusters where high electron currents are needed, neutralization is typically accomplished by thermionic emission in the form of a hollow cathode placed close to the thruster exhaust. This type of cathode needs to be fed with a significant amount of propellant to facilitate the outgoing flow of electron current, all while the actively emitting thermionic material is kept at temperatures normally between 1300-2000 °C. At those temperatures most types of molecules will completely disintegrate into their highly reactive atomic constituents. The materials commonly used for thermionic emission are typically fed with very pure and inert xenon, and the performance is very sensitive to changes in their surface chemical composition. Extensive exploration of the literature strongly indicates that hydrocarbons are not compatible with most common types of thermionic emitter materials [36–43]. While research on the Page 12 of 23 poisoning behavior of most emitters is scarce, emitters are likely to be inoperable in non-inert atmospheres because of their fragile and easily disrupted emission capacities. Alternative electron emission technologies, such as field emission (FE) array cathodes, exist. FE cathodes require no propellant feed or dedicated heating to operate, but they instead rely on a strong electric field to pull electrons from the surface of the material. The achievable emission output is typically limited to only a few mA cm-2, whereas high-performance hollow cathodes can be made to emit tens to hundreds of amperes. Recent advances have been made in operating resistant FE array cathodes based on carbon nanotubes in the hazardous environment of Low Earth Orbit, as part of the KITE mission conducted by the Japanese space agency JAXA [44,45]. Their achieved current output was quite limited, but other researchers have demonstrated optimized CNT array emitters reaching close to 1 A cm-2 [46]. In the future, FE cathodes like these could become viable for use with thrusters operated on alternative propellants like the AHs suggested here. 4.4 Safety Considerations and Environmental Impact AHs have been linked to several kinds of adverse health effects in humans, both acute and chronic [49], but studies have shown that the toxicity of unsubstituted AHs generally decreases with molecular weight, except for the highly carcinogenic benzene [50]. Naphthalene is classified as harmful if swallowed and suspected to be cancerogenic according to the European Chemicals Agency (ECHA [51]. The ECHA classification for 1-fluoronaphthalene only states that it causes serious eye irritation, causes skin irritation and may cause respiratory irritation [52]. Fluorobenzene is only classified to cause serious eye damage, although it has been identified that it may cause damage to organs through prolonged or repeated exposure [53]. Naphthalene is very toxic to aquatic life with long-lasting effects. The corresponding effects are less severe for fluorobenzene and 1-fluoronaphthalene as indicated by their ECHA classifications [51-53]. It would be necessary to implement appropriate safety procedures for the handling of AHs by ground facility personnel. However, it should be noted that the potential health hazards of the AHs are much less severe than for the highly toxic and carcinogenic monopropellant hydrazine and therefore the safety precautions for AHs would be much less restrictive. In addition, the AHs are not classified as explosives, and handling procedures would be simplified compared to those for monopropellants or xenon under high pressure. As for potential environmental impact, whereas halogenated hydrocarbons of the heavier halogens may have a damaging effect on the ozone layer when introduced into the stratosphere, it is known that atmospheric fluorine does not lead to depletion of ozone [47]. Fluorocarbons (such as the fluorinated compounds of this study) can contribute to global warming, mainly by absorbing 1000-1400 cm-1 IR radiation where the atmosphere is otherwise relatively transparent [48]. It should be noted that the lifetime of the AHs in this study is expected to be relatively short at altitudes above the stratosphere due to high levels of UV-radiation. Thus, the AHs are not likely to contribute to global warming. 5. CONCLUSIONS We have evaluated the aromatic hydrocarbon (AH) molecules fluorobenzene (C6H5F), naphthalene (C10H8), and 1-fluoronaphthalene (C10H7F) for use as alternative propellants in electric propulsion thrusters for in-space propulsion by comparing environmental and safety aspects, material compatibility, storability, cost and predicted ionization efficiency. Neither naphthalene nor the fluorinated AHs pose a risk of depleting the ozone layer if they were to reach the stratosphere. While appropriate safety procedures would have to be implemented Page 13 of 23 for the handling of AHs, they are not classed as explosives and do not require the safety precautions associated with high-pressure gas and monopropellants. A literature survey indicates that AHs are incompatible with available emitter materials used in high-current hollow cathode neutralizers, limiting the propellant flow rates and thrust achievable with AH propellants in electrostatic thrusters; however no material compatibility issues are foreseen for cathodeless electromagnetic thrusters. The AHs are liquid or solid at common satellite propulsion subsystem pressures and temperatures, and while their density is lower compared to high-pressure xenon or propellants like iodine and mercury, they do avoid the added mass and safety considerations of the highpressure propellant storage equipment required by the former and many material incompatibilities of the latter. The solid/liquid-vapor phase transition properties of the AHs show that they will be compatible with a low-power sublimation-based propellant feed system. Ionization efficiency was estimated by calculation of the ionization cross section and measurement of the ionization-induced fragmentation. Quantum chemical calculations and Binary Encounter Bethe (BEB) theory were used to calculate the ionization cross section of these AHs. The calculated ionization cross sections for the naphthalenes are more than five times higher than that of xenon. BEB calculations further indicate that 25 eV electrons and electrons from a 7 eV Maxwellian plasma deposit similar amounts of energy into the molecules, allowing estimation of the fragmentation behavior of the molecules in a 7 eV plasma thruster using 25 eV EI-MS. These measurements for the AHs show very low fragmentation with 81 - 86 % of the observed ions originating from intact molecules, compared with 34 % for adamantane. Considering that the ionization energy of the AHs is 4 eV lower than that of xenon, the higher ionization cross section together with the low degree of post-ionization dissociation indicate that the ionization efficiency of the AHs could be similar to or greater than that of xenon. The investigated AHs are considered viable candidates as an alternative propellant to xenon due to their similar mass, lower storage pressure, lower cost, and potentially comparable or higher ionization efficiency. Page 14 of 23 1. APPENDIX A (TABLES AND FIGURES) Table. 1 aromatic hydrocarbons (AH) identified as potential EV propellant candidates together with relevant physical properties reported at standard conditions. Adamantane and xenon are included at the bottom for easy comparison. Mw=molecular weight, TM=melting point, TV=boiling point, ρ=density, IEa is the adiabatic ionization energy, and T(ṁ) the temperature corresponding to an estimated evaporation/sublimation rate of ~10 mg cm-2 s-1 into vacuum as calculated with Eq. 1 and 2 (Appendix B) using Antoine constants found in Table. 3 Chemical formula Mw g mol-1 TM (TV) °C ρ kg m-3 1st IEa eV T(ṁ) °C Bulk price4 € kg-1 benzene C6H6 78.1 5.5 (80.1) 876 9.24 -57* 38.9 (100L) fluorobenzene C6H5F 96.1 -42 (85) 1020 9.20 -57* 39.4 (200kg) cyanobenzene C6H5CN 103.1 -10 (191) 1000 9.73 11* 47.9 naphthalene C10H8 128.2 86.3 (217) 1140 8.14 41 46.8 1-fluoronaphthalene C10H7F 146.2 -13 (215.2) 1132 8.15 - 93.3 2-fluoronaphthalene C10H7F 146.2 58 (211.6) 1240 8.23 - - fluorene1 C13H10 166.2 116 (298) 1200 7.91 86 73.8 (>98%) phenanthrene C14H10 178.3 101 (332) 1180 7.89 102 147.4 anthracene C14H10 178.3 216.4 (341) 1250 7.44 136 83.1 hexafluorobenzene C 6F 6 186.1 5.2 (80.2) 1610 9.90 -574 333.7 pyrene C16H10 202.3 146 (404) 1270 7.43 1424 112.1 (>98%) 6.92 2504 12984.6 (>95%) >2000 Anthanthrene2 C22H12 276.3 261 (556) 1377 coronene C24H12 300.3 437.3 (525) 1371 7.29 3094 adamantane xenon C10H16 Xe 136.2 131.3 279 -111.8 (-108) 1080 >15003 9.25 12.13 - 1Not fully aromatic but often considered an AH 2Commonly referred to as Dibenzo[def,mno]chrysene 3Storage density can be increased through pressurization 4Prices obtained in the middle of June 2021 and correspond to order size of 100 kg and ≥99 % purity unless otherwise stated (estimates produced by Chemtronica AB, www.chemtronica.com/en) 4Temperature estimated by extrapolation outside the valid range defined for the Antoine constants Page 15 of 23 Table. 2 Relative ion intensities for the spectra shown in Fig. 4 together with relative ionization cross sections calculated using Binary Encounter Bethe theory T (eV) 𝐓 𝟕𝟎 𝐈𝐌 + /𝐈𝐌 + 𝐈𝐌+ /𝐈𝐭𝐨𝐭 𝐈∑[𝐌−𝐇𝐱]+ /𝐈𝐭𝐨𝐭 𝟕𝟎 𝐓 /𝐈𝐭𝐨𝐭 𝐈𝐭𝐨𝐭 𝛔𝐓𝐁𝐄𝐁 /𝛔𝟕𝟎 𝐁𝐄𝐁 naphthalene 25 40 70 0.874 0.958 1.000 0.853 0.567 0.478 0.925 0.673 0.559 0.490 0.809 1.000 0.583 0.879 1.000 1-fluoronaphthalene 25 40 70 0.814 0.911 1.000 0.858 0.578 0.474 0.897 0.642 0.523 0.451 0.747 1.000 0.550 0.857 1.000 fluorobenzene 25 40 70 0.769 0.943 1.000 0.808 0.559 0.490 0.844 0.608 0.537 0.467 0.827 1.000 0.523 0.841 1.000 adamantane 25 40 70 0.974 1.066 1.000 0.339 0.212 0.170 0.413 0.263 0.212 0.489 0.854 1.000 0.569 0.877 1.000 Table. 3 Antoine constants for AHs and benzene analogues and the temperature ranges over which they are defined. No data for 1-fluoronaphthalene could be found in the literature Temperature range (K) (°C) A B (K) C (K) Benzene1 280-374 7-101 5.98523 1184.236 -55.623 Fluorobenzene2 255-357 -18-84 4.36225 1409.848 -34.792 Cyanobenzene2 301-464 28-191 4.85410 2110.572 -28.331 Naphthalene1 310-353 368-523 37-80 95-250 8.70592 6.13555 2619.91 1733.71 -52.5 -71.291 Fluorene1 307-348 348-388 34-75 75-115 5.33196 8.91036 1547.377 3245.362 -133.083 -45.893 Phenanthrene1 306-321 356-650 33-48 83-377 11.631 6.37081 4873.4 2329.54 0.05 -77.87 Anthracene1 299-430 26-157 10.5899 4903.3 -1.58 Hexafluorobenzene2 278-378 5-105 4.16054 1229.449 -57.503 Pyrene1 298-401 25-128 10.7545 5072.78 0.0 Anthanthrene1 465-495 192-222 12.014 7060 0.0 Coronene1 430-560 157-287 8.80062 5491.689 -48.484 Pressure given in 1kPa [54] 2102 kPa [55] Page 16 of 23 Fig. 5 Vapor pressures of examined cyclic aromatic hydrocarbons as a function of temperature obtained using data in Table 2 and Eq. 1. The melting point (measured at 25 °C and 100 kPa) for each compound is indicated by vertical lines and the points corresponding to an evaporation mass flow rate of ~10 mg cm-2 s-1 as calculated using Eq. 2 (where α=1 and Pc=0) are marked with a black cross if they are within their respective Antoine temperature range, or a grey cross if they are based on extrapolation outside the range of the source data. These temperatures (and vapor pressures for benzene and the fluorobenzenes) are indicated numerically in the figure Page 17 of 23 Fig. 6 Mass spectra of adamantane (A) naphthalene (B) 1-fluoronaphthalene (C), collected by electron ionization mass spectrometry (EIMS) and gas chromatography mass spectrometry (GCMS) using T=70 eV incident electron kinetic energy. A lower detection limit of m/z=39 amu was present for the GCMS measurements, therefore the figure above does not include any of the fragments detected with EIMS below that point Page 18 of 23 2. APPENDIX B (EQUATIONS) Eq. 1-2 Relations for calculating vapor pressures and mass flows by sublimation/evaporation from solid/liquid phase log10 (𝑃𝑣 ) = 𝐴 − 𝑚̇ = 𝐵 𝑇𝑠 + 𝐶 𝛼𝐴𝑠 (𝑃𝑣 − 𝑃𝑐 ) √2𝜋𝑘𝑏 𝑇𝑠 𝑚𝑝−1 (1) (2) where A, B, C are Antoine constants, 𝑚̇ is the mass flow rate (kg m-2s-1), Pv and Pc is the respective vapor pressure and surrounding gas pressure (Pa), kb is the Boltzmann constant, Ts is the surface temperature (K), mp is the molecular mass (kg), As is the surface area (m2) and α is a sticking coefficient. Eq. 3 Binary Encounter Bethe theory [24,25] used for calculating absolute single-collision (e,2e) ionization cross sections by taking the sum over all (occupied) orbitals i of the target atom or molecule 𝜎𝐵𝐸𝐵 = ∑ 𝜎𝑖 = ∑ 𝑖 4𝜋𝑎02 𝑁(𝑅/𝐵𝑖 )2 ln 𝑡 1 1 ln 𝑡 (1 − 2 ) + 1 − − [ ] 𝑡 𝑡 𝑡+1 𝑡+𝑢+1 2 𝑖 (3) where N is the orbital occupancy number, R the Rydberg constant, 𝑎0 the Bohr radius, Bi the negative orbital binding energy (i.e., the vertical ionization energy to afford a specific electronic state), Ui the orbital kinetic energy, T the kinetic energy of the incident electron, and u=U/B and t=T/B are the reduced versions of U and T, respectively. The procedure for calculating B and U is detailed in the method section of this article. Note that 𝜎𝑖 = 0 if 𝑇 < 𝐵 as there is not enough energy to eject an electron from the orbital. Eq. 4 Probability distribution for the energy deposition E into a target molecule or atom resulting from successful electron ionization through the (e,2e) process, as presented in the paper by K. Irikura [56] 𝑃(𝐸, 𝑇) = ∑𝑖 𝛿(𝐸 − 𝐵𝑖 )𝜎𝑖 (𝑇) ∑𝑖 𝜎𝑖 (𝑇) (4) where Bi and 𝜎𝑖 are defined in Eq. 3, E is the energy deposited into the ionization target, and 𝛿 is the Dirac delta function defined by 𝛿(𝑥) = 1 when 𝑥 = 0 and 𝛿(𝑥) = 0 when 𝑥 ≠ 0. Eq. 5 Modified version of Eq. 4 used to estimate the probabilistic energy deposition caused by (e,2e) ionization by electrons with energies following a Maxwellian distribution PMB 𝑃(𝐸, 𝑇) = ∑𝑖 𝛿(𝐸 − 𝐵𝑖 )𝑃𝑀𝐵 (𝑇)𝜎𝑖 (𝑇) ∑𝑖 𝑃𝑀𝐵 (𝑇)𝜎𝑖 (𝑇) (5) Page 19 of 23 Eq. 6 Probabilistic Maxwell-Boltzmann energy distribution derived for massive, non-relativistic particles 1 𝑇 𝑇 2 𝑃𝑀𝐵 (𝑇) = 2 ( 3 ) exp (− ) 𝑇𝑒 𝜋𝑇𝑒 (6) where Te is the electron temperature (in eV), and T is (same as Eq. 3-5) the electron kinetic energy. Eq. 7 Gaussian smoothing function 𝐸 − 𝐵𝑖 2 𝐺 = exp⁡(( ) 𝜌 (7) where 𝜌 = 1.5⁡𝑒𝑉 is the variance used for all calculations presented in this work, and E and Bi are defined in Eq. 3 and 4, respectively. Page 20 of 23 References [1] Szabo J, Robin M, Paintal S, Pote B, Hruby V. High density Hall thruster propellant investigations. 48th AIAA/ASME/SAE/ASEE Joint Propulsion Conference and Exhibit 2012 2012:1–15. https://doi.org/10.2514/6.2012-3853. [2] Szabo J, Robin M, Hruby V. Bismuth Vapor Hall Effect Thruster Performance and Plume Experiments. 35th International Electric Propulsion Conference (IEPC-25), 2017, p. 1–13. [3] Szabo J, Robin M, Duggan J, Hofer RR. Light Metal Propellant Hall Thrusters. 31st International Electric Propulsion Conference (IEPC-138), 2009, p. 1–12. [4] Kieckhafer A, King LB. Energetics of propellant options for high-power hall thrusters. J Propul Power 2007;23:21–6. https://doi.org/10.2514/1.16376. [5] Schwertheim A, Knoll A. 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Selective Akt Inhibitors Synergize with Tyrosine Kinase Inhibitors and Effectively Override Stroma-Associated Cytoprotection of Mutant FLT3-Positive AML Cells Ellen Weisberg1*., Qingsong Liu2., Xin Zhang2, Erik Nelson1, Martin Sattler1, Feiyang Liu3, Maria Nicolais1, Jianming Zhang2, Constantine Mitsiades1, Robert W. Smith1, Richard Stone1, Ilene Galinsky1, Atsushi Nonami1, James D. Griffin1*, Nathanael Gray2 1 Department of Medical Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, United States of America, 2 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, United States of America, 3 High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei, Anhui, P.R. China Abstract Objectives: Tyrosine kinase inhibitor (TKI)-treated acute myeloid leukemia (AML) patients commonly show rapid and significant peripheral blood blast cell reduction, however a marginal decrease in bone marrow blasts. This suggests a protective environment and highlights the demand for a better understanding of stromal:leukemia cell communication. As a strategy to improve clinical efficacy, we searched for novel agents capable of potentiating the stroma-diminished effects of TKI treatment of mutant FLT3-expressing cells. Methods: We designed a combinatorial high throughput drug screen using well-characterized kinase inhibitor-focused libraries to identify novel kinase inhibitors capable of overriding stromal-mediated resistance to TKIs, such as PKC412 and AC220. Standard liquid culture proliferation assays, cell cycle and apoptosis analysis, and immunoblotting were carried out with cell lines or primary AML to validate putative candidates from the screen and characterize the mechanism(s) underlying observed synergy. Results and Conclusions: Our study led to the observation of synergy between selective Akt inhibitors and FLT3 inhibitors against mutant FLT3-positive AML in either the absence or presence of stroma. Our findings are consistent with evidence that Akt activation is characteristic of mutant FLT3-transformed cells, as well as observed residual Akt activity following FLT3 inhibitor treatment. In conclusion, our study highlights the potential importance of Akt as a signaling factor in leukemia survival, and supports the use of the co-culture chemical screen to identify agents able to potentiate TKI anti-leukemia activity in a cytoprotective microenvironment. Citation: Weisberg E, Liu Q, Zhang X, Nelson E, Sattler M, et al. (2013) Selective Akt Inhibitors Synergize with Tyrosine Kinase Inhibitors and Effectively Override Stroma-Associated Cytoprotection of Mutant FLT3-Positive AML Cells. PLoS ONE 8(2): e56473. doi:10.1371/journal.pone.0056473 Editor: Ivan Cruz Moura, Institut national de la santé et de la recherche médicale (INSERM), France Received November 6, 2012; Accepted January 9, 2013; Published February 21, 2013 Copyright: ß 2013 Weisberg 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: Grant Support: QL and NG are supported by National Institutes of Health (NIH) Library of Integrated Network-Based Cellular Signatures (LINCS) grant HG006097. FL is supported by a Chinese Academy of Science High Magnetic Field Laboratory training grant. JDG is supported by NIH grant CA66996. FL is supported by CAS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: JDG has a financial interest with Novartis Pharma AG. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: ellen_weisberg@dfci.harvard.edu (EW); james_griffin@dfci.harvard.edu (JDG) . These authors contributed equally to this work. concentrations by HS-5 stroma (including SCF, IL-6, IL-8, IL11, M-CSF and GM-CSF), were able to partially protect TKItreated chronic myeloid leukemia (CML) cells and AML cells [2,3]. A subset of AML cells expresses a mutated form of the class III receptor tyrosine kinase FLT3 (Fms-Like Tyrosine kinase-3; STK1, human Stem Cell Tyrosine Kinase-1; or FLK-2, Fetal Liver Kinase-2) [4], which has inspired the development of a number of small molecule inhibitors of mutant FLT3. However, FLT3 inhibitors tested thus far, including PKC412 (midostaurin) [5], which is in late stage (Phase III) clinical trials, and the highly potent and selective FLT3 inhibitor, AC220 (quizartinib) [6], which is in early phase clinical trials, generally at best induce Introduction Resistance to TKIs in leukemia patients presents a significant clinical challenge. As small numbers of leukemia cells have been observed to persist in the bone marrow of TKI-treated patients, despite rapid and dramatic clearance of peripheral blood blasts, there is growing interest in determining the role of the bone marrow microenvironment in the long-term survival of leukemic stem cells. Indeed, the number of existing leukemic stem cells that exhibit high survival ability on bone marrow stromal layers has proven to be a significant prognostic indicator [1]. Of relevance, we have found that media conditioned by human HS-5 stromal cells, as well as a cocktail of cytokines secreted in high PLOS ONE | www.plosone.org 1 February 2013 | Volume 8 | Issue 2 | e56473 Akt and FLT3 Inhibition for Treatment of AML partial and transient clinical responses in patients when used alone. In addition, we have found that bone marrow-derived stroma diminishes the activity of both PKC412 and AC220 [7]. There is thus a need for identification and development of novel therapies that can be effectively combined with TKIs to delay or suppress leukemia progression, override stroma-associated drug resistance, and increase patient survival. We have recently identified the multi-targeted kinase inhibitor, dasatinib, and dasatinib-like compounds as being able to potentiate the activity of TKIs PKC412 and AC220 against mutant FLT3-expressing cells cultured in the presence of cytoprotective and cytokine-abundant stromal-conditioned media (SCM) by performing a combinatorial drug screen using the KIN001 library (Dr. Nathanael Gray) [7]. Our study also highlighted the potential of Jak inhibitors to synergize with PKC412 and AC220 as well as enhance their apoptotic activity against mutant FLT3-expressing cells cultured in the presence of SCM [7]. While the significance of stromal-derived growth factors in viability enhancement and cytoprotection of leukemic stem cells cannot be denied, not all hematologic malignancies can be rescued from programmed cell death by secreted cytokines in the absence of direct communication with the stromal cells themselves. As examples, protection of AML cells and B-lineage ALL cells from spontaneous and/or drug-induced apoptosis was observed to depend on direct bone marrow fibroblast cell:leukemic cell interaction [8–10]. Similarly, protection of CLL cells from apoptosis depends on adherence of these cells to bone marrow stromal layers [11], and adhesion between bone marrow stroma and myeloma cells is necessary for protection of these cells from drug-induced apoptosis [12]. Thus, the direct interaction between stromal cells and leukemic cells is important to fully understand the mechanisms driving stromal-mediated chemoresistance, as well as for identification of integral signaling molecules as potential therapeutic targets for overriding drug resistance. To address this, we used an adherent stroma-based co-culture system, as opposed to the SCM-based system used previously, as the basis for a combinatorial drug screen designed to identify novel kinase inhibitors able to potentiate the apoptosis-inducing effects of PKC412 against adherent stroma-protected mutant FLT3positive cells (see schematic in Figure S1, which illustrates both the adherent stroma-based screen used in this study as well as the SCM-based chemical screen [7]). In parallel to the KIN001 kinase inhibitor library, we also screened the LINCS kinase inhibitor library, which is composed of inhibitors characterized as being relatively potent and selective toward a limited range of kinase targets. Here, we identified selective Akt inhibitors, such as MK2206, as able to effectively combine with FLT3 inhibitors, including PKC412 and AC220, against mutant FLT3-expressing cell lines or primary AML cells cultured in a cytoprotective stromal environment. This synergy occurs both in the absence as well as the presence of stroma or stromal-derived cytokines, and could thus potentially be further investigated as a therapeutic for AML as well as possibly delay/eradicate residual disease. In addition, p38 MAPK inhibitors also positively combined with PKC412 against mutant FLT3-expressing cells protected by stroma. Our findings suggest that the combination of kinase inhibitorenriched chemical libraries and the leukemia cell:stromal cell coculture assay could be useful for discovery of novel therapeutic combinations for AML. This technical approach could also be employed for identification of protein kinases with potential to be exploited as novel therapeutic targets. PLOS ONE | www.plosone.org Materials and Methods Kinase Inhibitor Focused Libraries (KIN001 and LINCS) Two Kinase Inhibitor Focused Libraries (KIN001 and LINCS) were chosen for screening to identify single agents with little-to-no appreciable efficacy but that are able to synergize with PKC412 against the human mutant FLT3-expressing AML cell line, MOLM14-luc+, cultured in the presence of adherent HS-5 stroma. The KIN001 Library was developed by Dr. Nathanael Gray’s lab and is comprised of 188 commercially-available kinase inhibitors as well as in-house developed diverse pharmacophorebased kinase inhibitors targeting either active or inactive kinase conformations. The chemical screening concentration was 660 nM, which is the same screening concentration as was used previously when this library was used to identify kinase inhibitors able to synergize with FLT3 inhibitors in the presence of SCM [7]. The LINCS library is available from Harvard Medical School/ NIH LINCS program (https://lincs.hms.harvard.edu/) and contains 202 known selective and potent kinase inhibitors. Cell Lines and Cell Culture The human AML-derived, FLT3-ITD-expressing cell lines, Molm14 [13] and MV4;11 [14], were provided to us by Dr. Scott Armstrong, Dana Farber Cancer Institute, Boston, MA. MOLM14 cells were transduced with the FUW-Luc-mCherrypuro lentivirus as previously described [15]. Within the past six months, mutant FLT3 expression and integrity in this line was confirmed. The human AML-derived, FLT3-ITD-expressing cell line, MOLM-13, was obtained from DSMZ (German Resource Centre for Biological Material). MOLM-13 cells were also engineered to express luciferase fused to neomycin phosphotransferase (pMMP-LucNeo) by transduction with a VSVG-pseudotyped retrovirus (MOLM13-luc+ cells), as previously described [16]. The IL-3-dependent murine hematopoietic cell line, Ba/F3, was transduced with FLT3-ITD-containing MSCV retroviruses harboring a neomycin selectable marker, and selected for resistance to neomycin [17]. Mutant FLT3-transduced cells were selected for growth in G418 (1 mg/ml). Within the past six months, mutant FLT3 expression and integrity in this line was confirmed. The HS-5 stromal cell line was purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). All cell lines were cultured with 5% CO2 at 37uC, at a concentration of 26105 to 56105/mL in RPMI (Mediatech, Inc., Herndon, VA) with 10% fetal bovine serum and supplemented with 2% L-glutamine and 1% penicillin/streptomycin. AML Patient Cells Mononuclear cells were isolated from AML patients (Table S1). Mononuclear cells were isolated by density gradient centrifugation through Ficoll-Plaque Plus (Amersham Pharmacia Biotech AB, Uppsala, Sweden) at 2000 rpm for 30 minutes, followed by two washes in 1X PBS. Freeze-thawed cells were then cultured in liquid culture (DMEM, supplemented with 20% FBS) and then tested in the presence of 50% SCM with different concentrations of single and combined agents. All blood and bone marrow samples from AML patients were obtained through written consent under approval of the Dana Farber Cancer Institute Institutional Review Board. The ethics committees approved the consent procedure. Chemical Compounds and Biologic Reagents PKC412 was synthesized by Novartis Pharma AG, Basel, Switzerland, and was dissolved in DMSO to obtain a 10 mM stock 2 February 2013 | Volume 8 | Issue 2 | e56473 Akt and FLT3 Inhibition for Treatment of AML Trypan Blue exclusion assay to quantify cells for cell seeding, and Cell Titer Glo for proliferation studies. Cell viability was expressed as the function of growth affected (FA) drug-treated versus control cells; data were analyzed by Calcusyn software (Biosoft, Ferguson, MO and Cambridge, UK), using the Chou-Talalay method [18]. The combination index = [D]1 [Dx]1+ [D]2/[Dx]2, where [D]1 and [D]2 are the concentrations required by each drug in combination to achieve the same effect as concentrations [Dx]1 and [Dx]2 of each drug alone. Values less than one indicate synergy, whereas values greater than one indicate antagonism. Calcusyn combination indices can be interpreted as follows: CI ,0.1 indicate very strong synergism; values 0.1–0.3 indicate strong synergism; values 0.3–0.7 indicate synergism; values 0.7– 0.85 indicate moderate synergism; values 0.85–0.90 indicate slight synergism; values 0.9–1.1 indicate nearly additive effects; values 1.10–1.20 indicate slight antagonism; values 1.20–1.45 indicate moderate antagonism; values 1.45–3.3 indicate antagonism; values 3.3–10 indicate strong antagonism; values .10 indicate very strong antagonism. Note: For some experiments, namely those in which there was no observed single agent activity due to stromal protection, combination indices could not be reliably calculated using the Calcusyn software. solution. Serial dilutions were then made, to obtain final dilutions for cellular assays with a final concentration of DMSO not exceeding 0.1%. Dasatinib and AC220 were purchased from Haoyuan Chemexpress (Shanghai, China). KIN112 and KIN113 were developed in Dr. Gray’s lab (DFCI). KIN001 or LINCS library compounds identified in the screen as able to synergize with PKC412 in the presence of adherent HS-5 stroma were as follows: HMSL10035 (KIN001-102; Akt inhibitor); KIN001-200 (VX-702; p38 MAPK inhibitor); HMSL10168 (LG168, VX0745; p38 MAPK inhibitor). Akt and p38 MAPK inhibitors tested to assess the significance of drugs identified in the chemical screen were as follows: HMSL10167 (SB 203580; RWJ 64809, PB 203580; p38 MAPK inhibitor); HMSL10060 (TAK-715; p38a inhibitor); HMSL10154 (AT7867; Akt inhibitor); HMSL10128 (GSK 690693; Akt inhibitor); and HMSL10057 (MK2206; Akt inhibitor) (Table S2). The selective Akt inhibitors used in our study are very wellcharacterized research tools that have been widely used in different contexts (for inhibitor characteristics and background, please see Table S2). Cell Proliferation, Viability and Cell Cycle Analysis The trypan blue exclusion assay (for quantifying cells prior to seeding), Annexin-V-Fluos Staining Kit (Boehringer Mannheim, Indianapolis, IN) (for apoptosis), and cell cycle analysis were carried out as previously described [5]. Due to technical convenience, SCM was used instead of adherent stroma for the apoptosis and cell cycle assays. The Cell Titer Glo assay (Promega, Madison, WI) (for proliferation) was used for proliferation studies, and carried out according to manufacturer instructions. Human Adherent Stroma Validation Experiments HS-5 human stromal cells (10,000/well) were determined in a pilot study to be sufficient for maximum cytoprotection of PKC412-treated MOLM14-luc+ cells (Figure S2). Stromal cells were seeded 24 hours in advance of seeding MOLM14-luc+ cells (4000/well), followed by drug treatments. The Bright Glo assay (Promega, Madison, WI) was performed for co-culture assays to selectively measure leukemia cell viability and was carried out according to manufacturer’s instructions. Antibodies and Immunoblotting For analysis of phospho- and total Akt and phospho- and total GSK3b, MOLM14-luc+ cells were treated with drugs for two hours before they were collected at 1100 rpm for 8 minutes. Cells were lysed using M-PER lysis buffer (Pierce) supplemented with phosphatase inhibitors and protease inhibitors (Roche) according to the manufacturers’ instructions. Equivalent amounts of proteins were loaded. For analysis of phospho- and total STAT5, phosphoand total S6K, and phospho- and total MAPK, immunoblotting was carried out as previously described [5]. The following primary antibodies were purchased from Cell Signaling Technology (Danvers, MA): Anti-phospho-Akt (T308), anti-phospho-Akt (S473), and anti-Akt. The following primary antibodies were purchased from Sigma-Aldrich (St Louis, MO): Anti-tubulin, anti-GSK3b, and anti-phospho-GSK3b. All antibodies were used at 1:1000 dilution, except for anti-tubulin, which was used at 1:5000. The following primary antibodies (for data shown in supporting data section) were purchased from Cell Signaling Technology (Danvers, MA): Phospho-STAT5 (Tyr 694) (C11C5) (rabbit, #9359), used at 1:500, total STAT5 (rabbit, #9363), used at 1:1000, phospho-p70 S6K (Thr389) (1A5) (mouse, #9206), used at 1:300, total p70 S6K (49D7) (rabbit, #2708), used at 1:1000, phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (rabbit, #9101), used at 1:1000, total p44/42 MAPK (Erk1/2) (3A7) (mouse, #9107), used at 1:1000. HRP conjugated secondary antibodies were purchased from Promega and were used at a dilution of 1:5000. Results Chemical Screen Identification of Inhibitors able to Potentiate Effects of PKC412 against Mutant FLT3expressing Cells co-cultured with Adherent Human Stromal Cells In the present study, which is a direct and intentional extension of our previous work [7], we set out to compare the use of SCM and adherent stroma as the basis for a chemical screen geared toward identification of drugs capable of overriding drug resistance due to stromal influences. Specifically, we conducted an unbiased combinatorial screen of 188 compounds comprising the KIN001 chemical library in an attempt to identify kinase inhibitors able to synergize with PKC412 against mutant FLT3positive cells co-cultured with adherent stroma. Similar to previous findings using HS-5 SCM [7], three dual Src/Abl inhibitorsdasatinib, KIN112, and KIN113- were identified as being able to positively combine with PKC412 against MOLM14-luc+ cocultured with adherent HS-5 stroma cells as a replacement for SCM (Figure S3). In addition to confirming previously published findings, these results also validate the use of either SCM or adherent stroma as part of a chemical screen approach to identify agents able to override drug resistance due to a cytoprotective microenvironment. We also identified library-derived inhibitors of major signaling pathways, including the allosteric Akt inhibitor, KIN001-102, as able to positively combine with PKC412 against adherent stromaprotected mutant FLT3-expressing cells (Figure 1A). In order to validate whether or not Akt as a therapeutic target was important for the observed higher percentage of killing of stromal-protected cells when used in combination with PKC412, we tested a panel of Drug Combination Studies For drug combination studies, single agents were added simultaneously at fixed ratios to mutant FLT3-expressing cells cultured in the presence of adherent HS-5 stroma, 50–80% SCM, or RPMI+10% FBS. Cell viability was determined using the PLOS ONE | www.plosone.org 3 February 2013 | Volume 8 | Issue 2 | e56473 Akt and FLT3 Inhibition for Treatment of AML selective Akt inhibitor analogs against MOLM14-luc+ cells under the same co-culture conditions. Similar to KIN001-102, the selective Akt inhibitors, AT7867, GSK690693, and MK2206 positively combined with PKC412 against MOLM14-luc+ cells cultured in either the presence of adherent HS-5 stroma (Figure 1B, C) or HS-5 SCM (Figure 2), with combination indices at ED75-ED90 suggestive of synergy (Figures 1 and 2). To further validate the co-culture model for the combination drug screen, we investigated the effects of single agents and combination treatments on adherent stromal cells. This would establish whether or not stromal cell killing (and hence removal of the source of protective secreted cytokines) played a role in the observed synergy between PKC412 and Akt inhibitors. To address this, selective Akt inhibitors were tested against adherent HS-5 stroma directly. Compared to inhibitor effects against MOLM14luc+ cells, inhibitor activity against adherent stroma was considerably weaker (Figure 1B, D). In addition, whereas PKC412 (40 nM) and selective Akt inhibitors (660 nM) were highly effective alone and combined against Ba/F3 cells expressing mutant FLT3, the same drugs at the same concentrations displayed little-to-no appreciable effects against parental Ba/F3 cells and displayed little activity in the presence of 15% WEHI as a source of IL-3 (Figure S4). These data, taken together, suggest that drug activity observed against mutant FLT3-expressing cells is due to on-target effects. In addition to Akt inhibitors, positive hits from the chemical library screens also included inhibitors of p38 MAPK inhibitors, which positively combined with PKC412 against MOLM14-luc+ cells cultured in the presence of adherent HS-5 stroma (Figure S5). However, the ability of p38 MAPK inhibitors to positively combine with PKC412 was substantially diminished when mutant FLT3-expressing cells were cultured in the presence of HS-5 SCM as opposed to adherent stroma (Figure S5). There exists the possibility that high levels of stromal-secreted cytokines may negatively influence the synergizing potential of p38 MAPK inhibitors with FLT3 inhibitors. Hence, Akt inhibitors may be superior in terms of their overall combination potential and general ability to override stromal-mediated drug resistance and were therefore our main focus in this study. Figure 1. Selective inhibitors of AKT positively combine with PKC412 in the presence of adherent HS-5 stroma against MOLM14luc+ cells. (A) Approximately two-day proliferation study performed with MOLM14-luc+ cells cultured in the presence of adherent HS-5 stroma testing the combination of PKC412 and KIN001-102 versus each agent alone. (B) MOLM14-luc+ cells cultured in the presence of adherent HS-5 stroma for approximately two days: PKC412 (40 nM)+/2 Akt inhibitors (660 nM). (C) Approximately two-day PKC412 treatment of MOLM14-luc+ cells cultured in the absence and presence of human stroma. (D) Approximately two-day treatment of adherent HS-5 stroma: PKC412 (40 nM) +/2 Akt inhibitors (660 nM). (E) Calcusyn combination indices derived from 4-point concentration proliferation experiments. The cut-off for nearly additive effects (C.I.: 1.1) is marked by a dashed line. doi:10.1371/journal.pone.0056473.g001 PLOS ONE | www.plosone.org 4 February 2013 | Volume 8 | Issue 2 | e56473 Akt and FLT3 Inhibition for Treatment of AML Figure 2. Selective inhibitors of AKT positively combine with PKC412 in the presence of SCM against MOLM14-luc+ cells. (A–D) Approximately two-day proliferation studies performed with selective AKT inhibitors in combination with PKC412 in the presence of HS-5 SCM. (E) Approximately two-day PKC412 treatment of MOLM14-luc+ cells cultured in the absence or presence of HS-5 SCM (n = 2). (F) Calcusyn combination indices derived from the 4-point concentration proliferation experiments shown in A-D. The cut-off for nearly additive effects (C.I.: 1.1) is marked by a dashed line. doi:10.1371/journal.pone.0056473.g002 cells (Table 1 and Figures S6 Part I and II). These results suggest that induction of apoptosis, more than cell cycle arrest, contributes to the observed synergy between PKC412 and KIN001-102 against mutant FLT3-expressing cells cultured in a cytoprotective stromal environment. Synergy was observed between PKC412 and selective Akt inhibitors against MOLM14-luc+ cells cultured in the presence of RPMI+10% FBS (Figure 3). Synergy was also observed between selective Akt inhibitors and the highly potent and selective FLT3 inhibitor, AC220, against mutant FLT3-positive leukemia cells cultured in RPMI+10% FBS (Figure 4). The ability of selective Akt inhibitors to positively combine with FLT3 inhibitors against mutant FLT3-positive AML cells in the presence of RPMI+10% FBS correlated well with induction of apoptosis, as the combination of PKC412 and KIN001-102 showed the highest percentages of cell killing as compared to single agent effects (Table 2 and Figure S7 Part I and II). After 48 hrs in RPMI+10% FBS, however, the combination of PKC412 and KIN001-102 did not lead to greater G1 arrest than PKC412 alone (40 nM) for MOLM14-luc+ cells (Table 2). Synergy between the selective Akt inhibitors and PKC412 was additionally observed in Ba/F3-FLT3-ITD cells and the two FLT3 Inhibitor and Akt Inhibitor Combination Effects on Cell Cycle Progression and Apoptosis of Stromaprotected AML Cells Synergy observed between PKC412 and KIN001-102 against MOLM14-luc+ cells cultured in the presence of 50% SCM correlated with induction of apoptosis, as drug combinationtreated cells showed the highest percentages of apoptotic cells (Table 1). An increase in the G1 population was observed for MOLM14-luc+ cells cultured for 24 hr in the presence of 50% SCM and treated with PKC412 alone (approximately 86% of cells were in G1/G0). Combination treatments led to approximately 89% of cells in G1/G0 (Table 1), which is a comparatively small increase in percentage. In contrast, compared to PKC412 alone, combination treatment of MOLM14-luc+ cells for 48 hr resulted in substantially increased apoptosis (PKC412 alone: 27.1% apoptosis, versus combination treatments: 41.3%-48.9% apoptosis) (Table 1 and Figure S6 Part I and II). Stromal protection was evidenced by the fact that PKC412 treatment of MOLM14-luc+ cells in RPMI+10% FBS led to 47% viable cells (Table 2 and Figure S7 Part I and II), whereas PKC412 treatment of MOLM14-luc+ cells in the presence of SCM led to 71% viable PLOS ONE | www.plosone.org 5 February 2013 | Volume 8 | Issue 2 | e56473 Akt and FLT3 Inhibition for Treatment of AML Table 1. Effects of PKC412 and KIN001-102, alone and combined, on MOLM14-luc+ cell cycle progression (following 24 hours of treatment) and apoptosis (following 48 hours of treatment) when cells are cultured in the presence of 50% HS-5 SCM. Cell Cycle Progression Cell Viability MOLM14-luc+Treatment % G1/G0 % G2M %S % Viable % Apoptotic % Necrotic DMSO Control 67.95 6.270 25.78 84.70 14.00 1.400 PKC412 (40 nM) 86.24 3.070 10.68 71.00 27.10 2.000 KIN001-102 (165 nM) 71.27 5.090 23.64 81.50 17.30 1.200 KIN001-102 (330 nM) 73.81 4.750 21.44 80.90 17.70 1.400 KIN001-102 (660 nM) 77.79 4.910 17.30 77.80 20.30 2.000 PKC412 (40 nM)+KIN001-102 (165 nM) 88.87 2.980 8.150 57.30 41.30 1.400 PKC412 (40 nM)+ KIN001-102 (330 nM) 89.47 2.310 8.220 52.10 45.70 2.200 PKC412 (40 nM)+ KIN001-102 (660 NM) 89.11 3.880 7.010 47.30 48.90 3.900 Details of the assays used for these studies are provided in the Materials and Methods section. doi:10.1371/journal.pone.0056473.t001 mutant FLT3-expressing human cell lines, MOLM13-luc+ and MV4,11, cultured in the presence of RPMI+10% FBS (Figures 5 and 6, Table 3, Figure S8). Partial protection of PKC412-treated MOLM13-luc+ cells was observed when cells were cultured in the presence of HS-5 SCM (Figure 5), and combinations of selective Akt inhibitors and PKC412 were synergistic against MOLM13luc+ cells cultured in the presence of HS-5 SCM (Figure 5, Table 3). Figure 3. Selective inhibitors of AKT positively combine with PKC412 in RPMI+10% FBS against MOLM14-luc+ cells. (A–D) Approximately two-day proliferation studies performed with selective AKT inhibitors in combination with PKC412 in RPMI+10% FBS. (E) Calcusyn combination indices. The cut-off for nearly additive effects (C.I.: 1.1) is marked by a dashed line. doi:10.1371/journal.pone.0056473.g003 PLOS ONE | www.plosone.org 6 February 2013 | Volume 8 | Issue 2 | e56473 Akt and FLT3 Inhibition for Treatment of AML Figure 4. Selective inhibitors of AKT positively combine with AC220 in RPMI+10% FBS against MOLM14-luc+ cells. (A–D) Approximately two-day proliferation studies performed with selective AKT inhibitors in combination with AC220 in RPMI+10% FBS. (E) Calcusyn combination indices. The cut-off for nearly additive effects (C.I.: 1.1) is marked by a dashed line. doi:10.1371/journal.pone.0056473.g004 Table 2. Effects of PKC412 and KIN001-102, alone and combined, on MOLM14-luc+ cell cycle progression (following 24 hours of treatment) and apoptosis (following 48 hours of treatment) when cells are cultured in the presence of RPMI+10% FBS. Cell Cycle Progression MOLM14-luc+ Treatment % G1/G0 % G2M Cell Viability %S % Viable % Apoptotic % Necrotic DMSO Control 56.10 9.320 34.58 92.50 6.900 0.600 PKC412 (40 nM) 86.94 1.50 11.56 46.90 52.70 0.400 KIN001-102 (165 nM) 59.60 7.360 33.04 92.00 7.800 0.300 KIN001-102 (330 nM) 61.03 9.010 29.96 91.80 8.000 0.200 KIN001-102 (660 nM) 61.93 7.030 31.05 90.90 8.800 0.300 PKC412 (40 nM)+KIN001-102 (165 nM) 85.22 3.430 11.35 31.50 68.30 0.200 PKC412 (40 nM)+ KIN001-102 (330 nM) 86.92 2.760 10.32 18.10 81.70 0.100 PKC412 (40 nM)+ KIN001-102 (660 NM) 81.11 3.160 15.73 16.90 82.80 0.300 Details of the assays used for these studies are provided in the Materials and Methods section. doi:10.1371/journal.pone.0056473.t002 PLOS ONE | www.plosone.org 7 February 2013 | Volume 8 | Issue 2 | e56473 Akt and FLT3 Inhibition for Treatment of AML Figure 5. Selective inhibitors of AKT positively combine with PKC412 in the absence and presence of HS-5 SCM against MOLM13luc+ cells. (A–B) Approximately two-day proliferation studies performed with selective AKT inhibitors in combination with PKC412 in the presence of RPMI+10% FBS. (C–D) Approximately two-day proliferation studies performed with selective AKT inhibitors in combination with PKC412 in 80% HS5 SCM. (E) Treatment of MOLM13-luc+ cells with PKC412 in either RPMI+10% FBS or 80% HS-5 SCM. doi:10.1371/journal.pone.0056473.g005 2MK2206 (165 nM) (Figure S9). Each agent was tested at respective concentrations that led to a substantial combination effect in proliferation studies (Figure 3). PKC412 alone and PKC412 combined with MK2206 decreased phosphorylation of STAT5 to similar extents in these cells, and no significant changes were observed in expression of phosphorylated MAPK between single agent-treated and drug combination-treated cells (Figure S9). Phospho-Akt Mediates Synergy Observed between Allosteric Akt Inhibitor, KIN001-102, and PKC412 In order to verify observed combination effects in terms of signaling, we examined the phosphorylation status of Akt following either single agent treatment or combined drug treatment. Immunoblots demonstrated that pAKT levels were inhibited to a greater extent in MOLM14-luc+ cells cultured in the presence of 50% HS-5 SCM upon treatment with a combination of KIN001102 and PKC412, as compared to either drug alone (Figure 7). The expression of GSK3b was additionally investigated as GSK3b is a direct substrate of protein kinase Akt. These results suggest that Akt activity is critical for maintaining stromal cytoprotection under these conditions. In contrast to the robust drug combination effect observed against phospho-Akt (S473) at lower concentrations of KIN102, there was no apparent combination effect observed between PKC412 (40 nM) and KIN102 (165 nM) against phospho-S6K for MOLM14-luc+ cells cultured in the presence of SCM (data not shown). Similarly, no significant changes were observed in expression of phosphorylated S6K in MOLM14-luc+ cells cultured in RPMI+10% FBS and treated for 1 hr with PKC412 (5 nM)+/ PLOS ONE | www.plosone.org Ability of Akt Inhibitors to Potentiate the Activity of PKC412 or AC220 against Primary AML Patient Cells Cultured in the Presence of Cytoprotective SCM In order to validate the stromal cell co-culture screening model, we tested the lead drugs from the screening on primary AML patient cells cultured in the presence of cytoprotective HS-5derived SCM. Several of these samples were confirmed to express FLT3-ITD. Combination studies between PKC412 and KIN001102, AT7867, MK2206, and GSK690693, respectively, showed the highest degree of cell killing in combination-treated, SCMprotected primary AML cells as compared to any single agent (a representative dose-response experiment for a highly drug-re8 February 2013 | Volume 8 | Issue 2 | e56473 Akt and FLT3 Inhibition for Treatment of AML Figure 6. Selective inhibitors of AKT positively combine with PKC412 in RPMI+10% FBS against MV4,11 and Ba/F3-FLT3-ITD cells. (A–C) Approximately two-day proliferation studies performed with selective AKT inhibitors in combination with PKC412 in RPMI+10% FBS against MV4,11 cells. (D–F) Approximately two-day proliferation studies performed with selective AKT inhibitors in combination with PKC412 in RPMI+10% FBS against Ba/F3-FLT3-ITD cells. doi:10.1371/journal.pone.0056473.g006 performed in the presence of SCM, which provides cytoprotective cytokines and dampens inhibitor efficacy. sistant AML patient sample (#2) is shown in Figure 8A). Analysis of the combinatorial effect with Calcusyn revealed synergy (ED75:0.39202, ED90:0.55992) for PKC412+AT7867 against mutant FLT3-positive AML#2. Given the multiple targets of PKC412, we investigated the ability of AC220, which has a very high selectivity index, to combine with Akt inhibitors. A positive combination effect was observed between AC220 and selective inhibitors of Akt against MOLM14-luc+ cells cultured in the presence of HS-5-derived SCM (Figure 8B). AC220 similarly combined positively with Akt inhibitors against MOLM14-luc+ cells cultured in RPMI+10% FBS with combination indices at ED75 and ED90 suggestive of synergy (Figure 8C). Selective Akt inhibitors were thus also tested for their ability to potentiate the effects of AC220, as compared to PKC412, against primary AML patient cells cultured in the presence of HS-5derived SCM. As shown in Figures 8D–F, PKC412 and AC220, which were tested in parallel, resulted in the highest degree of patient cell killing when either drug was used in combination with Akt inhibitors as compared to any single agent against a panel of primary AML samples. Of note, all drug treatments were PLOS ONE | www.plosone.org Discussion Previous studies of ours suggest that TKI-dependent combination therapy likely represents a potentially useful approach to counteracting both intrinsic and stroma-associated drug resistance in leukemia patients [7,19,20,21]. With the recent discovery of numerous FLT3 inhibitor-responsive serine/threonine and tyrosine phosphorylation sites uncovered in primary AML patient bone marrow samples [22], identification of protein kinase inhibitors that are able to enhance the potency of FLT3 inhibitors makes intuitive sense. Here, selective inhibitors targeting kinases involved in PI3K/ Akt and Ras/MEK/MAPK signaling were identified in a chemical screen as synergizing with PKC412 against mutant FLT3expressing cells in the presence of adherent stroma. Akt inhibitors synergized with FLT3 inhibitors in the presence of either SCM or adherent stroma, as compared to p38 MAPK inhibitors, which synergized with FLT3 inhibitors only in the presence of adherent stroma. One possibility for this may be traced to the nature of 9 February 2013 | Volume 8 | Issue 2 | e56473 Akt and FLT3 Inhibition for Treatment of AML Table 3. Calcusyn software-derived combination indices. Drug Combination Cell Line ED25 ED50 ED75 ED90 PKC412+MK2206 Ba/F3-FLT3- 0.73063 ITD 0.71787 0.70932 0.70100 PKC412+AT7867 Ba/F3-FLT3- 0.62418 ITD 0.65624 0.68996 0.72540 PKC412+GSK690693 Ba/F3-FLT3- 0.64287 ITD 0.72479 0.81965 0.92849 PKC412+MK2206 (2-day) MOLM13 0.54469 0.52206 0.55200 0.71660 PKC412+MK2206 (3-day) MOLM13 0.15758 0.22911 0.34332 0.55817 PKC412+AT7867 (2-day) MOLM13 0.95116 1.02639 1.11056 1.20483 PKC412+AT7867 (3-day) MOLM13 0.76889 0.26391 0.40983 0.64279 PKC412+GSK690693 MOLM13 (2-day) 0.97412 0.29241 0.33779 0.56141 PKC412+GSK690693 MOLM13 (3-day) 1.11399 0.79787 0.62777 0.57590 PKC412+MK2206 (SCM) MOLM13 0.75681 0.23956 0.26180 0.29207 PKC412+GSK690693 MOLM13 (SCM) 0.91433 0.39599 0.51463 0.72551 PKC412+MK2206 MV4,11 0.84669 0.39562 0.43775 0.76686 PKC412+AT7867 MV4,11 0.45640 0.67869 1.08529 1.84914 Figure 7. Phospho-Akt mediates synergy observed between allosteric Akt inhibitor, KIN001-102, and PKC412. Immunoblots of protein lysates prepared from MOLM14-luc+ cells treated for 2 hours with PKC412 (40 nM), KIN001-102 (165, 330, 660 nM), or a combination of the two agents in the presence of 50% SCM. Data shown are representative of two independent experiments in which similar results were achieved. doi:10.1371/journal.pone.0056473.g007 Data shown here correspond to dose-response curves shown in Figures 5 and 6 and Figure S8. Interpretation of combination indices is provided in the Materials and Methods section. doi:10.1371/journal.pone.0056473.t003 nib plus the farnesyltransferase inhibitor lonafarnib [28]. Inhibitors of Akt and Erk combined respectively with nilotinib diminished resistance. In contrast to our findings, however, inhibition of p38 MAPK in this study increased TKI (nilotinib) resistance [29]. Importantly, we observed synergy between selective Akt inhibitors and FLT3 inhibitors in the absence of stroma as well as its presence, suggesting that this synergy is not specific to leukemia cells growing in a cytoprotective microenvironment. Of significance, there are reports that have been and that are continuing to be published that support the potential clinical importance of inhibiting components of major signaling pathways in combination with TKIs as a way to treat AML. The identification of Akt and p38 MAPK inhibitors as able to potentiate the effects of FLT3 inhibitors is at least in part attributable to the use of the LINCS library to identify comparatively ‘‘clean’’ kinase inhibitors, in contrast to the chemical library screened previously [7], which included a number of multi-kinase inhibitors such as dasatinib. A chemical library composed of relatively selective inhibitors offers a significant technical advantage in that it translates into easier elucidation of mechanism of inhibition by a single agent and synergy between agents as the drug targets are more well-defined and easier to validate. Our in vitro findings with cell lines and primary patient samples, which closely reflect the genetic heterogeneity amongst AML patients, warrant further testing and validation in preclinical models of progressive leukemia and minimal residual disease. In vivo models that reflect stromal cell interactions, however, are fairly complex and are beyond the scope of this study. We are planning to address these questions in future studies. stromal protection by SCM, characterized by highly concentrated levels of stromal-derived cytokines. Of relevance, studies have implicated Akt- and MAPKmediated signaling in stromal enhancement of leukemia cell viability. For instance, co-culture of leukemia cells and bone marrow-derived stroma has been shown to lead to activation of the MAPK/ERK pathway and integrin-linked kinase (ILK), which phosphorylates Akt [22]. ILK/Akt is likely critical for leukemia cell survival in bone marrow, and thus inhibitors of ILK have been proposed as an approach to simultaneously target both leukemia cells and leukemia-activated stromal cells [23]. Additionally, p38 MAPK activation has been found to play a role in stromadependent survival of B-CLL cells [24] and ALL cells [25]. In addition, continuous FLT3 inhibitor treatment leads to the development of drug-resistant cells characterized by constitutive activation of parallel downstream PI3K/Akt and/or Ras/MEK/ MAPK signaling pathways, which is believed to compensate for the loss of FLT3 activity in terms of survival and growth [26]. In support of this, constitutive activation of ERK/Akt/STAT pathways has been observed in AML despite small molecule inhibition of FLT3-ITD activity, suggesting that optimal treatment of AML may require FLT3 inhibition combined with inhibition of additional signaling pathways [27]. Dual inhibition of FLT3 and Akt-mediated signaling, such as that conferred by the multiple kinase inhibitor, KP372-1, has indeed been found to inhibit primary AML cell growth with minimal effect on normal progenitor cells [28]. Consistent with our results is the finding that Akt, p38MAPK, and Erk activation correlates with development of resistance of BCR-ABL-positive acute lymphoblastic leukemia (ALL) to nilotiPLOS ONE | www.plosone.org 10 February 2013 | Volume 8 | Issue 2 | e56473 Akt and FLT3 Inhibition for Treatment of AML Figure 8. Ability of Akt inhibitors to positively combine with PKC412 or AC220 against AML patient samples in the presence of cytoprotective SCM. (A) Approximately two-day proliferation study performed with a selective Akt inhibitor in combination with PKC412 in the presence of HS-5 SCM against mutant FLT3-positive AML#2. (B) Approximately two-day combination studies: AC220 (0.4 nM) +/2 selective AKT inhibitors (660 nM) against MOLM14-luc+ cells in the presence of 50% HS-5 SCM. (C) Approximately two-day combination studies: AC220 (0.4 nM) +/ 2 selective AKT inhibitors (660 nM) against MOLM14-luc+ cells in the presence of RPMI+10% FBS. (D) Approximately two-day combination studies: PKC412 (40 nM)+/2 selective AKT inhibitors (660 nM) against primary AML patient cells in the presence of 50% HS-5 SCM. (E) Approximately two-day combination studies: AC220 (0.4 nM) +/2 selective AKT inhibitors (660 nM) against primary AML patient cells in the presence of 50% SCM. (F) Ability of Akt inhibitors to positively combine with PKC412 or AC220 against primary AML cells in the presence of cytoprotective SCM. Patient information is provided in Table S1. doi:10.1371/journal.pone.0056473.g008 Figure S2 Co-culture pilot study. Approximately 1500 MOLM14-luc+ cells were tested in a two-day assay in the presence and absence of HS-5 stroma seeded at 10,000 cells/well, 20,000 cells/well, and 40,000 cells/well. (TIF) In conclusion, selective inhibition of kinases such as Akt in combination with FLT3 inhibitors in mutant FLT3-positive AML patients may represent a novel approach to improving treatment effects and patient survival. Findings presented here may provide novel options for adjunctive therapy. Figure S3 Coculture chemical screen identification of KIN001 library compound, dasatinib, and dasatinib-like compounds, KIN112 and KIN113, as able to synergize with PKC412 in the presence of adherent HS-5 stroma against MOLM14-luc+ cells. (A–C) Approximately two-day assays, validating the combination potential of the KIN001 coculture chemical screen identified agents (dasatinib, KIN112, KIN113) to synergize with PKC412 against MOLM14-luc+ cells in the presence of adherent HS-5 stroma. Approximately 5000 MOLM14-luc+ cells were seeded/well; approximately 10,000 HS-5 stromal cells were seeded/well. (D) PKC412 Supporting Information Figure S1 Schematic of kinase inhibitor-focused chemical screen approaches. Stromal-conditioned media (SCM)- or adherent stroma-based chemical libraries are used to identify agents that are able to potentiate the effects of FLT3 inhibitors against mutant FLT3-expressing cells cultured in a cytoprotective microenvironment. (TIF) PLOS ONE | www.plosone.org 11 February 2013 | Volume 8 | Issue 2 | e56473 Akt and FLT3 Inhibition for Treatment of AML Figure S8 Selective inhibitors of AKT positively combine with PKC412 in RPMI+10% FBS against MOLM13luc+ cells. (A–C) Approximately three-day proliferation studies performed with selective AKT inhibitors in combination with PKC412 in RPMI+10% FBS against MOLM13-luc+ cells. (TIF) treatment of MOLM14-luc+ cells cultured in the absence or presence of adherent HS-5 stroma (n = 2). (E) Calcusyn combination indices. The cut-off for nearly additive effects (C.I.: 1.1) is marked by a dashed line. (TIF) Figure S4 Treatment of parental Ba/F3 cells and Ba/ F3-FLT3-ITD cells with PKC412, alone and in combination with selective inhibitors of Akt. (A) Approximately three-day drug treatment of parental Ba/F3 cells cultured in the presence of IL-3 and Ba/F3-FLT3-ITD cells cultured in the absence of IL-3. (B) Approximately three-day drug treatment of Ba/F3-FLT3-ITD cells cultured in the presence of IL-3. PKC412 was used at 40 nM and selective AKT inhibitors were each used at 660 nM. (TIF) Figure S9 Investigation of phosphorylation of signaling molecules downstream of FLT3. Immunoblots of protein lysates prepared from MOLM14-luc+ cells treated for 1 hour with PKC412 (5 nM), MK2206 (165 nM), or a combination of the two agents in RPMI+10% FBS. (TIF) Table S1 Patient sample information. Patients shown here were cultured in the presence of 50% HS-5 SCM, and treated with different combinations of kinase inhibitors. *Patient information for AML patients 2 and 7 has been previously published (Weisberg et al, 2012a, Leukemia). (DOC) Figure S5 Selective inhibitors of p38 MAPK positively combine with PKC412 against MOLM14-luc+ cells cultured in the presence of adherent HS-5 stroma, however not HS-5 SCM. Calcusyn combination indices. The cut-off for nearly additive effects (C.I.: 1.1) is marked by a dashed line. (TIF) Table S2 Selective AKT and p38 MAPK inhibitors. *Hirai H, Soontome H, Nakatsuru Y, Miyama K, Taguchi S, Tsujioka K et al. MK-2206, an allosteric Akt inhibitor, enhances antitumor efficacy by standard chemotherapeutic agents or molecular targeted drugs in vitro and in vivo. Mol Cancer Ther 2010;9:1956-67. **Levy DS, Kahana JA, Kumar R. AKT inhibitor, GSK690693, induces growth inhibition and apoptosis in acute lymphoblastic leukemia cell lines. Blood 2009;113:1723-9. ***Grimshaw KM, Hunter LJ, Yap TA, Heaton SP, Walton MI, Woodhead SJ, et al. AT7867 is a potent and oral inhibitor of AKT and p70 S6 kinase that induces pharmacodynamic changes and inhibits human tumor xenograft growth. Mol Cancer Ther 2010;9:1100-10. (DOC) Figure S6 Part 1. Annexin/pi staining corresponding to data shown in Table 1: Effects of PKC412 (40 nM) and KIN001-102 (165, 330, 660 nM), alone and combined, on MOLM14-luc+ cell apoptosis (following 48 hours of treatment) when cells are cultured in the presence of 50% HS-5 SCM. Cells labeled ‘‘dying’’ are in early apoptotic phase, and cells labeled ‘‘apoptotic’’ are in late apoptotic phase. Part 2. Quantitative values corresponding to data shown in Figure S6 (part 1): Effects of PKC412 (40 nM) and KIN001-102 (165, 330, 660 nM), alone and combined, on MOLM14-luc+ cell apoptosis (following 48 hours of treatment) when cells are cultured in the presence of 50% HS-5 SCM. Cells labeled ‘‘dying’’ are in early apoptotic phase, and cells labeled ‘‘apoptotic’’ are in late apoptotic phase. (DOC) Author Contributions Responsible for generation of research findings reported in paper (design/ performance of experiments), integrity and analysis of the data, writing of the manuscript: EW. Responsible for generation of research findings reported in paper (design/performance of experiments), integrity and analysis of the data, editing of the manuscript: QL. Performed Akt, GSKbeta, tubulin immunoblotting experiments: XZ. Assisted with proliferation studies: EN. Offered valuable scientific feedback and helped with conception of research reported in paper: MS. Assisted with the chemical screening: FL MN JZ AN. Offered valuable scientific feedback and helped with conception of research reported in paper: CM. Carried out flow cytometry analysis and helped with cell cycle and apoptosis studies: RWS. Provided AML patient samples used in this study as well as patient information: RS IG. Responsible for conception of research reported in paper, integrity and analysis of the data: JDG NG. Conceived and designed the experiments: EW QL MS CM AN JDG NG. Performed the experiments: EW XZ EN FL MN. Analyzed the data: EW QL RWS. Contributed reagents/materials/analysis tools: JZ RS IG. Wrote the paper: EW QL MS. Figure S7 Part 1. Annexin/pi staining corresponding to data shown in Table 2: Effects of PKC412 (40 nM) and KIN001-102 (165, 330, 660 nM), alone and combined, on MOLM14-luc+ cell apoptosis (following 48 hours of treatment) when cells are cultured in the presence of RPMI+10% FBS. Cells labeled ‘‘dying’’ are in early apoptotic phase, and cells labeled ‘‘apoptotic’’ are in late apoptotic phase. Part 2. Quantitative values corresponding to data shown in Figure S7 (part 1): Effects of PKC412 (40 nM) and KIN001-102 (165, 330, 660 nM), alone and combined, on MOLM14-luc+ cell apoptosis (following 48 hours of treatment) when cells are cultured in the presence of RPMI+10% FBS. Cells labeled ‘‘dying’’ are in early apoptotic phase, and cells labeled ‘‘apoptotic’’ are in late apoptotic phase. (DOC) References 5. Weisberg E, Boulton C, Kelly LM, Manley P, Fabbro D, et al. (2002) Inhibition of mutant FLT3 receptors in leukemia cells by the small molecule tyrosine kinase inhibitors PKC412. Cancer Cell 1: 433–443. 6. 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Analytical Technology in Nutrition Analysis Edited by Jose M. Miranda Printed Edition of the Special Issue Published in Molecules www.mdpi.com/journal/molecules Analytical Technology in Nutrition Analysis Analytical Technology in Nutrition Analysis Special Issue Editor Jose M. Miranda MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin Special Issue Editor Jose M. Miranda Universidade de Santiago de Compostela Spain Editorial Office MDPI St. Alban-Anlage 66 4052 Basel, Switzerland This is a reprint of articles from the Special Issue published online in the open access journal Molecules (ISSN 1420-3049) (available at: https://www.mdpi.com/si/molecules/Nutrition analysis). For citation purposes, cite each article independently as indicated on the article page online and as indicated below: LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year, Article Number, Page Range. ISBN 978-3-03928-764-2 (Hbk) ISBN 978-3-03928-765-9 (PDF) c 2020 by the authors. Articles in this book are Open Access and distributed under the Creative  Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND. Contents About the Special Issue Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Jose M. Miranda Analytical Technology in Nutrition Analysis Reprinted from: Molecules 2020, 25, 1362, doi:10.3390/molecules25061362 . . . . . . . . . . . . . 1 Larissa Gabrielly Barbosa Lima, Julia Montenegro, Joel Pimentel de Abreu, Millena Cristina Barros Santos, Talita Pimenta do Nascimento, Maiara da Silva Santos, Antônio Gilberto Ferreira, Luiz Claudio Cameron, Mariana Simões Larraz Ferreira and Anderson Junger Teodoro Metabolite Profiling by UPLC-MSE, NMR, and Antioxidant Properties of Amazonian Fruits: Mamey Apple (Mammea Americana), Camapu (Physalis Angulata), and Uxi (Endopleura Uchi) Reprinted from: Molecules 2020, 25, 342, doi:10.3390/molecules25020342 . . . . . . . . . . . . . . 5 Ahmidin Wali, Atikan Wubulikasimu, Sharafitdin Mirzaakhmedov, Yanhua Gao, Adil Omar, Amina Arken, Abulimiti Yili and Haji Akber Aisa Optimization of Scorpion Protein Extraction and Characterization of the Proteins’ Functional Properties Reprinted from: Molecules 2019, 24, 4103, doi:10.3390/molecules24224103 . . . . . . . . . . . . . 23 Emmanuel Martinez, Jose A. Rodriguez, Alicia C. Mondragon, Jose Manuel Lorenzo and Eva M. Santos Influence of Potato Crisps Processing Parameters on Acrylamide Formation and Bioaccesibility Reprinted from: Molecules 2019, 24, 3827, doi:10.3390/molecules24213827 . . . . . . . . . . . . . 39 Yonghang Ma, Zhengcheng Zeng, Lingchang Kong, Yuanxin Chen and Pingli He Determination of N-Carbamylglutamate in Feeds and Animal Products by High Performance Liquid Chromatography Tandem Mass Spectrometry Reprinted from: Molecules 2019, 24, 3172, doi:10.3390/molecules24173172 . . . . . . . . . . . . . 49 Alexis Ayala-Ni ˜no, Gabriela Mariana Rodr´ıguez-Serrano, Luis Guillermo Gonz´alez-Olivares, Elizabeth Contreras-López, Patricia Regal-López and Alberto Cepeda-Saez Sequence Identification of Bioactive Peptides from Amaranth Seed Proteins (Amaranthus hypochondriacus spp.) Reprinted from: Molecules 2019, 24, 3033, doi:10.3390/molecules24173033 . . . . . . . . . . . . . 63 Li Duan, Chenmeng Zhang, Chenjing Zhang, Zijing Xue, Yuguang Zheng and Long Guo Green Extraction of Phenolic Acids from Artemisia argyi Leaves by Tailor-Made Ternary Deep Eutectic Solvents Reprinted from: Molecules 2019, 24, 2842, doi:10.3390/molecules24152842 . . . . . . . . . . . . . 77 Thomas Rosemary, Abimannan Arulkumar, Sadayan Paramasivam, Alicia Mondragon-Portocarrero and Jose Manuel Miranda Biochemical, Micronutrient and Physicochemical Properties of the Dried Red Seaweeds Gracilaria edulis and Gracilaria corticata Reprinted from: Molecules 2019, 24, 2225, doi:10.3390/molecules24122225 . . . . . . . . . . . . . . 89 v Gretel Dovale-Rosabal, Alicia Rodrı́guez, Elyzabeth Contreras, Jaime Ortiz-Viedma, Marlys Muñoz, Marcos Trigo, Santiago P. Aubourg and Alejandra Espinosa Concentration of EPA and DHA from Refined Salmon Oil by Optimizing the Urea–Fatty Acid Adduction Reaction Conditions Using Response Surface Methodology Reprinted from: Molecules 2019, 24, 1642, doi:10.3390/molecules24091642 . . . . . . . . . . . . . 103 Aroa Lopez-Santamarina, Jose Manuel Miranda, Alicia del Carmen Mondragon, Alexandre Lamas, Alejandra Cardelle-Cobas, Carlos Manuel Franco and Alberto Cepeda Potential Use of Marine Seaweeds as Prebiotics: A Review Reprinted from: Molecules 2020, 25, 1004, doi:10.3390/molecules25041004 . . . . . . . . . . . . . 115 Marcos Flores, Carolina Saravia, Claudia E. Vergara, Felipe Avila, Hugo Valdés and Jaime Ortiz-Viedma Avocado Oil: Characteristics, Properties, and Applications Reprinted from: Molecules 2019, 24, 2172, doi:10.3390/molecules24112172 . . . . . . . . . . . . . 141 vi About the Special Issue Editor Jose M. Miranda (Ph.D.) Prior to his academic and scientific activities, he worked for several years as a health inspector. Currently, he is working as assistant professor in the University of Santiago de Compostela (Spain). He has published over 100 scientific articles in indexed journals. His areas of expertise are food safety, food chemical analysis, characterization of compounds with nutritional interest, and metagenomics. vii molecules Editorial Analytical Technology in Nutrition Analysis Jose M. Miranda Laboratorio de Higiene Inspección y Control de Alimentos, Departamento de Química Analítica, Nutrición y Bromatología, Universidade de Santiago de Compostela, 27002 Lugo, Spain; josemanuel.miranda@usc.es; Tel.: +34-982-252-231 (ext. 22407); Fax: +34-982-254-592 Received: 9 March 2020; Accepted: 11 March 2020; Published: 17 March 2020 The great challenge facing humanity in the coming decades is to secure food for the 9.8 billion people who are expected to inhabit the planet by around 2050 and 11.2 billion in 2100 [1]. To increase the food production by traditional methods to meet this demand for food is very difficult. Additionally, traditional methods of food production, both of plant and animal origin, present specific problems that make it difficult for them to meet such an ambitious increase target. In this sense, terrestrial agriculture presents a problem because fresh water (an essential resource) is an increasingly scarce commodity, and progressive desertification of the Earth’s surface is taking place and will probably be aggravated in the future by global warming. About 45% of the world’s land surface is currently considered drylands, while 12 million hectares of land are degraded yearly through a lack of water and related processes. According to the Food and Agriculture Organization of the United Nations [2], agricultural productivity is persistently declining at over 1% per year. With respect to food production of animal origin, this also presents specific challenges, such as the fact that intensive production methods require large amounts of land, water and feed, and some livestock (such as ruminants) produce high levels of greenhouse gas emissions. Thus, intensive methods of animal production have serious drawbacks from the point of view of environmental care. In order to properly feed such a large population, it will be necessary to increase food production while respecting ecosystems and natural resources. The current high demand for animal proteins requires that livestock is reared in large numbers over diminishing land resource which is not possible and, therefore, alternative substitutes for animal proteins needs to be embraced to overcome this problem [3]. The abovementioned fact means that demand for food produced from non-traditional sources is expected to rise in the coming decade [4]. Fortunately, nowadays increasing acceptance for novel foods is also being observed, not only in developing but also in developed countries, which is mainly influenced by consumer awareness of the nutritional benefits linked to these kinds of foods [3]. As it can be seen in Figure 1, the investigation about novel foods has experienced a dramatic increase in the last decade (about three-fold). In addition to the need for an increase in food production, nowadays, in most countries of the world, there is a growing prevalence of chronic non-communicable diseases, many of which are diet-related [5]. As a result, there is widespread consumer demand for foods with a nutritional composition more in line with current nutritional guidelines, and which include a greater proportion of the nutrients that have a potential beneficial effect on human health, or fewer of those components that have a negative effect on human health [6]. Therefore, both because of the need to ensure food safety in food, especially in those that do not have a history of safe use. In addition, there is also a need for analytical methodologies to reliably determine both the presence of specific nutrition-related components in foods, and the effects of these dietary components on human health, in areas as diverse as lipidomics, proteomics, transcriptomics, genomics, epigenomics, or metagenomics [7,8]. To meet these needs, it is essential that we in the international scientific community work intensively to ensure safe, effective and honest food production and to protect the health of consumers. Molecules 2020, 25, 1362; doi:10.3390/molecules25061362 1 www.mdpi.com/journal/molecules Molecules 2020, 25, 1362 For this reason, from Molecules it was recognized the need to propose the Special Issue “Analytical Technologies in Nutrition Analysis”. This Special Issue was aimed to offer an appropriate opportunity to all the contributors to make their results and techniques more visible, and to present the most recent findings. Figure 1. Results analysis for Scopus query “novel foods” in title, keywords or abstract section of the articles between 2009 and 2019. This Special Issue has received remarkably positive feedback, with many contributions submitted by numerous geographically diverse scientists, resulting in a collection of 10 publications, including two exhaustive review articles [9–18]. Among the contributing authors, authors can be found from Asia (China, India, and Uzbekistan), Europe (Spain), South America (Brazil and Chile) and North America (Mexico). The published articles include findings related to the comprehensive bioactive compound profile and antioxidant capacities of mamey apple (Mammea americana), camapu (Physalis angulata), and uxi (Endopleura uchi) that can contribute to their economic exploitation [9]. Another article described the multifunctional activity of amaranth (Amaranthus hypochondriacus spp.) proteins, opening the possibility that amaranth hydrolyzed with alcalase and flavourzyme to be used as a value-added ingredient with multi-functional bioactive properties [10]. Another article aimed to find an efficient extraction method and investigate some of physical and chemical parameters, like water solubility, emulsification, foaming properties, and oil-holding capacity of obtained scorpion proteins. The results obtained suggest that scorpion proteins can be considered as an important ingredient and raw material for the creation of water-soluble supramolecular complexes for drugs [11]. A fractional factorial design was used to evaluate the effects of temperature, frying time, blanching treatment and the thickness of potato slices on a very relevant potential toxic compound (acrylamide) content in crisps. The findings obtained demonstrate that acrylamide concentration remained at 70% in fried chips, and reductions took place, mainly at the intestinal phase, as a result of reaction with nucleophilic compounds [12]. N-carbamylglutamate, a synthetic analogue of N-acetylglutamate, is an activator of blood ammonia conversion and endogenous arginine synthesis. This study will provide a solid foundation for the evaluation of availability and metabolic mechanism of N-carbamylglutamate in animals [13]. The Artemisia argyi leaf has been used as a traditional medicine and food supplement in Asian countries for hundreds of years. Phytochemical studies disclosed that Artemisia argyi leaf contains various bioactive constituents, mainly phenolic acids, which have great potential as possible alternatives to those organic solvents in health-related areas such as food and pharmaceuticals [14]. Regarding the use of seaweeds as alternative dietary fibre sources to terrestrial vegetables, in this Special Issue 2 Molecules 2020, 25, 1362 an article is presented evaluating the nutritional composition and physicochemical properties of two dried commercially interesting edible red seaweeds, Gracilaria corticata and G. edulis. In view of the results, both G. corticata and G. edulis contain important nutrients for human health and are possible natural functional foods [15]. More generally, a wide review about the current knowledge surrounding the impacts of seaweeds and their derived polysaccharides on the human microbiota is also presented, in which potential benefits against chronic non-transmissible diseases were discussed [16]. Finally, two articles describing potentially beneficial food sources of fat for humans are presented. In one on them, it was concluded that refined commercial salmon oil can be transformed into a profitable source of eicosapentaenoic and docosapentaenoic acids, thus leading to a product with higher commercial value, and that this process can be optimized by using response surface methodology [17]. The last article of the Special Issue consists of a review article about avocado oil, including discussion about the extraction methods, chemical composition, and various applications of avocado oil in the food and medicine industries. Based on the available data, avocado oil has established itself as an oil that has a very good nutritional value at low and high temperatures, with multiple technological applications that can be exploited for the benefit of its producers [18]. This Special Issue is accessible thought the following link: https://www.mdpi.com/journal/ molecules/special_issues/Nutrition_analysis. As Guest Editor for this Special Issue, I would like to thank all the authors and co-authors for their contributions and all the reviewers for their effort in carefully and rapid evaluating the manuscripts. Last but not least, I would like to appreciate the hard work done by the editorial office of the Molecules journal, as well as their kind assistance in preparing this Special Issue. Funding: This research received no external funds. Conflicts of Interest: The author declares no conflict of interest. References 1. 2. 3. 4. 5. 6. 7. 8. 9. United Nations, Department of Economics and Social affairs, Population Division. World Population Prospects 2019: Highlights; United Nations: New York, NY, USA, 2019. FAO. The Future of Food and Agriculture–Trends and Challenges; Food and Agriculture Organization of the United Nations: Rome, Italy, 2017. Imathiu, S. Benefits and food safety concerns associated with consumption of edible insects. NFS J. 2020, 18, 1–11. [CrossRef] Selenius, O.; Korpela, J.; Salminen, S.; Gallego, C.G. Effect of chitin and chitooligosaccharide on in vitro growth of Lactobacillus rhamnosus GG and Escherichia coli TG. Appl. Food Biotechnol. 2018, 5, 163–172. Miranda, J.M.; Anton, X.; Redondo-Valbuena, C.; Roca-Saavedra, P.; Rodriguez, J.A.; Lamas, A.; Franco, C.M.; Cepeda, A. Egg and egg-derived foods: Effects on human health and use as functional foods. Nutrients 2015, 7, 706–709. [CrossRef] [PubMed] Roca-Saavedra, P.; Mendez-Vilabrille, V.; Miranda, J.M.; Nebot, C.; Cardelle-Cobas, A.; Franco, C.M.; Cepeda, A. Food additives, contaminants and other minor components: Effects on human gut microbiota—A review. J. Physiol. Biochem. 2018, 74, 69–83. [CrossRef] [PubMed] Lamas, A.; Regal, P.; Vázquez, B.I.; Miranda, J.M.; Franco, C.M.; Cepeda, A. Transciptomics: A powerful tool to evaluate the behavior of foodborne pathogens in the food production chain. Food Res. Int. 2019, 15, 108543. [CrossRef] [PubMed] Ramon Vidal, D. Model organisms and “OMIC” technologies: New tools for the development of healthy foods. Curr. Opin. Biotechnol. 2013, 24, S19. [CrossRef] Barbosa Lima, L.G.; Montenegro, J.; de Abreu, J.P.; Barros santos, M.C.; Pimenta do Nascimento, T.; da Silva santos, M.; Ferreira, A.G.; Cameron, L.C.; Larraz Ferreira, M.S.; Teodoro, A.J. Metabolite Profiling by UPLC-MSE, NMR, and Antioxidant Properties of Amazonian Fruits: Mamey Apple (Mammea Americana), Camapu (Physalis Angulata), and Uxi (Endopleura Uchi). Molecules 2020, 25, 342. [CrossRef] [PubMed] 3 Molecules 2020, 25, 1362 10. 11. 12. 13. 14. 15. 16. 17. 18. Ayala-Niño, A.; Rodríguez-Serrano, G.M.; González-Olivares, L.G.; Contreras-López, E.; Regal-López, P.; Cepeda-Sáez, A. Sequence identification of bioactive peptides from Amaranth seed proteins (Amaranthus hypochondriacus spp.). Molecules 2019, 24, 3033. [CrossRef] [PubMed] Wali, A.; Wubulikasimu, A.; Mirzaakhmedov, S.; gao, Y.; Omar, A.; Arken, A.; Yili, A.; Aisa, H.A. Optimization of Scorpion protein extraction and characterization of the proteins’ functional properties. Molecules 2019, 24, 4103. [CrossRef] [PubMed] Martinez, E.; Rodriguez, J.A.; Mondragon, A.C.; Lorenzo, J.M.; Santos, E.M. Influence of potato crisps processing parameters on acrylamide formation and bioaccesibility. Molecules 2019, 24, 3827. [CrossRef] [PubMed] Ma, Y.; Zeng, Z.; Kong, L.; Chen, Y.; He, P. Determination of N-carbamylglutamate in feeds and animal products by high performance liquid chromatography tandem mass spectrometry. Molecules 2019, 24, 3172. [CrossRef] [PubMed] Duan, L.; Zhang, C.; Zhang, C.; Xue, Z.; Zheng, Y.; Guo, L. Green extraction of phenolic acids from Artemisia argyi leaves by Tailor-made ternary deep eutectic solvents. Molecules 2019, 24, 2842. [CrossRef] [PubMed] Rosemary, T.; Arulkumar, A.; Paramasivam, S.; Mondragon-Portocarrero, A.; Miranda, J.M. Biochemical micronutrient and physicochemical properties of the dried red seaweeds Gracilaria edulis and Gracilaria corticata. Molecules 2019, 24, 2225. [CrossRef] [PubMed] Lopez-Santamarina, A.; Miranda, J.M.; Mondraon, A.C.; Lamas, A.; Cardelle-Cobas, A.; Franco, C.M.; Cepeda, A. Potential use of marine seaweeds as prebiotics: A review. Molecules 2020, 25, 1004. [CrossRef] [PubMed] Dovale-Rosabal, G.; Rodriguez, A.; Contreras, E.; Ortiz-Viedma, J.; Muñoz, M.; Trigo, M.; Aubourg, S.P.; Espinosa, A. Concentration of EPA and DHA acid adduction reaction conditions using response surface methodology. Molecules 2019, 24, 1642. [CrossRef] [PubMed] Flores, M.; saravia, C.; Vergara, C.E.; Avila, F.; Valdés, H.; Ortiz-Viedma, J. Avocado oil: Characteristics, properties, and applications. Molecules 2019, 24, 2172. [CrossRef] [PubMed] © 2020 by the author. 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/). 4 molecules Article Metabolite Profiling by UPLC-MSE, NMR, and Antioxidant Properties of Amazonian Fruits: Mamey Apple (Mammea Americana), Camapu (Physalis Angulata), and Uxi (Endopleura Uchi) Larissa Gabrielly Barbosa Lima 1 , Julia Montenegro 1 , Joel Pimentel de Abreu 1 , Millena Cristina Barros Santos 2,3 , Talita Pimenta do Nascimento 2,3 , Maiara da Silva Santos 4 , Antônio Gilberto Ferreira 5 , Luiz Claudio Cameron 3 , Mariana Simões Larraz Ferreira 2,3 and Anderson Junger Teodoro 1, * 1 2 3 4 5 * Laboratory of Functional Foods, Nutrition Biochemistry Core, Food and Nutrition Graduate Program, Federal University of the State of Rio de Janeiro, UNIRIO. Av. Pasteur, 296, Rio de Janeiro 22290-240, Brazil; larissagabrielly_lima@hotmail.com (L.G.B.L.); juliamontenegro95@gmail.com (J.M.); pimenabreu@gmail.com (J.P.d.A.) Laboratory of Bioactives, Nutrition Biochemistry Core, Food and Nutrition Graduate Program, UNIRIO. Av. Pasteur, 296, Rio de Janeiro 22290-240, Brazil; barrosmillena@gmail.com (M.C.B.S.); talitapiment@gmail.com (T.P.d.N.); mariana.ferreira@unirio.br (M.S.L.F.) Center of Innovation in Mass Spectrometry, Laboratory of Protein Biochemistry, UNIRIO. Av. Pasteur, 296, Rio de Janeiro 22290-240, Brazil; cameron@unirio.br Fluminense Federal Institute of Education, Science and Technology, IFF, Av. Dário Viêira Borges, 235-Lia Márcia, Bom Jesus do Itabapoana, Rio de Janeiro 28360-000, Brazil; maiarasantos@yahoo.com.br Laboratory of NMR, Department of Chemistry, Federal University of São Carlos, UFSCar. Washington Luiz, s/n, São Carlos 13565-905, SP, Brazil; giba_04@yahoo.com.br Correspondence: atteodoro@gmail.com; Tel.: +55-21-25427236; Fax: +55-21-25427752 Received: 1 October 2019; Accepted: 12 December 2019; Published: 15 January 2020 Abstract: The metabolite profiling associated with the antioxidant potential of Amazonian fruits represents an important step to the bioactive compound s characterization due to the large biodiversity in this region. The comprehensive bioactive compounds profile and antioxidant capacities of mamey apple (Mammea americana), camapu (Physalis angulata), and uxi (Endopleura uchi) was determined for the first time. Bioactive compounds were characterized by ultra-performance liquid chromatography coupled to high resolution mass spectrometry (UPLC-MSE ) in aqueous and ethanolic extracts. Globally, a total of 293 metabolites were tentatively identified in mamey apple, campau, and uxi extracts. The main classes of compounds in the three species were terpenoids (61), phenolic acids (58), and flavonoids (53). Ethanolic extracts of fruits showed higher antioxidant activity and total ion abundance of bioactive compounds than aqueous. Uxi had the highest values of phenolic content (701.84 mg GAE/100 g), ABTS (1602.7 μmol Trolox g−1 ), and ORAC (15.04 μmol Trolox g−1 ). Mamey apple had the highest results for DPPH (1168.42 μmol TE g−1 ) and FRAP (1381.13 μmol FSE g−1 ). Nuclear magnetic resonance (NMR) spectroscopy results showed that sugars and lipids were the substances with the highest amounts in mamey apple and camapu. Data referring to chemical characteristics and antioxidant capacity of these fruits can contribute to their economic exploitation. Keywords: Amazonian fruits; antioxidant; phenolic compounds; UPLC-MSE ; bioactive compounds 1. Introduction The Amazonian region offers a wide variety of native fruits and most of them are typically obtained from nature or grown only for the local market supply in form of pulp or in natura. The economic Molecules 2020, 25, 342; doi:10.3390/molecules25020342 5 www.mdpi.com/journal/molecules Molecules 2020, 25, 342 exploitation is potentially of great importance for the region [1]. These native fruits have been studied as potential bioactive sources and many of them have shown high antioxidant capacity and elevated phenolic compounds content [2]. The antioxidant properties of Amazonian fruits have been the object of many researches, mainly due to the presence of natural antioxidants such as carotenoids and phenolic compounds. The group of antioxidant compounds found in fruits produced in Amazonian can protect the human body against toxic effects and preventing diseases such as chronic degenerative disorders, cardiovascular diseases, premature ageing, diabetes, and neurodegenerative diseases [3–5]. The Brazilian Amazonian region has a great biodiversity with approximately about 220 edible plant species producing fruit, representing 44% of native fruit diversity in Brazil. Some species such as mamey apple (Mammea americana), camapu (Physalis angulata), and uxi (Endopleura uchi) have widely appreciated flavors by Brazilian consumers, but still of moderate importance to the economy. They show potential for commercialization in both domestic and international markets [6]. Mamey apple (Mammea americana) is a fruit with a reddish yellow color, aromatic, and edible pulp, and is popularly known as abricó-do-pará, abricó, mammey apple or apricot from St. Domingo. This fruit has an important agroindustrial potential and this pulp can be used to produce different products such as syrup, juice, sherbet, jam, and pastes [7]. Physalis angulata L. is an herb indigenous of the Solanaceae family, and dispersed throughout tropical areas, including the Amazonian region [8]. This is popularly known as “camapu” and its juice is used as sedative, depurative, anti-rheumatic, and for the relief of earache, and is also used as a traditional medicine [9]. Uxi (Endopleura uxi) is an important fruit distributed in Pará and Amazonas, states of Northern Brazil. The only edible part of uxi presents a yellow-brownish color pulp with a rough-like texture, containing high content of fat (mainly oleic acid) and carotenoids, mostly trans-β-carotene, with an unique flavor [10]. Due to their peculiar biodiversity, knowledge of the species and functional property characterization of native Amazonian fruits such as mamey apple, camapu, and uxi present a major challenge to their appreciation, as many of these species are unexplored and their chemical properties remain unknown. Few studies in the literature were found to provide a comprehensive metabolomic analysis of the bioactive compounds combined to antioxidant capacity of these fruits from the Amazonian biome. However, some studies have shown the metabolomic composition of Amazonian native fruits. Paz et al. [11] studied Clavija lancifolia Desf. using liquid chromatography mass spectrometry (LC–MS/MS) and found some compounds of flavonoids for which kaempferol was the main compound. Souza et al. [12] identified the phenolic compounds cyanidin 3-O-rutinoside, chlorogenic acid, and rutin in Oenocarpus distichus fruits, using high-performance liquid chromatography (HPLC). It has been reported the presence of catechin, caffeic acid, rutin, orientin, quercetin, apigenin, luteolin, and kaempferol in Mauritia flexuosa L. f. (Arecaceae), another fruit from Amazonian biome [13]. Concerning the importance of the characterization of bioactive compounds from Amazonian fruits, the aim of this study was to assess the phytochemical profile and the total antioxidant capacity of mamey apple (Mammea americana), camapu (Physalis angulata), and uxi (Endopleura uchi). 2. Results and Discussion 2.1. Total Phenolic Compounds Content Mamey apple (MA), camapu (C), and uxi (U) were submitted for analysis of total phenolic compounds from extractions submitted with different vehicles solvents (water (W) and ethanol (E)) to determine the most efficient extraction solvent of the mentioned compounds. The results showed that the ethanolic extracts (MAE and UE) of the different Amazonian fruits showed a greater quantity of phenolic compounds when compared to the aqueous extracts (MAW and UW) (Figure 1). The preparation and extraction from this wide range of samples depends mostly on the nature of the sample matrix and the chemical properties of the phenolics, including molecular structure, polarity, 6 Molecules 2020, 25, 342 concentration, number of aromatic rings, and hydroxyl groups. Conventional solid-liquid using organic solvent extraction is the main method used to extract phenolics. The sample preparation, polarity of the solvent used, the technique employed and temperature are factors that can influence the extraction and contents of these compounds [14]. PJJDOLFDFLGJ      0$: 0$( &: &( 8: 8( Figure 1. Total phenolic content in aqueous mamey apple (MAW), ethanolic mamey apple (MAE), aqueous camapu (CW), ethanolic camapu (CE), aqueous uxi (UW), and ethanolic uxi (UE) extracts. Results are expressed by mean ± SD (n = 3) and were compared by the one-way ANOVA test with post-test Tukey s (* p < 0.01; ** p < 0.001). Uxi ethanolic extract (UE) showed the highest phenolic compound content (701.839 mg GAE/100 g), followed by mamey apple (MAE) (556.105 mg GAE/100 g), and camapu (CE) (237.39 mg GAE/100 g). The aqueous extracts of mamey apple (MAW) and uxi (UW) showed significant difference (p < 0.05) when compared to the respective ethanolic extracts. No significant differences (p > 0.05) were observed in the content of phenolic compounds between the aqueous and ethanolic extracts in the camapu sample. Aqueous and ethanolic extract showed similar fruits yield extracts, being dependent on the moisture percentage of each fruit. Uxi yield was significantly higher (UE-28.82% and UW-29.42%), followed by mamey apple (MAE-14.04% and MAW-13.13%) and camapu (CE-10.50% and CW-11.10%). Considering these results, the levels of phenolic compounds per mass of fruits for the highest yield would be 202.27, 78.08, and 24.92 mg GAE/100 g for uxi, mamey apple, and camapu, respectively. Péroumal et al. [15] studied the pulp of six mamey apple accessions and found values for total phenolic content between 90 and 143 mg GAE/100 g. It can be observed that the results obtained for uxi ethanolic extracts in this work are higher than some Amazonian fruits such as araça-boi (87.2 ± 3.0 mg GAE/100 g) and araça (129.1 ± 9.3 mg GAE/100 g), and lower than camu-camu (1797.2 ± 37.7 mg GAE/100 g) reported by Genovese et al. [16]. Comparing the results of this study with fruits from the Brazilian Cerrado biome, the Amazonian fruits present lower phenolic compounds in relation to sweet passion fruit (245.36 ± 3.70 mg GAE/100 g), soursop (281 ± 5.40 mg GAE/100 g), murici (334.37 ± 9.07 mg GAE/100 g), and marolo (739.37 ± 7.92 mg GAE/100 g) [17]. According to the classification proposed by Vasco et al. [18], MAE, uxi extracts were classified as fruits as having with medium phenolic content (100–500 mg GAE/100 g). 2.2. Phytochemical Profile by UPLC-MSE For the first time, bioactive compounds of Amazonian fruits like mamey apple, camapu, and uxi were elucidated by UPLC-MSE metabolomic approach. Globally, 293 compounds were tentatively identified from aqueous and ethanolic extracts of these fruits (Table S1) and relatively quantified taking account all extracts based on ion counting. Table 1 shows the number of identified compounds and their classification into eight chemical classes according to Phenol Explorer database [19]: phenolic acid, flavonoids, chalcones, coumarins, amino acid related compounds, fatty acid, and terpene related compounds. The main bioactive compounds in the three species were terpenoids (n = 61; 21%), 7 Molecules 2020, 25, 342 phenolic acids (n = 58; 20%), and flavonoids (n = 53; 18%). Other metabolites were also identified such as other polyphenols (n = 50; 17%), including lignans, coumarins and tannins, and also other metabolites (n = 56; 19%) such as amino acid related, alkaloids and polyketides, showing the extraction and LC-MS methods were suitable to characterize different polarities compounds. Table 1. Number of bioactive compounds distributed by classes and other compounds identified in aqueous (W) and ethanolic (E) extracts of mamey apple, camapu, and uxi. Compounds (%) Phenolic Acid Flavonoids Chalcones Coumarins Others phenolic compounds Amino acid related compounds Fatty acids related compounds Terpenoids Total of compounds Mamey Apple Camapu Uxi W E W E W E 43 (20.57%) 36 (17.22%) 2 (0.96%) 16 (7.66%) 27 (12.92%) 17 (8.13%) 22 (10.53%) 46 (22.01%) 209 (100%) 37 (19.68%) 30 (15.96%) 2 (1.06%) 15 (7.98%) 25 (13.30%) 14 (7.45%) 22 (11.70%) 43 (22.87%) 188 (100%) 44 (24.04%) 33 (18.03%) 2 (1.09%) 14 (7.65%) 23 (12.57%) 17 (9.29%) 11 (6.01%) 39 (21.31%) 183 (100%) 40 (21.51%) 29 (15.59%) 2 (1.08%) 14 (7.53%) 30 (16.13%) 20 (10.75%) 12 (6.45%) 39 (20.97%) 186 (100%) 38 (23.17%) 24 (14.63%) 3 (1.83%) 8 (4.88) 20 (12.20%) 18 (10.98%) 14 (8.54%) 39 (23.78%) 164 (100%) 35 (21.21%) 27 (16.36%) 3 (1.82%) 9 (5.45%) 26 (15.76%) 15 (9.09%) 15 (9.09%) 35 (21.21%) 165 (100%) The identification of different phenolic compounds in this study makes it relevant, since the presence of these compounds has a range of bioactivities, as already reported in vitro and in vivo studies [20]. In a recent study, extracts of phenolic compounds from jatobá-do-cerrado can inhibit α-amylase and α-glycosidase after in vitro digestion and modulate the glucose metabolism [21]. Mamey apple was characterized by high number of bioactive compounds. Among the identified metabolites, 209 (71%) and 188 (64%) were found in aqueous and ethanolic extracts of mamey apple, respectively. Furthermore, 183 (62%) and 186 (63%) compounds were found in the aqueous extract ethanolic extracts of camapu, respectively. In uxi, 164 (56%) and 165 (56%) compounds were tentatively identified in the aqueous and ethanolic extracts, respectively. Notably, 50 (17%) compounds were found only in mamey apple (e.g., droserone, norlichexanthone, procyanidin C1, 3,4-leucopelargonidin II, and piquerol A). Piquerol A is a sesquiterpene with low stability in nature and has only previously been tested as an insecticide and as an inhibitor of metabolism in cell cultures [22]. About 43 (15%) were tentatively identified only in camapu (e.g., myristicin, 11-deoxocucurbitacin, synapic acid I, and pseudopurpurin). Medina et al. [23] reported the presence of sinapic acid in Passiflora edulis Sims. Furthermore, about 36 (12%) compounds were found exclusively in the uxi extract (e.g., 4-coumaroylchiquimate, ferulic acid I, kaempferol II, jacareubin, 6-deoxyjacareubin, vanylactic acid II, nigakilactone A, and jasmonic acid). Kaempferol is an important flavonol identified in other fruits like hybrid grapes by Rosso et al. [24]. These metabolites are antioxidant inhibitors of mutagenic and carcinogenic compounds and are considered neuroprotective agents in neurodegenerative disorders, such as Parkinson s and Alzheimer s diseases [25]. The acid phenolic profiling was the most abundant subclass of phenolic compounds in all extracts evaluated, as shown in the Tables 1 and 2. Aqueous and ethanolic camapu extracts (CW and CE) showed about 24 and 22% of phenolic acids, respectively, followed by mamey apple aqueous (MAW) and ethanolic (MAE) extracts. In addition to phenolic acids, flavonoids showed high number of identification as well as elevated abundance relative. 8 Molecules 2020, 25, 342 Table 2. Flavonoids tentatively identified in mamey apple, camapu, and uxi extracts. Possible Identifications CAS m/z Exp Genistin 529-59-9 431.0978 RT (min) Fragment m/z 4.55 133.0294 (10.20) Error (ppm) Mamey Camapu W E W E W Uxi E −1.23 − − − − X X −2.86 X X − − − − Eriodictiol I 552-58-9 287.0552 3.05 81.0338 (5.46); 93.0339 (78.57); 119.0496 (100); 155.0342 (10.19); 163.0395 (60.20) Hesperidin 520-26-3 609.1875 0.43 79.0188 (0.66); 369.0671 (50.27); 488.1618 (10.04) 8.37 X X X X X X 9.66 X X X X X X X X X X 14259-46-2 579.1775 0.43 72.9924 (78.62); 79.0188 (0.58); 117.0187 (26.50); 135.0294 (87.65); 357.1033 (3.92); 369.0671 (44.06); 535.1514 (21.08) Pomiferin I 572-03-2 419.1505 2.62 nd 1.21 X X X X Ononin 486-62-4 429.1170 2.02 nd −4.89 X X X X Mammeisin 18483-64-2 405.1677 4.01 109.0653 (0.46); 154.0615 (0.72) −7.30 X X Quercitrin 522-12-3 447.0923 3.48 151.0030 (2.20); 285.0393 (5.36) −2.09 X X X −1.44 X X X Narirutin Quercetin 3-galactoside 482-36-0 463.0875 3.47 151.0030 (2.97); 255.0291 (5.58); 271.0241 (16.99); 285.0393 (7.25); 300.0266 (31.67) Kaempferol I 520-18-3 285.0396 4.90 nd −2.70 X X Dihydroquercetin 480-18-2 303.0505 1.42 147.0120 (100) −1.65 X X Luteoforol 24897-98-1 289.0733 0.56 109.0289 (12.30) 5.54 X X X X X X X X Terpenes were between the most abundant bioactive classes in all extracts. About 46 and 43 terpenes, representing 22% of identifications, were found in the MAW and MAE extracts, respectively. In UE extracts, 39 (23%) terpenes were tentatively identified too. Terpenoids are important secondary metabolites of plants and are extremely chemically diversified, being estimated at more than 40,000 substances. Its use has been described as flavoring agents, in addition to providing benefits to human health through its antioxidant potential [26,27]. Although the number of identifications between the extracts of the same fruit was not significantly different, the ethanolic extracts showed a greater relative abundance of compounds in relation to the aqueous extracts in all fruits studied (Figure 2). This fact can be explained by the large variability of the structures of these bioactive compounds and the proportion of organic solvents with water is required for better extraction. The principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied to explain possible difference among extracts (Figures 3 and 4). First, the PCA biplot represented both loading (metabolites) and scores (extracts). Such parameters distinguished the profile of bioactive compounds among the fruits evaluated in all extracts. Principal components (PC1 and PC2) demonstrated that the fruits extracts have different composition of bioactive compounds, but the use of aqueous and ethanolic extractors did not favor the removal of distinct metabolite profile in the species. The two major principal components (PC1 and PC2) explained more than 72% of the variance pattern (Figure 3). Discriminatory metabolites, which showed maximum variance (eigenvalues) among 9 Molecules 2020, 25, 342 extracts, were observed mainly in the PC2, including the following bioactive compounds bryophyllin A II, 1-O-2 -hydroxy-4 -methoxycinnamoyl-b-d-glucose I, mammeisin II, eriodictiol II, justicidin A, hallactone B II, 7,2 -dihydroxy-4 -methoxy-isoflavanol, eleganin I, 5-hydroxyferulic acid methyl ester II, lancerin II, 6-methoxytaxifolin II, zapoterin, salvinorin A, meconic acid, benzoic acid II, auriculoside, leucocyanidin II, lophophorine, lancerin I, vernodalol, visnagin, sinapic acid II, sinapyl alcohol II, 6-methoxytaxifolin I, isobrucein A, and syringin I. 5HODWLYHDEXQGDQFH      0$: 0( &: &( 8: 8( Figure 2. Relative abundance based on total ion counting of identified compounds from aqueous mamey apple (MAW), ethanolic mamey apple (MAE), aqueous camapu (CW), ethanolic camapu (CE), aqueous uxi (UW), and ethanolic uxi (UE) extracts. Results are expressed by mean ± SD (n = 3) and were compared by the one-way ANOVA test with post-test Tukey s (* p < 0.05; ** p < 0.01; *** p < 0.001). Figure 3. Principal component analysis (PCA) biplot (loadings and scores) of the bioactive compounds tentatively identified (loadings, empty circles) in the mamey (squares), camapu (triangles), and uxi (diamonds) fruits extracted with aqueous (W) and ethanolic (E) solvents. Then, HCA was applied to observe the similarity/dissimilarity among the abundance of the discriminatory polyphenols (Figure 4) in order to understand the profile of composition among extracts. The heatmap indicated that metabolites (20) found in uxi extracts showed higher abundance in comparison to other extracts, especially in the ethanol extracts. Mamey also showed a distinguished bioactive profile, with six compounds found exclusively. Among them, sinapoyl alcohol II is an exclusive metabolite with high abundance in the mamey apple extracts. One of the metabolites with highest abundance in these extracts was syringin. This compound was determined and reported from fresh jaboticaba methanolic extract by Wu et al. [28] and implicated as immunomodulator having an anti-allergic effect [29]. 10 Molecules 2020, 25, 342 Figure 4. Hierarchical cluster analysis (HCA) and heatmap of the bioactive compounds in the mamey apple, camapu, and uxi fruits extracted with aqueous (W) and ethanolic (E) solvents, which showed maximum variance (eigenvectors) among extracts. 2.3. In Vitro Antioxidant Activity Due to the multifunctional characteristics of the phenolic compounds found in Amazonian fruits, the effectiveness of measuring the antioxidant capacity of a pulp extract is better evaluated when using commonly accepted tests. The ethanolic and aqueous fruits extracts was determined by four different methods with different action mechanism (ABTS, DPPH, FRAP, and ORAC) (Table 3). According to Barros et al. [14], a single assay does not accurately account for all of the groups of antioxidant compounds, because of complexity of fruit matrices, and because of these methodologies can suffer interferences. ABTS assay revealed values ranged between 263.67 ± 23.90 and 1602.7 ± 30 μmol Trolox g−1 . The highest antioxidant capacity was presented in UE samples (1602.7 ± 30.16 μmol Trolox g−1 ). Freitas et al. [30] analyses fresh pulp of uxi and found 51.6 mg TE/100 g for ABTS. In general, the ethanolic extracts of fruits showed the higher antioxidant capacity for this methodology. However, the aqueous extract from camapu fruit was an exception with a value of 432.74 ± 16.17 μmol Trolox g−1 (Table 3). Schiassi et al. [31] studied the activity antioxidant of methanolic extracts of araça (10.92 ± 0.11 μmol Trolox g−1 ), buriti (6.03 ± 0.00 μmol Trolox g−1 ), cagaita (29.32 ± 0.69 μmol Trolox g−1 ), yellow mombin (5.55 ± 0.01 μmol Trolox g−1 ), and marolo (132.16 ± 1.40 μmol Trolox g−1 ) and found values lower than the present study. 11 Molecules 2020, 25, 342 Table 3. Antioxidant activity of the mamey apple, camapu, and uxi samples by ABTS, DPPH, FRAP, and ORAC assays. Assay ABTS (μmol Trolox g−1 ) DPPH (μmol Trolox g−1) FRAP (μmol ferrous sulphate g−1 ) ORAC (μmol Trolox g−1 ) Mamey Apple Camapu Uxi Aqueous Ethanolic Aqueous Ethanolic Aqueous Ethanolic 263.67 ± 23.90 a 336.60 ± 3.05 a 564.18 ± 18.90 a 5.17 ± 0.56 a 937.66 ± 218.49 b 1168.42 ± 218.56 b 1381.13 ± 189.95 b 8.88 ± 0.52 b 432.74 ± 16.17 c 386.24 ± 116.99 c 970.60 ± 28.92 c 12.30 ± 1.15 c 419.43 ± 18.55 c 705.77 ± 100.74 d 1183.98 c ± 46.62 b 11.15 ± 0.42 c 271.86 ± 22.14 a 46.95 ± 17.17 e 376.66 ± 1.81 d 14.33 ± 1.36 d 1602.7 ± 30.16 d 509.27 ± 26.95 f 448.68 ± 41.97 e 15.04 ± 0.84 d Results are expressed as mean ± standard deviation. Different letters on the same line show significant difference. Results were compared by the One-way ANOVA test with Tukey post-test (p < 0.05). Antioxidant capacity was also evaluated by DPPH• radical scavenging method (Table 3) and expressed in aqueous and ethanolic extract concentration μmol Trolox g−1 sample. The highest activity was found in MAE (1168.42 μmol Trolox g−1 ), followed by CE (705.771 μmol Trolox g−1 ) and UE (509.68 μmol Trolox g−1 ). According to Huchin et al. [32], the DPPH method can analyze hydrophilic and lipophilic compounds, and ethanol is one of the most used solvents for bioactive compounds extraction due, because proposes some benefits, such as low toxicity, good extraction produce, it is safe for human consumption and allows the extracts to be used in the food industry [33]. Antioxidant activity by FRAP method followed the same pattern of the DPPH analysis, where higher values were found for the ethanolic extracts. It is probable that the antioxidant compounds of the samples detected by FRAP are the same with those evaluated by DPPH. MAE also presented the highest activity (1381.13 μmol ferrous sulphate g−1 ) These results was lower than obtained by Barros et al. [14] for aqueous extracts of achachairu (712.35 ± 6.61 μmol ferrous sulphate g−1 ), araça-boi (798.92 ± 1.52 μmol ferrous sulphate g−1 ), and bacaba (6567.45 ± 4.25 μmol ferrous sulphate g−1 ). Regarding to ORAC assay, the results revealed that UE and UW obtained the highest value (15.04 ± 0.84 and 14.33 ± 1.36 μmol Trolox g−1 ), followed by CE and CW (11.15 ± 0.42 and 12.30 ± 1.15 μmol Trolox g−1 ). No significant differences (p > 0.05) were observed between the aqueous and ethanolic extracts of uxi and camapu sample. MAE (8.88 ± 0.52 μmol Trolox g−1 ) and MAW (5.17 ± 0.56 μmol Trolox g−1 ) showed the lowest values (p > 0.05) between extracts. Santos et al. [34] reported to Amazonian fruits such as bacaca (195.00 ± 10.00 μmol Trolox g−1 ), buriti (83.00 ± 6.00 μmol Trolox g−1 ), inajá (26.00 ± 2.00 μmol Trolox g−1 ), pupunha (94.00 ± 1.00 μmol Trolox g−1 ), and tucumã (64.00 ± 4.00 μmol Trolox g−1 ) results higher than camapu, mamey apple and uxi extracts. 2.4. Correlation between Total Phenolic Compounds and Antioxidant Capacity The results of Pearson correlation coefficients (r) between total phenolic compounds and antioxidant capacity of aqueous and methanolic extracts suggest that the total phenolic compounds contributed to the in vitro antioxidant capacity of the extracts according to the method used. The aqueous extract showed a good association of total phenolic compounds with ABTS (r = 0.843) and ORAC (r = 0.752) for camapu fruit. ORAC assay has been largely applied to the assessment of free radical scavenging capacity of pure antioxidant compounds and antioxidant plant extracts [35]. The radical cation ABTS [2,29-]azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) is one of the spectrophotometric methods used to measure water-soluble as well as lipid-soluble antioxidants, pure compounds, and food extracts. The pre-formed radical monocation of 2,29-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•1 ) is generated by oxidation of ABTS with potassium persulfate and is reduced in the presence of such hydrogen-donating antioxidants [36]. 1-O-2 -Hydroxy-4 -methoxycinnamoyl-b-d-glucose, a compound present in CW (Figure 4), this compound belongs to the sub class of organic compounds known as hydroxycinnamic acid glycosides. Hydroxycinnamic acids are natural antioxidants found in fruits, vegetables, and cereals [37]. Regarding to mamey-apple, the ethanolic extract presented positive correlation with total phenolic compounds in relation to DPPH assay (r = 0.910). The 2,2-diphenylpicrylhydrazyl (DPPH) assay is widely used in plant biochemistry to evaluate their properties for scavenging free radicals. The method 12 Molecules 2020, 25, 342 is based on the spectrophotometric measurement of DPPH concentration change resulting from the reaction with an antioxidant [38]. According to Figure 4, the acids syrigin I and visnagin were components with high relative abundancy. Visnagin is an antioxidant furanocoumarin derivative and is a furanochromone that is furo [3,2-g]chromen-5-one which is substituted at positions 4 and 7 by methoxy and methyl groups, respectively. Syringic acid is a dimethoxybenzene that is 3,5-dimethyl ether derivative of gallic acid. It has a role as a plant metabolite, a member of benzoic acids, and derives from a gallic acid. Previous reports described that hydroxybenzoic acids itself and its derivatives showed antioxidant properties against different type of free radicals and can prevent or decrease overproduction of reactive species. The main structural feature responsible for the antioxidative and free radical scavenging activity in the case of phenolic derivatives is the phenolic hydroxyl group. Phenols are able to donate the hydrogen atom of the phenolic OH to the free radicals, thus stopping the propagation chain during the oxidation process. This effect is modulated by the ring substituents, so that electron-withdrawing groups increase the bond-dissociation enthalpy, due to the stabilization of the phenol by a polar structure that leaves a positive charge on the OH group [39–41]. In uxi ethanolic extract, some bioactive compounds were identified with great relative abundance, such as auriculoside, that is a flavan glycoside. Most natural flavans are lipid-soluble and prominent in fruit skin or peel and leaf surfaces, and are usually found at higher concentrations in immature fruits compared to mature fruits [42]. Lancerin a plant metabolite found in UE is a member of xanthones, a C-glycosyl compound and a polyphenol. Among the polyphenols, the xanthones derivatives comprise an important class of the oxygenated heterocycles with a diversity of substitution patterns that have been described for their antioxidant activity, show to act as metal chela-tors, free radical scavengers, as well as inhibitors of lipid peroxidation [43]. The presence of these compounds may have contributed to the strong correlation (r = 0.910) found between total phenolic compounds and ORAC assay. These results demonstrate the importance of do different methods to available the antioxidant activity, above all in complex matrices. The positive correlation between compound phenolics and ORAC, can be explained due to this assay may be considered a more exact method, because it uses a biologically relevant radical source (peroxyl) and allows the measurement of total antioxidant capacity through the combination of the antioxidant capacity of hydrophilic and lipophilic fractions [44]. According to the methodology used, these results suggest that phenolic compounds may be one of the main factors responsible for the antioxidant capacity of mamey apple, camapu, and uxi. 2.5. NMR Profile Table 4 described the identified compounds for each fruit, with the chemical shift and signal multiplicity, as well as coupling constant (J), and the quantification in mg/g of freeze-dried fruit. Camapu had the greatest number of different compounds, indicating that it has a large variety of nutrients and metabolites, even more than could be extracted and identified. Uxi had the least amount, probably due to the great quantity of lipophilic compounds that could not be extracted, so this result does not mean that there are few compounds in uxi. Sugars were most abundant compounds in those fruits. Camapu showed the highest amount of sucrose, and mamey apple showed the highest amount of fructose and glucose, both α and β. In the uxi extracts no sugars were found. This result indicates that mamey apple and camapu are sweeter than uxi, and probably would be more accepted by consumers. The sugars present in mamey apple were sucrose (1.50 ± 0.10 mg/g), fructose (0.85 ±0.03 mg/g), α-glucose, and β-glucose (0.49 ± 0.02 and 0.40 ± 0.01 mg/g, respectively). The sugars found in camapu were sucrose (4.09 ± 0.46 mg/g), followed by fructose, α-glucose, and β-glucose (0.11 ± 0.00, 0.04 ± 0.00, and 0.04 ± 0.00 mg/g respectively). Fruit and vegetable flavor depend upon taste (given by sweetness, sourness, acidity, and astringency) and aroma (concentrations of odor-active volatile compounds). Sweetness is determined by the concentrations of the predominant sugars, which are ranked relative to sucrose (fructose > sucrose > glucose). Sourness or acidity is determined by the predominant organic acids, which 13 Molecules 2020, 25, 342 are ranked relative to citric acid (citric > malic > tartaric). Some amino acids, such as aspartic and glutamic, may also contribute to sourness. In general, consumer acceptance is related to soluble solids concentration (sugars, organic acids, soluble pectin, some phenolic compounds, and ascorbic acid) or the ratio of soluble solids to titratable acidity [45]. Table 4. NMR fingerprinting identification and quantification of mamey apple, camapu, and uxi. Fruit Compound δ 1 H (ppm) Multiplicity (J) Mass (mg/g) Mamey Apple Formic acid Shikimic acid Sucrose α-glucose β-glucose Fructose Choline Ethanol 8.44 6.48 5.42 5.22 4.64 4.10 3.19 1.18 s m d (3.88) d (3.76) d (7.94) d (3.42) s t (7.09) 0.01 ± 0.00 0.11 ± 0.01 1.51 ± 0.11 0.49 ± 0.02 0.40 ± 0.02 0.85 ± 0.04 0.01 ± 0.00 0.01 ± 0.00 Camapu Formic acid Sucrose α-glucose β-glucose Fructose Choline Aspartic acid Acetic acid GABA Alanine Lactic acid Ethanol Valine 8.44 5.42 5.23 4.65 4.11 3.20 2.82 1.94 1.89 1.47 1.33 1.18 1.04 s d (3.91) d (3.73) d (7.92) d (3.42) s dd (17.4; 3.78) S quin (7.44) d (7.2) d (6.68) t (7.09) d (7.07) 0.01 ± 0.00 4.09 ± 0.46 0.04 ± 0.01 0.04 ± 0.01 0.12 ± 0.01 0.01 ± 0.00 0.04 ± 0.01 0.01 ± 0.00 0.02 ± 0.01 0.02 ± 0.01 0.01 ± 0.00 0.02 ± 0.001 0.01 ± 0.00 Uxi Linoleic acid Acetic acid Alanine Ethanol Valine 5.32 1.94 1.45 1.16 1.05 M S d (7.20) t (7.10) d (7.05) 1.79 ± 0.11 0.16 ± 0.01 0.04 ± 0.01 0.15 ± 0.02 0.05 ± 0.001 It has been shown that mamay apple has ethanol (0.01 ± 0.00 mg/g), which is possibly a fruit sugar fermentation product. It was also found choline (0.01 ± 0.00 mg/g) and organic acids, such as formic acid and shikimic acid (0.01 ± 0.00 and 0.11 ± 0.00 mg/g) in mamey apple. Shikimic acid (3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid) is natural acid from the plant metabolism and common in berries fruits. It is a precursor for the biosynthesis of primary metabolites such as aromatic amino acids and folic acid, and a great many other aromatic compounds. The benzene ring is formed through the shikimate pathway, and shikimic acid is an extremely essential compound in plants and microbes. Shikimic acid has been found to occur in many tissues of a variety of plants, with a sufficiently high percentage. Moreover, its content and accumulation in different tissues depends on the rate of metabolic processes taking place in them. Shikimic acid is utilized for industrial synthesis of the oseltamivir (antiviral), zeylenone (employed as a preparation for chemotherapy of cancer), and monopalmityloxy shikimic acid (anticoagulant activity). In addition, shikimic acid derivatives represent a great interest for agriculture because many of them are used as herbicides and antibacterial agents [46]. Choline is a natural amine that can be synthesized in human body, but this production usually is not enough to meet human needs in men and postmenopausal women. Some important functions of choline are it is a part of the neurotransmitter acetylcholine; it is a part of the predominant phospholipids in membranes; it forms betaine, which is an important osmolyte in the kidney glomerulus and helps with the reabsorption of water from the kidney tubule. Eggs and liver are the main sources of choline, but many other foods contain significant amounts of choline and esters of choline [47]. 14 Molecules 2020, 25, 342 Ethanol (0.02 ± 0.00 mg/g) was also present in camapu, indicating fermentation. The organic acids found in camapu were lactic acid, acetic acid and formic acid (0.01 ± 0.00, 0.01 ± 0.00, and 0.01 ± 0.00). Besides those organic acids, it was found γ-amino butyric acid (GABA) in camapu (0.02 ± 0.00 mg/g). GABA is a four-carbon nonprotein amino acid and is widespread in bacteria, animals, and plants. GABA is naturally present in small quantities in plants, and is produced in response to anaerobic conditions, γ-radiation, low pH, low or high temperatures, and darkness, and by mechanical manipulation. Some functions of GABA in plants are: regulation of cytosolic pH, protection against oxidative stress, defense against insects, and the regulation of pollen tube growth and guidance [48]. In vertebrates is a neurotransmitter that is deficient in the brain of people with Alzheimer disease [49]. Camapu has been shown to be effective in delaying the development of this disease and in its treatment, by increasing the proliferation of neural stem cells in vivo [50] and also shown an anxiolytic effect [51] which the authors suppose is due to the presence of GABA agonists. Several amino acids such as aspartic acid, alanine, and valine (0.03 ± 0.00, 0.02 ± 0.00, and 0.01 ± 0.08 mg/g) and choline (0.01 ± 0.00 mg/g) were also present in camapu in sufficient quantities to be identified. For uxi, the compound with in the highest amount was linoleic acid (1.79 ± 0.10 mg/g), which is the main fatty acid of ω-6 group. This result shows that there are fatty acids that are sufficient to appear in a spectrum of fingerprints, even though other lipids may not have been completely extracted from the sample. There is probably a low amount of sugar in uxi fruit because there are high amounts of lipids and usually sugar and lipids in the pulp of fruits are inversely proportional. However, it was found ethanol (0.15 ± 0.01 mg/g), indicating that some sugar that was present before it was fermented. It was also found enough amount of acetic acid (0.15 ± 0.00 mg/g) valine and alanine amino acids (0.05 ± 0.00 and 0.04 ± 0.00 mg/g, respectively). 3. Material and Methods 3.1. Standards and Chemicals 2,2-Diphenyl-1-picrylhydrazyl (DPPH• ), 2,2 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), gallic acid, quercetin, 2 ,7 -dichlorofluorescin diacetate (DCFH-DA), L-(+)-ascorbic acid (AA). Phenolic standards (caffeic acid, (+)-catechin, ellagic acid, (−)-epicatechin, gallic acid, gentisic acid, 4-hydroxybenzoic acid, myricetin, pyrogallol, quercetin, and quercetin 3-O-glucoside) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All the solvents used for the UPLC-MSE analysis were of LC-MS purity grade and were also purchased from Sigma-Aldrich (St. Louis, MO, USA). 3.2. Samples The fruits in natura of mamey apple (Mammea americana), camapu (Physalis angulata), and uxi (Endopleura uchi) were obtained from producing regions of Pará State, in the months of January and February 2017. They were transported in vacuum plastic containers to Federal University of the State of Rio de Janeiro (UNIRIO, RJ, Brazil) and stored at −18 ◦ C. The part of the fruit used for extraction was carried out according to their consumption, for mamey apple only pulp, for camapu the whole fruit and for uxi pulp and bark. 3.3. Sample Preparation Samples were extracted by means of 2 extraction solvents: (I) ethanol 70% and (II) water, from 5 g of sample and 100 mL of extraction solvent, followed by homogenization at room temperature (−20 ◦ C) in a homogenizer TE-420 (Tecnal, São Paulo, Brazil) for 10 min in the absence of light. The samples were then centrifuged (Thermo Fisher Scientific, Bartlesville, CA, USA) (5000× g, 5 min, 20 ◦ C) and filtered through analytical filter paper. The extraction solvents were evaporated using a rotary evaporator under vacuum (Savant, Thermo Scientific) and the extracts were then frozen at −80 ◦ C in an ultra-freezer 15 Molecules 2020, 25, 342 and lyophilized (Terroni® LD 300, São Carlos, SP, Brazil) for 24 h. After this process, extracts were frozen at −20 ◦ C until use in the experiments. For UPLC-MSE analysis, the extraction solvents were evaporated using a rotary evaporator under vacuum (Savant, Thermo Scientific, USA) at 40 ◦ C and thereafter ressuspended in methanol/acetonitrile/water (2:5:93; v/v). Stock solutions (500 ppm) of 11 standards (caffeic acid, (+)-catechin, ellagic acid, (−)-epicatechin, gallic acid, gentisic acid, 4-hydroxybenzoic acid, myricetin, pyrogallol, quercetin and quercetin 3-O-glucoside from Sigma-Aldrich were prepared individually by dissolving accurately weighed amounts of standards in aqueous methanol an aliquot of each stock solution was mixed to achieve a mixed standard solution with a final concentration of 10 mg/L for each compound. Finally, extracts and standards were filtered through a 0.22 μm syringe filter and stored at −80 ◦ C until UPLC-MSE analysis. 3.4. Quantification of Total Phenolic Compounds Total phenolic content was determined by the Folin–Ciocalteu method, which was adapted from Rocchetti et al. [52]. The extract and 2.5 mL Folin-Ciocalteu reagent solution 10% were combined and then mixed well using a Vortex. The mixture was allowed to react for 5 min then 2 mL of sodium carbonate 4% solution was added and mixed well the solution was incubated at room temperature (25 ◦ C) in the dark for 2 h. The absorbance was measured at 750 nm using a spectrophotometer (Turner® 340, Haverhill, MA, USA) and the results were expressed in mg gallic acid equivalents (GAE) per 100 g of extract using a gallic acid (2,5–50 μg/μL) standard curve [53]. 3.5. Phytochemical Profile Characterization by UPLC-MSE The UPLC-MSE analyses were carried out according to Santos et al. (2018) [54] with slightly modifications. Two uL of extracts and standards were injected in triplicate into an Acquity UPLC (Waters, Milford, MA, USA) coupled to Xevo G2-S QTOF-MS/MS (Waters, Manchester, UK) system equipped with an electrospray ionization source (ESI) operating in negative ion mode. The column used was a UPLC HSS T3 C18 (100 mm × 2.1 mm, 1.8 μm) (Waters, Wexford, Ireland). The flow rate was 0.6 mL/min and the mobile phase gradient elution consisted of acidified water (5 mM ammonium formate and 0.3% formic acid, v/v) (pump A) and acetonitrile containing 0.3% formic acid (pump B), as follows: 97% A at 0 min, 50% A at 6.8 min, 15% A at 7.4–8.5 min, followed by an additional equilibration step 97% A at 9.1–12 min. Data were acquired using a multiplexed MS/MS acquisition in the extended mode with alternating low and high energy acquisition (MSE ) 30–55 eV using ultra-high pure argon (Ar). The capillary and cone voltage were set at 3.0 kV and 30 V, respectively. The desolvation gas (N2 ) was set at 600 L/h at 450 ◦ C, the cone gas was set at 50 L/h and the source temperature at 120 ◦ C. Data acquisition was performed by using MassLynx 4.1 (Waters Corporation, Milford, MA, USA). To ensure accuracy and reproducibility, all acquisitions were performed by infusing lock mass calibration with leucine-enkephaline (Waters Corporation, Milford, MA, USA) (m/z 554.2615) at a concentration of 1.0 ng/L in acetonitrile: H2 O (50:50, v/v) with 0.1% (v/v) formic acid at a flow rate of 10 μL/min. The raw data of all replicates were processed with Progenesis QI v2.1 (Nonlinear Dynamics, Waters Corporation, UK) with the following conditions: centroid data, resolution full-width at half maximum (FWHM) of 50,000, deprotonated molecule [M − H]− . The identification of phenolic compounds was performed by searching for polyphenols with MetaScope, using a customized database of polyphenol compounds from PubChemID by using the following parameters: precursor and fragment mass error tolerance (5 and 10 mg/L, respectively), retention time limit 7.5 min. Target analysis was also applied for identification of the phenolic compounds by comparing the run parameters of phenolic standards such as the retention time, exact mass, mass error, and the MS-MS spectra, besides the other above mentioned parameters. The processed data were exported to XLSTAT software (Addinsoft, Paris, France), where the values of relative abundance obtained from each compound based on ion mass spectra counting were used to relative quantification and then to the multivariate statistical evaluation where Principal Components Analysis (PCA) biplot was elaborated. The Metaboanalyst 3.0 web server 16 Molecules 2020, 25, 342 was used for analysis of multivariate data (HCA, hierarchical cluster analysis and heat map) where the eigenvectors of the correlation matrix was used to select the discriminatory bioactive compounds between the samples analyzed. 3.6. Analysis by Nuclear Magnetic Resonance Spectroscopy For the NMR analyses, samples were prepared in triplicate. It was done by weighing 30.0 (±0.5) mg of freeze-dried samples (for each repetition), which was subjected during 10 min to shaking in a vortex with 0.8 mL of extractor solvent (D2 O for abrico and camapu; methanol-d4 for uxi), with TMSP-d4 at concentration 0.02% (m/v) as internal reference and mass standard and, after, to ultrasonic bath during 10 min. Next, samples were centrifuged (13,000 RPM) at room temperature for more 10 min. A 0.6 mL supernatant aliquot was analyzed. The 1 H-NMR spectra, as well as the 2D experiments, were conducted in the Bruker Avance III 9.4 Tesla (400 MHz for hydrogen frequency) spectrometer equipped with a PABBI probe (5 mm) and with an Automatic Tuning and Matching (ATMA) unit located in NMR Laboratory of the Chemistry Department at Federal University of São Carlos-UFSCar, Brazil. The TMSP-d4 was used to determine the 0 point in the chemical shift scale. 1H NMR spectra were registered at temperature of 300 K using 64 K data points and the standard Bruker pulse sequence (zgps). All spectra were obtained through pre-saturation pulse sequence in order to suppress the residual water sign in the solvent. Spectra were acquired through 16 scans (NS), spectral window 15.0191 mg/L with 128,000 data points, receiver gain (RG) 80.6, relation delay of 50 s (D1) and acquisition time of 10.9 s (AQ). The exponential line broadening function value adopted for spectral apodization (LB) was 0.30 Hz. The NMR data was analyzed in TopSpin Brucker Software. Quantification was performed by comparison of the compound s signals area and the area of the TMSP-d4 signal to find out the compound s mass since the TMSP-d4 mass is known. The equation used was: analyte mass = (analyte area × TMSP-d4 1 H Nº × TMSP-d4 mass × analyte MM)/(TMSP-d4 area × analyte 1 H Nº × TMSP-d4 MM). The quantification is expressed in mg/g of freeze-dried fruit. 3.7. ABTS For ABTS assay, the procedure followed the method of Thaipong et al. [55]. The stock solutions included 7.0 mM ABTS solution and 140 mM potassium persulfate solution. The working solution was then prepared by mixing the two stock solutions, 5 mL ABTS solution in 88 μL potassium persulfate solution, and allowing them to react for 16 h at room temperature in the dark. The solution was then diluted by mixing 1 mL ABTS solution with ethanol necessary to obtain an absorbance of 0.709–0.701 units at 734 nm using the spectrophotometer (Turner® 340, Haverhill, MA, USA). The fruits extracts were allowed to react with 2.5 mL of the ABTS solution for 6 min in a dark condition. The standard curve was linear between 0 and 2000 μM Trolox equivalents. Results are expressed in μM Trolox equivalents g−1 of extract. 3.8. DPPH The antioxidant activity of all fruits extracts in relation to the DPPH (2,2-diphenyl-1-picrilidrazil) radical was quantified by using a protocol described by Brand-Williams and Berset [56], using wave 515 nm in spectrophotometer (Turner® 340, Haverhill, MA, USA). The working solution was obtained by dissolving 2.4 mg DPPH reagent with 100 mL methanol and stored in the dark until needed. The extracts were allowed to react with 2.5 mL of the DPPH solution for 30 min in the dark. The standard curve was linear between 0 μM and 2000 μM Trolox. Results are expressed in μmol Trolox g−1 of extract. 17 Molecules 2020, 25, 342 3.9. FRAP The FRAP assay was done according to Thaipong et al. [55]. The stock solutions included 0.3 M acetate buffer, pH 3.6, 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution, and 20 mM ferric chloride solution. The fresh working solution was prepared by mixing 25 mL acetate buffer, 2.5 mL TPTZ solution, and 2.5 mL ferric chloride solution. Fruits extracts were allowed to react with 2.7 mL of the FRAP solution for 30 min in the dark condition and warmed at 37 ◦ C. Readings of the colored product were then taken at 595 nm in spectrophotometer (Turner® 340, Haverhill, MA, USA). The standard curve was linear between 500 μM and 2000 μM ferrous sulphate. Results are expressed in μmol ferrous sulphate g−1 of extract. 3.10. ORAC The ORAC procedure used an automated plate reader (SpectraMax i3x, Molecular Devices, USA) with 96-well plates [54–57]. Analyses were conducted in phosphate buffer pH 7.4 at 37 ◦ C. Peroxyl radical was generated using 2, 2 -azobis (2-amidino-propane) dihydrochloride which was prepared fresh for each run. Fluorescein was used as the substrate. Fluorescence conditions were as follows: excitation at 485 nm and emission at 520 nm. The standard curve was linear between 1 μM and 90 μM Trolox. Results are expressed as μM TE g−1 of extract. 3.11. Statistical Statistical comparisons were carried out by ANOVA and post hoc Tukey s test using Graph Pad Prism 5.0 and the differences were considered significant when p < 0.05 in total phenolic content and relative abundance of identified compounds. The correlation coefficients between different results of evaluation antioxidant capacity of the samples, ABTS, DPPH, FRAP, ORAC and total phenolic compounds (PC) were obtained. Principal component analysis (PCA) and heatmap was used to interpret data, using Stat graphics software. 4. Conclusions This work highlights the significantly concentration of phenolic compounds and antioxidant properties of Amazonian fruits. A total of 293 phenolic compounds were tentatively identified in mamey apple, camapu, and uxi by comparison with standards and by fragmentation patterns. Mamey apple extracts presented major number of phenolic compounds, being the phenolic acids and terpenoids the classes more identified. ABTS, DPPH, FRAP, and ORAC assays revealed that the ethanol extract presents a higher antioxidant activity and aqueous extract presents a high correlation with phenolic compounds content in Amazonian fruits. The findings of this study highlight the potential of mamey apple, camapu, and uxi as a valuable source of natural antioxidants. Supplementary Materials: The following are available online. Table S1: Bioactive compounds detected and characterized in mamey apple, camapu and uxi fruits extracted with aqueous (W) and ethanolic (E) solvents. Author Contributions: Conceptualization, L.G.B.L., A.G.F., L.C.C., M.S.L.F., and A.J.T.; methodology, L.G.B.L., J.M., J.P.d.A., M.C.B.S., T.P.d.N., M.d.S.S., A.G.F, M.S.L.F., and A.J.T. data curation, L.G.B.L., J.M., J.P.d.A., M.C.B.S., T.P.d.N., M.d.S.S., A.G.F, M.S.L.F., and A.J.T. writing—original draft preparation, L.G.B.L., J.M., M.C.B.S., T.P.d.N., M.d.S.S., M.S.L.F., and A.J.T. writing—review and editing, L.G.B.L., M.C.B.S., T.P.d.N., M.S.L.F., and A.J.T. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by FAPERJ grant number 248660, 246295, 216954 and 246296. Acknowledgments: This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Foundation for Research Support of the State of São Paulo (FAPESP). 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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/). 22 molecules Article Optimization of Scorpion Protein Extraction and Characterization of the Proteins’ Functional Properties Ahmidin Wali 1,2 , Atikan Wubulikasimu 1,2 , Sharafitdin Mirzaakhmedov 3 , Yanhua Gao 1 , Adil Omar 1,2 , Amina Arken 1,2 , Abulimiti Yili 1, * and Haji Akber Aisa 1 1 2 3 * Key Laboratory of Plant Resources and Chemistry in Arid Regions, Xinjiang Technical, Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, Xinjiang, China; ahmidin@ms.xjb.ac.cn (A.W.); obuatikam@sina.com (A.W.); gaoyh@ms.xjb.ac.cn (Y.G.); adilomar@126.com (A.O.); ak950208@163.com (A.A.); haji@ms.xjb.ac.cn (H.A.A.) University of Chinese Academy of Science, Beijing 100039, China Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent 100125, Uzbekistan; mirzaakhmedov@mail.ru Correspondence: abu@ms.xjb.ac.cn; Tel.: +86-991-3835679 Academic Editor: Jose M. Miranda Received: 26 September 2019; Accepted: 6 November 2019; Published: 13 November 2019 Abstract: Scorpion has long been used in traditional Chinese medicine, because whole scorpion body extract has anti-cancer, analgesic, anti-thrombotic blood anti-coagulation, immune modulating, anti-epileptic, and other functions. The purpose of this study was to find an efficient extraction method and investigate some of physical and chemical parameters, like water solubility, emulsification, foaming properties, and oil-holding capacity of obtained scorpion proteins. Response surface methodology (RSM) was used for the determination of optimal parameters of ultrasonic extraction (UE). Based on single factor experiments, three factors (ultrasonic power (w), liquid/solid (mL/g) ratio, and extraction time (min)) were used for the determination of scorpion proteins (SPs). The order of the effects of the three factors on the protein content and yield were ultrasonic power > extraction time > liquid/solid ratio, and the optimum conditions of extraction proteins were as follows: extraction time = 50.00 min, ultrasonic power = 400.00 w, and liquid/solid ratio = 18.00 mL/g. For the optimal conditions, the protein content of the ultrasonic extraction and yield were 78.94% and 24.80%, respectively. The solubility, emulsification and foaming properties, and water and oil holding capacity of scorpion proteins were investigated. The results of this study suggest that scorpion proteins can be considered as an important ingredient and raw material for the creation of water-soluble supramolecular complexes for drugs. Keywords: scorpion (Buthus martensii Karsch) protein; ultrasonic extraction; response surface methodology; scanning electron microscopy; functional properties 1. Introduction Scorpions are Chelicerate arthropods and members of the class Arachnida. There are about 1500 known scorpion species, and only 30 among them produce venom considered potentially dangerous to humans, which might be lethal without medical treatment [1,2]. Scorpion venoms are rich sources of complex mixtures of substances, including toxic peptides, free amino acids, enzymes, nucleotides, lipids, amines, mucoproteins, heterocyclic components, and inorganic salts, which affect the ion channels of both excitable and non-excitable cells [3,4]; some scorpion venoms also contains bioactive compounds, like trimethylamine, nucleosides, and betaine. Therefore, it has anti-thrombosis, anticoagulant, fibrinolysis, analgesic, anti-tumor, anti-epileptic, and several pharmacological effects [5–8]. A number of studies indicate that scorpion venom is comprised of more than 300 toxins, most of which are less Molecules 2019, 24, 4103; doi:10.3390/molecules24224103 23 www.mdpi.com/journal/molecules Molecules 2019, 24, 4103 than 10 kDa in molecular mass [9]. Scorpions have been used in traditional medicine in Asia and Africa for thousands of years, and large-scale commercial farming has achieved good social and economic benefits [10,11]. The body parts of scorpions are effective for the treatment of cancer [12–14]. In this work, our main attention is focused on investigating whole scorpion body extract, not directed to venomous part. With the emergence of several novel methods for the study and identification of scorpion body parts and venom components, several bioactive peptides have been proved effective to treat a variety of diseases [15–17]. The whole body part of a scorpion is used as medicinal material in traditional Chinese medicine. Although there are previous studies on the extraction, isolation, and activity of scorpion toxin [18,19], few reports are available on the extraction and functional evaluation of scorpion total protein. Scorpion bodies are a main medicinal recourse, but few studies on the bioactive peptides from scorpion proteins have been mentioned [20]. Most of the functional characteristics of proteins play a significant role in the protein’s physical and chemical properties in the processing, storage, preparation, and sale phase at different areas of economy [21,22]. Many factors, such as the protein’s size, pH, temperature, protein’s shape, ionic strength, structure, amino acid sequence, composition, and charge distribution are very important factors of protein functionality. The conditions of the proteins at extraction and each stage of purification and drying are factors that must be taken into consideration [23,24]. Therefore, the aim of this study was to investigate some of physical and chemical parameters, like water solubility, emulsification, foaming properties, and oil-holding capacity of scorpion proteins. In order to reach of this goal, response surface methodology for the optimization of three parameters of ultrasonic extraction (UE)—ultrasonic power, solid/liquid ratio, and extraction time—are also investigated. 2. Materials and Methods 2.1. Materials and Chemicals Scorpions (Buthus martensii Karsch) were collected (about 1000 pieces) in April in the Turpan region, Xinjiang, China, and after killing, they were put into plastic bottles and kept in a refrigerator. A Pierce BCA Protein Assay Kit was purchased from Thermo Scientific; electrophoresis reagents ware purchased from Biosharp Corporation (Beijing, China). All other chemicals were purchased from local suppliers. The freeze drier was an FDU-1110 (EYELA Company, Tokyo, Japan), the high-speed refrigerated centrifuge was a CR22N (Hitachi Koki Co., Ltd., Tokyo, Japan), the refrigerated centrifuge was an 5417R (Eppendorf, Germany), and the large-capacity oscillator was an HY-8A (Millipore, Burlington, MA, USA). A Spectra Max M5 enzyme labeling analyzer (Bio-Tec Co., Ltd., USA) was also used. 2.2. Total Extraction of Proteins from Scorpion Bodies Ten grams of dried scorpion were ground and separated through a 40-mesh separator. After that, separated powders (8.2 g) were subjected to de-oiling by petroleum ether (150 mL) in a continuous soxhlet extractor, until the solvent became colorless. The final residue was collected and air-dried. According to the following procedure, scorpion proteins (SPs) were extracted by (1) distilled water, (2) sodium chloride (0.5 M), (3) phosphate buffer (PBS; 50 mM, pH = 8.0), and (4) isoelectric precipitation (adjusted pH to 9.0 with 1.0 N NaOH, and adjusted pH to 4.5 with 1.0 N HCl to precipitate SPs) using an ultrasonic extraction method and a stirring extraction method (as a comparison) for 1 h, respectively. This method was used for the first time by us. Scorpion proteins were extracted with an ultrasonic generator, and the ultrasonic power was 200 w. The extraction steps were repeated three times and dialyzed against distilled water for 48 h at 4 ◦ C (F0136-1 Dialysis Membrane, MWCO 1000Da, United States). The obtained dialysates were lyophilized using a freeze drier (FDU-1110, EYELA Company, Tokyo, Japan) and kept at −20 ◦ C until use. 24 Molecules 2019, 24, 4103 2.3. Methods for the Determination of Protein Extraction Indexes 2.3.1. Determination of Protein Contents (%) The protein content of lyophilized SPs was determined by the BCA (bicinchoninic acid) method [25]. Bovine serum albumin (BSA) was used as a standard. A total of 1.0 mL of distilled water was used to dissolve 1.0 g of sample, which was clarified through centrifugation at 4500× g for 15 min at 4 ◦ C. According to the specification of the BCA protein measuring kit, the absorbance was read by an enzyme labeling instrument (Spectra Max M5 enzyme labeling analyzer) at 562 nm. The average value of three parallel measurements was used to calculate the protein content. 2.3.2. Determination of Yields (%) The yield of SPs was determined using Equation (1): mSe × 100% mSd Yield (%) = (1) where mSe is the weight of scorpion protein extraction (mg), and mSd is the weight of the defatted scorpion (mg). 2.4. Single-Factor Experiments The influence of ultrasonic power (w), extraction time (min) and liquid/solid ratio (mL/g) on protein content and yield of the UE were investigated. Based on the ultrasonic extraction and stirring extraction experiments, scorpion protein was extracted with 0.5 M NaCl buffer by the ultrasonic extraction method. The ultrasonic extraction time (20, 30, 40, 50, and 60 min), liquid/solid ratio (5, 10, 15, 20, and 25 mL/g), and ultrasonic power (50, 100, 200, 300, and 400 w) were tested. 2.5. Gel Electrophoresis Analysis Gel electrophoresis (SDS-PAGE) of the SPs was done using 15% acrylamide gel (Bio-Rad Mini-PROREAN Tetra System) [26]. Gel electrophoresis was carried out at 10 v/cm constant voltage. Coomassie Brilliant Blue R-250 dye was used for staining the protein bands for 1.5 h, which were then de-stained in a decoloring solution for 1.5 h via shaking by large capacity oscillator (HY-8A). 2.6. Box–Behnken of RSM and Statistical Analysis RSM is a statistical tool for solving multivariable problems by using reasonable experimental design method and obtaining certain data through experiments, and by analyzing regression equations to determine the optimal technological parameters [27]. In this study, the RSM and Box–Behnken design was used to optimize the extraction conditions. Based on the single-factor experiment, the complete design was made up of 17 runs, and these were done in triplicate to optimize the levels of the selected variables (extraction time, ultrasonic power, and liquid/solid ratio). For the statistical analysis, the three independent variables were coded as X1 , X2 , and X3 , respectively using Equation (2): xi = Xi − X0 ΔXi (2) where xi is the coded value of an independent variable, Xi is the real value of an independent variable, X0 is the real value of an independent variable at the center point, and Xi is the step change value. The quadratic equation of the variables is as follows: Y = β0 +  βi xi +  25 βii x2i +  βij xi x j (3) Molecules 2019, 24, 4103 where Y is the predicted response variable; B0 , βi , βii , and βij are the constant regression coefficients of the model; and xi and xj (i = 1, 3; j = 1, 3, i  j) represent the independent variables. The accuracy and fitness of the above model were evaluated by the coefficient of determination (R2 ) and the F-value. Based on the above results, the second order polynomial coefficients were undertaken using Design Expert (Version 8.0.6, USA) software. The model was performed to evaluate the analysis of variance (ANOVA). 2.7. Functional Properties The functional properties of de-oiled scorpion flour (DSF), stirring extraction (SE), and ultrasonic extraction (UE) have been tested at the optimal conditions. 2.7.1. Protein Solubility Analyses Using the method from [28], with slight modification, the solubility of the protein was determined. A total of 200 mg of SP was dissolved in 20 mL deionized water, and the pH was adjusted to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 by 1.0 mol/L HCl and 1.0 mol/L NaOH. After the pH was stabilized, it was stirred for 0.5 h at a speed of 8000 rpm/min for 20 min; centrifugation was done to obtain the supernatant. Using the Bradford method [29], the protein content of the supernatant was determined. The value of each sample was measured three times, and the average value was taken. Protein solubility was calculated using the following formula: Protein Solubility % =  m mT  × 100% (4) where m is the protein content of the supernatant (mg/g), and mT is the content of total protein in SP (mg/g). 2.7.2. Foaming Properties The foaming capacity (FC) and foaming stability (FS) were carried out using the methods described previously, with minor modifications [24,30]. Using these methods, 0.2 g of SP was dissolved in 20mL of distilled water (w/v = 1.0%), and the solution (V 0 ) was stirred for 2.0 min. The mixture was transferred immediately into a 50 mL tube, the initial foam volume was measured (V 1 ), and the foam volume after standing for 30 min was also measured (V 3 ). The FC was calculated using Equation (5):  FC (%) = V1 + V2 − V0 V0  × 100% (5) where V 2 is the volume of the liquid remaining just after stirring. FS is calculated using the following formula: FS (%) = V3 × 100%. V1 (6) 2.7.3. Water Absorption Capacity (WAC) Using the method from [31], with slight modifications, water absorption capacity (WAC) was determined. Here, 1 g (W 0 ) of SP was weighed together with the centrifugal tube (W 1 ). Thereafter, 10 mL of distilled water was added in the tube and mixed. After being held at room temperature for 30 min, the emulsion was centrifuged at 2100× g for 15 min. In the end, the supernatant was decanted, and the tube with sediment was weighed (W 2 ). Using Equation (7), the WAC was calculated: WAC = W2 − W1 . W0 26 (7) Molecules 2019, 24, 4103 2.7.4. Oil Absorption Capacity (OAC) The oil absorption capacity (OAC) was determined using the method reported previously [31], with minor modifications. One gram (F0 ) of SP was weighed together with a centrifugal tube (F1 ) and mixed with 5 mL of corn oil. The emulsion was incubated at room temperature for 30 min, and then centrifuged (Eppendorf centrifuge 4530R, Germany) at 3500 rpm/min for 15 min under the same conditions. The tube was reweighed (F2 ) after the removal of the supernatant. The OAC was determined using Equation (8): F2 − F 1 (8) OAC = F0 2.7.5. Emulsifying Properties The emulsifying activity (EA) of the UE and SE was determined by the method described by Wu et al. [32], with slight modification. Here, 1.0 g of each SP was weighed and dissolved in 20 mL of distilled water and 20 mL of corn oil, and mixed at a high speed for 1 min at room temperature. The mixture was centrifuged at 4000 rpm for 5 min. The EA was calculated using Equation (9): EA (%) = h × 100% H (9) where h is the height of the emulsified layer and H is the height of the tube contents. The emulsion stability (ES) of UE and SE were determined using the method from [30], with slight modifications. One gram of each SP was weighed and dissolved with 20 mL of distilled water and 20 mL of corn oil, and thereafter mixed at a high speed for 2 min at room temperature. The mixture was centrifuged at 4000 rpm for 5 min. After preparation, 30 mL of emulsions was then transferred into test tubes, and the emulsions were stored at room temperature; they separated into a top oil layer and a bottom serum layer over time. The ES was evaluated using Equation (10): ES (%) = Hs × 100% Ht (10) where Hs is the height of bottom serum layer (mm) and Ht is the total height of emulsion in the tube (mm). 2.8. Scanning Electron Microscopy (SEM) Analysis To study the effect of scorpion protein extraction technology on protein content and yield, we analyzed SPs by scanning electron microscopy (SEM; SUPRA 55VP, ZEISS). The morphology and surface characteristics of the SPs were observed and recorded by SEM after the samples were fixed on silicon wafers and sputtered by gold. 2.9. Statistical Analysis The experiments were performed with three independent trials, and all the determinations were triplicated. The results were represented as mean ± standard deviation. Analysis of variance (ANOVA) was performed to identify significant differences (p < 0.05). 3. Results and Discussion 3.1. Analysis of Scorpion Protein Extraction Method In this study, the effects of diverse buffer solutions for the extraction of scorpion protein were studied. Meanwhile, the effects of ultrasonic extraction (this method is used to increase the yield of protein), as well as stirring extraction of the yield (%) and protein content (%) were compared. Figure 1 and Table 1 show that the order of the effects of four buffer solutions on yield and protein 27 Molecules 2019, 24, 4103 content was 0.5 M NaCl > 20 mM PBS > 0.02 M NaOH > water. The 0.5 M NaCl buffer solution (yield 14.64 ± 0.08%, protein content 79.06 ± 0.05%) with ultrasonic was better than other buffers for extracting scorpion protein, followed by 20 mM PBS (yield 18.29 ± 0.05%, protein content 60.98 ± 0.07%). Figure 1. SDS-PAGE electrophoresis of scorpion proteins with different extractions and buffer conditions. M refers to the marker; S1, S2, S3, and S4 are scorpion proteins extracted by stirring with water, 0.5 M NaCl, 20 mM phosphate buffer (PBS), alkali extraction, and isoelectric precipitation, respectively; U1, U2, U3, and U4 are scorpion proteins extracted by ultrasonic with water, 0.5 M NaCl, 20 mM PBS, alkali extraction, and isoelectric precipitation, respectively. Table 1. Effects of ultrasonic and stirring methods on extraction of total proteins from scorpion body. Yield (%) Extraction Method Water 0.5 M NaCl 20 mM PBS 0.02 M NaOH Protein Content (%) Ultrasonic Extraction Stirring Extraction Ultrasonic Extraction Stirring Extraction 34.85 ± 0.06 14.64 ± 0.08 18.29 ± 0.05 7.70 ± 0.11 34.15 ± 0.09 11.80 ± 0.03 13.45 ± 0.10 7.57 ± 0.13 31.14 ± 0.04 79.06 ± 0.05 60.98 ± 0.07 50.87 ± 0.07 18.08 ± 0.06 35.26 ± 0.08 37.25 ± 0.09 51.91± 0.17 3.2. Analysis of a Single Factor Results 3.2.1. Effect of Extraction Time on Yield and Protein Content Extraction time plays a key role in protein extraction [32]. In this experiment, the effect of extraction time on the yield and protein content is examined. For that, ultrasound power was 200 W, the liquid/solid ratio was 15, and extraction time was carried out in a range between 20–60 min. The results obtained are shown in Figure 2A. Obtained data show that with the increase of ultrasonic extraction time, the yield and protein content rapidly increased and reached a maximum at 50 min, at 17.95% and 66.35%, respectively. A further increase of time did not affect the yield and amount of protein. Figure 2. Effects of extraction time (A), ultrasonic power (B), and liquid/solid ratio (C) on yield and protein content. 28 Molecules 2019, 24, 4103 3.2.2. Effect of Ultrasonic Power on Yield and Protein Content In this experiment, the effect of ultrasonic power on the yield and protein content was examined. For that, the extraction time was 50 min, the liquid/solid ratio (mL/g) was 15, and the ultrasonic power was evaluated in the range 50–400 w. The results obtained are shown in Figure 2B. The obtained data show that with an increase in power, the yield and protein content increased rapidly, and reached their maximums at 300 w, at 23.60% and 75.30%, respectively; in addition, a further increase of time did not affect the yield and amount of protein. 3.2.3. Effect of Liquid/Solid Ratio on Yield and Protein Content In this experiment, the effect of the liquid/solid ratio on the yield and protein content is examined. For that, ultrasound power was 200 w, extraction time was 50 min, and the liquid/solid ratio was evaluated in the range between 5–25 mL/g. The results obtained are shown in Figure 2C. The obtained data show that with the increase of liquid/solid ratio to the 15 mL/g the yield, protein content rapidly increased, and when the ratio reached to 20 mL/g, a slower increase can be seen, reaching maximums of 19.21% and 55.32%, respectively. A further increase of time did not affect the yield and amount of protein. 3.3. Optimization of Extraction Parameters by RSM Table 2 shows that there was a significant change in protein content and yield at different values of the selected parameters. Using Equation (3), the results were analyzed. After multiple regressions fitting with Design-Expert V8.0.6 software, the regression model equation is as follows: Y1 = 56.43 + 7.25A + 0.066B + 7.69C + 4.80AB + 4.97AC + 4.67BC + 2.14A2 + 1.28B2 − 3.18C2 (11) Y2 = 19.46 + 0.86A + 0.75B + 1.93C + 1.14AB + 1.80AC + 1.72BC + 0.31A2 − 1.64B2 − 0.26C2 (12) where Y1 and Y2 are the predicted conversion (%) of protein content and yield, respectively; and A, B, and C are the extraction time (X1 , min), liquid/solid ratio (X2 , mL/g), and ultrasonic power (X3 , w), respectively. When the factor sign is positive, it shows that the amount of the response variable increases with an increase in its value, and vice versa. Figure 3A,C can also be used to compare the model predicted and the experimental results. Figure 3B,D is the normal probability chart indicating that the points follow a narrow linear pattern. The analysis of variance of the regression equation is shown in Table 3. F is the value of the regression model, where the protein content and yield were 17.49 and 34.12 and the p-values were 0.0005 and <0.0001, respectively. These values show that the model obtained was significant. The F-value and p-value for the lack of fit of the protein content and yield were 1.54 and 2.04, respectively, and 0.3354 and 0.2506, respectively, which shows that the equation has a good fitting degree and high reliability. In addition, the decision coefficients (R2 ) of the model were 0.9574 and 0.9777, respectively, and the adjusted coefficients of determination (Adj-R2 ) were 0.9027 and 0.9491, respectively. The values of R2 and adjusted R2 for the models are shown in Table 3, which shows that the regression equation can predict the result of extracting scorpion protein accurately. 29 Molecules 2019, 24, 4103 Figure 3. Standard statistical diagrams for model verification. (A,C) Model-predicted values versus actual data. This figure shows a comparison between the model-predicted values and the actual values that have been obtained by the experiments. (B,D) Normal probability plot of the residuals. Monotonous distribution and linearity of the data, which are obvious from these figures, confirm the validity of the model and its capability to predict the results. Table 2. RSM experimental design and results for the three-factor/three-level Box–Behnken design (BBD). Runs Extraction Time (min) X 1 Liquid/Solid Ratio (mL/g) X 2 Ultrasonic Power (W) X 3 Protein Content/% Yield /% 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 60 40 20 40 40 60 40 40 60 40 40 60 40 20 20 20 40 15 20 10 15 15 10 20 10 15 10 15 20 15 20 15 15 15 400 400 300 300 300 300 200 200 200 400 300 300 300 300 200 400 300 75.23 69.01 59.31 55.98 56.39 64.09 44.01 49.4 50.17 55.70 55.70 70.00 53.58 46.01 45.49 50.66 60.52 24.32 21.41 17.76 19.53 18.89 16.62 14.84 16.25 17.04 17.74 19.31 20.78 19.52 17.37 18.31 18.39 20.04 30 Molecules 2019, 24, 4103 Table 3. Least-squares fit and parameter estimates (significance of regression coefficient). Source Model X1 X2 X3 X1 × 2 X1 × 3 X2 × 3 X1 2 X2 2 X3 2 Lack of Fit R2 Adj-R2 Protein Content Yield F-Value p-Value F-Value p-Value 17.49 53.50 4.46 60.17 11.73 12.57 11.11 2.45 0.88 5.43 1.54 0.9574 0.9027 0.0005 0.0002 0.9486 0.0001 0.0111 0.0094 0.0125 0.1617 0.3800 0.0526 0.3354 34.12 23.98 18.15 118.71 20.67 51.76 25.77 1.67 45.25 1.12 2.04 0.9777 0.9491 < 0.0001 0.0018 0.0037 < 0.0001 0.0026 0.0002 0.0014 0.2378 0.0003 0.3256 0.2506 3.3.1. Analysis of the Influence of Various Factors on the Extraction of Scorpion Proteins The degree of influence of each factor on the test index can be compared by the F-value (Table 4): F (X1 ) = 53.50, 23.98; F (X2 ) = 4.46, 18.15; F (X3 ) = 60.17, 118.71. These values indicate that the order of influence of each factor on the extraction of scorpion protein was ultrasonic power (w) > extraction time (min) > liquid/solid ratio (mL/g). The response surface can describe the interaction between variables and predict the optimal conditional values of each factor [33]. Figure 4A–F shows the effect of operational variables on protein content and yield protein content and yield. The effect of extraction time on the protein process is greater than that of the ratio of the liquid/solid, and that of ultrasonic power is greater than that of extraction time. According to the above analysis, the order of the influence of each variable on the extraction technology of scorpion protein was ultrasonic power > extraction time > liquid/solid ratio. 3.3.2. Interactions of Variables Contour plots for each of the fitted models that display the effects of the three variables (to visualize the combined effects of the three factors on protein content and yield) were generated. Figure 4 illustrates the two-dimensional (2D) plots of the binary interactions of the variables on protein content and yield via the contour plots. The interaction between the extraction time and the liquid/solid ratio is shown in Figure 4A,D. This plot indicates that protein content and yield depend more on X1 than on X2. Figure 4A,D reveals that at low values of X1, maximum protein content and yield occurs at higher values of X2. However, at higher values of X1, maximum protein content and yield occurs at lower values of X2. As seen in Figure 4A,D, the interaction between the two factors is weak. The interaction between the extraction time and ultrasonic power is shown in Figure 4B,E. This plot indicates that protein content and yield depend more on X3 than on X1. In Figure 4C,F, the effect of the interaction between the liquid/solid ratio and ultrasonic power is depicted. Figure 4C,F shows that increasing the liquid/solid ratio at different ultrasonic power levels has no important effect on the protein content and yield, so the plot shows that protein content and yield depends more on X3 than on X2. Therefore, the optimum values of X1, X2, and X3, as determined by the software, are 50 min, 400 w, and 18 mL/g, respectively. 31 Molecules 2019, 24, 4103 Figure 4. Effects of operational variables on protein content and yield. Interaction between (A) liquid/ solid ratio and extraction time, (B) ultrasonic power and extraction time, and (C) ultrasonic power and liquid/solid ratio to protein content. Also depicted are the interaction between the (D) liquid/solid ratio and extraction time, (E) ultrasonic power and extraction time, and (F) ultrasonic power and liquid/solid ratio to yield. 3.3.3. Determination and Validation of Optimal Extraction Conditions Optimum conditions, which were obtained by Design-Expert V8.0.6 software, were as follows: extraction time = 47.68 min; ultrasonic power = 395.84 w; and solid/liquid ratios = 18.01 mL/g. In view of the feasibility of the experiment, the optimum conditions of extraction proteins were adjusted as follows: extraction time = 50.00 min, ultrasonic power = 400 w, and solid/liquid ratio = 18.00 mL/g. 32 Molecules 2019, 24, 4103 After several tests (n > 3), the protein content and yield were 78.94%, and 24.80%, respectively. It is shown that the regression equation and the optimal conditions obtained by the response surface method are reliable. 3.4. Functional Properties of Scorpion Proteins 3.4.1. Protein Solubility (PS) The protein solubility (PS) profiles of de-oiled scorpion flour (DSF), ultrasonic extraction (UE), and stirring extraction (SE) in the water at different pH ranges (2–12) are shown in Figure 5. The PS of DSF, UE, and SE were significantly different, and Figure 5 shows the same U-shaped curves. When the pH value is in the range of 2–4, the solubility of DSF, UE, and SE decreased; however, when in the pH range of 6–10, the solubility of DSF, UE, and SE significantly increased. The minimum protein solubility of DSF, UE, and SE were presented at pH 4, with values of 8.05%, 15.25%, and 18.75%, respectively. The maximum protein solubility was presented at pH 12, with values of 13.5%, 70.15%, and 79.5%, respectively. Therefore, results indicate that scorpion protein extracted by ultrasonic method shows exceptional solubility at an alkaline pH. Figure 5. Protein solubility profiles of de-oiled scorpion flour (DSF), stirring extraction (SE), and ultrasonic extraction (UE) at different pH levels. 3.4.2. Water and Oil Absorption Capacity The obtained results for the water absorption capacity (WAC) and oil absorption capacity (OAC) of proteins extracted by ultrasonic and stirring are presented in Table 4. The WAC and OAC from both methods were significantly different (p < 0.05), with UE having the higher WAC (40.3 ± 0.28 g/g) and OAC (27.70 ± 0.14 g/g) than SE (with WAC = 33.45 ± 0.07 g/g and OAC = 18.80 ± 0.15 g/g) at pH 7.0. 33 Molecules 2019, 24, 4103 Table 4. Functional properties of scorpion proteins. Properties Ultrasonic Extraction Stirring Extraction Water holding capacity (g/g) Oil holding capacity (g/g) Emulsifying activity (%) Emulsion stability (%) Foam capacity (%) Foam stability (%) 25.25 ± 0.21 38.50 ± 0.14 45.55 ± 0.64 85.50 ± 0.28 40.30 ± 0.28 21.70 ± 0.14 20.45 ± 0.07 30.65 ± 0.64 40.25 ± 0.07 69.45 ± 0.35 33.45 ± 0.07 18.80 ± 0.15 3.4.3. Emulsifying Properties Charged or non-charged polar amino acids in proteins can affect the proteins’ emulsifying and surfactant properties. Results of emulsifying properties of UE and SE are shown in Table 4. At pH 7.0, the emulsifying properties of UE and SE were significantly different from one another, with values of 45.55 ± 0.64% and 40.25 ± 0.07%, respectively. However, the emulsifying property of UE was significantly different and was higher (85.50 ± 0.28%) than that of SE (69.45 ± 0.35%). The UE had pronounced effects on the emulsifying properties, since it might be exposing more hydrophobic groups to the water and oil interface, giving rise to increased emulsifying capacity and more stable emulsion. 3.4.4. Foaming Properties Foam formation is similar to emulsion formation. In foam formation, water molecules surround air droplets, which is a nonpolar phase. At pH 7, the foam capacity (FC) of UE was considerably higher than that of SE, with values of 40.30 ± 0.28% and 33.45 ± 0.07%, respectively. The foam stability (FS) of UE was significantly higher than that of SE, with values of 21.70 ± 0.14% and 18.80 ± 0.15%, respectively (Table 4). The results obtained show that the UE, compared to SE, has more flexible protein structure in aqueous solution, and forms more stable foams for stronger interaction on the air–water interface. The variations in foaming properties of UE and SE might be explained with the difference in their protein concentrations. A high protein concentration will improve foam capacity and stability, increasing the viscosity and promoting the formation of the interfacial multilayer membrane [34]. 3.5. Scanning Electron Microscopy Analysis Protein samples obtained using various extraction methods were investigated by the SEM method. Figure 6 shows the surface states of protein samples extracted from whole-body, de-oiled protein (DSF) powder, stirred extraction (SE), and ultrasonic extraction (UE). These studies focused on the study of the surface layer of protein molecules. Accordingly, in Figure 6A, adhesive surface layers of protein molecules in dried DSF can be seen. This state has a collapsed form, which shows the density of protein molecules in relation to one other; therefore, these molecules have a higher crystalline degree than other states, and this will result in lower water solubility. In Figure 6B, on the surface of dried SE protein layers, a plurality of elements similar to “wood shavings” can be seen, in contrast to Figure 6A. These elements formed as a result of separation from the collapsed surface. This means that during drying after SE, from the crystalline state shown in Figure 6A, the protein molecules transition from a crystalline to amorphous state, as shown in Figure 6B; the outcome of this results in an increase in the solubility of the protein molecules in water. Also, in Figure 6C, you can see the results of the drying of protein molecules obtained after UE. This picture clearly shows the increase the number of “wood shavings” on the surface, compared to Figure 6B, and how the transformation collapse forms into thin film pieces. Therefore, UE leads to the formation of scorpion protein molecules from a crystalline state to an amorphous one, which results in an increase of their water solubility. 34 Molecules 2019, 24, 4103 Figure 6. Scanning electron microscope images of (A) DSF, (B) SE, and (C) UE. (1.00 KX magnifications, bar 10 μm). 4. Conclusions Studies have shown that the sum of proteins extracted from scorpion bodies had good emulsifying and foaming properties. At the same time, when they were subjected to extraction under different conditions, UE was found to be the best way to obtain the sum of proteins with improved water solubility. According to SEM analysis, the improvement of water solubility of the sum of protein components is accompanied by the transition of these macromolecules from the collapsed and crystalline form to a more amorphous and friable phase. For the extraction of scorpion body parts, some simple ways, i.e., cold water, hot water, and ethanol extraction methods used previously [35,36] were used, and SPs obtained by these methods were physiologically effective for the treatment of various diseases. However, we think that using hot water or ethanol solvents may generally result in the denaturation of native compounds and reduce the real activities of the extracted SPs. Therefore, using UE in an alternative way, at the optimal conditions of UE (i.e., X1 , X2 , and X3 are 50 min, 400 w, and 18 mL/g, respectively) resulted in increased water solubility and improvement of its emulsifying and foaming properties. In our opinion, the SPs obtained by the UE way should keep a number of their above-mentioned activities, and will be planned for use in creating medicines on that basis in TCM. On the other hand, UE could be the basis for the creation of water-soluble supramolecular complexes for those drugs that are poorly soluble in water. Obtaining such drugs in this way will contribute in the 35 Molecules 2019, 24, 4103 future to (1) an increase the drugs’ water solubility, (2) a reduction their therapeutic doses, (c) increase pharmacological action, and (d) reduce toxicity and side effects. Since the sum of proteins from the scorpion body are not individual components, it might be promising, if created in their base gel drug forms, that they could be used through the skin. Author Contributions: Project administration, A.Y., H.A.A.; Investigation, A.W.(Ahmidin Wali), A.A. and A.O.; Methodology, A.W.(Atikan Wubulikasimu) and Y.G.; Formal analysis, S.M. 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Liu, T.H.; Zhang, C.X.; Zhuang, F.J.; Xu, X.P.; Chi, Y.S. A Review on the Chemical Components and the Edible and Medicinal Value of Scorpion. Hans. J. Food Nutr. Sci. 2017, 6, 165–174. [CrossRef] Wu, F.L.; Dong, Q.H.; Wang, H.; Tan, J.; Lin, H.Q.; Li, P.Y.; Li, Y.J. Research Progress of Chinese Scorpion. J. Liaoning Univ. TCM 2018, 20, 108–111. Sample Availability: Samples of the compounds are available from the authors. © 2019 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/). 38 molecules Article Influence of Potato Crisps Processing Parameters on Acrylamide Formation and Bioaccesibility Emmanuel Martinez 1 , Jose A. Rodriguez 1 , Alicia C. Mondragon 2 , Jose Manuel Lorenzo 3 and Eva M. Santos 1, * 1 2 3 * Area Academica de Quimica, Universidad Autonoma del Estado de Hidalgo, Carr. Pachuca-Tulancingo Km. 4.5, Mineral de la Reforma, 42184 Hidalgo, Mexico; qaemmanuel@gmail.com (E.M.); josear@uaeh.edu.mx (J.A.R.) Laboratorio de Higiene, Inspeccion y Control de Alimentos, Departamento de Quimica Analitica, Nutricion y Bromatologia, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain; alicia.mondragon@deinal.es Meat Technology Centre of Galicia, Rúa Galicia Nº 4, Parque Tecnológico de Galicia, San Cibrao das Viñas, 32900 Ourense, Spain; jmlorenzo@ceteca.net Correspondence: emsantos@uaeh.edu.mx Academic Editor: Jose M. Miranda Received: 24 September 2019; Accepted: 21 October 2019; Published: 23 October 2019 Abstract: A fractional factorial design was used to evaluate the effects of temperature, frying time, blanching treatment and the thickness of potato slices on acrylamide content in crisps. The design was used on freshly harvested and four-month stored potatoes. The critical factors found were temperature and frying time, and the interaction between blanching treatment and slice thickness. Once frying conditions were selected, an acrylamide content of 725 and 1030 mg kg−1 was found for non-stored and 4-month stored tubers, with adequate textural parameters in both cases. The difference in concentration is related to storage conditions, which must be controlled in order to control acrylamide levels. Bioaccesibility studies demonstrated that acrylamide concentration remained at 70%, and reductions took place mainly at the intestinal phase, as a result of reaction with nucleophilic compounds. Keywords: acrylamide; crisps; temperature; frying time; blanching; thickness 1. Introduction Acrylamide, a thermal processing contaminant with a low molecular weight, which is soluble in water, is formed when carbohydrate-rich foods are subjected to temperatures above 120 ◦ C in low-moisture conditions, such as frying, roasting or baking. Several studies consider Maillard reactions to be the main pathway for acrylamide, 5 hydroxymethylfurfural, methylglyoxal–lysine dimers, Nε–carboxymethyl–lysine and pyrraline formation in processed foods, particularly reactions between the carbonyl group of reducing sugars and amino acids [1,2]. Initial reports showed relatively high concentrations of acrylamide in high-carbohydrate foodstuffs, such as crispy bread, breakfast cereals, pastries, coffee, French fries and crisps. In general, potato products present higher acrylamide contents (250–4000 μg kg−1 ) compared to other food products, because of the higher concentration of asparagine in potato tubers [3,4]. Acrylamide has been classified by the Agency for Research on Cancer as “probably carcinogenic to humans” (Group 2A) and, in the latest report from the European Food Safety Authority Expert Panel on Contaminants in the Food Chain (CONTAM, 2015), the margins of exposure for acrylamide lead to concerns regarding their neoplastic effects, based on animal evidence [5]. Therefore, the European Commission recently established “indicative” levels for the presence of acrylamide in food, suggesting Molecules 2019, 24, 3827; doi:10.3390/molecules24213827 39 www.mdpi.com/journal/molecules Molecules 2019, 24, 3827 a limit of 750 μg kg−1 for crisps [6]. Previously, the European Union and FDA had encouraged food industries to reduce the presence of this contaminant. In response to this, the food industry has been forced to apply different strategies to reduce acrylamide formation, applying processing modifications according to recommendations published by the European Food and Drink Federation in the document “Acrylamide Toolbox”, which includes the following variables: temperature and time during frying, blanching treatment, and thickness of potato slices [7]. However, the main challenge for the food industries is in producing potato chips with low acrylamide levels without affecting their sensory properties [8]. Although it is important to evaluate the effect of pre-treatments on the frying process on acrylamide mitigation, it is also necessary to determine their influence on its bioavailability. Potato chips are an important part of the snack food market in many countries, and few studies have been published about the bioavailability of acrylamide after crisps have been through the digestion process [8,9]. Acrylamide intake in humans occurs mainly via food ingestion, where the total amount of this compound does not necessarily reflect the available amount to the body. There are chemical changes in the food when it enters the digestive tract, because of pH variations, and the action of several enzymes in every stage of the digestion process [10–12]. In this sense, bioaccessibility is used to evaluate the amount of a chemical compound available for absorption after it is released from the food matrix into the gastrointestinal tract. Several methodologies can be used to assess the bioaccessibility of contaminants or nutrients: 1) in vitro, 2) ex vivo, 3) in situ and 4) in vivo models [13,14]. Among the techniques mentioned before, in vitro models are more commonly used than in vivo models, because of their simplicity, lower cost, lack of ethical issues and good reproducibility under controlled conditions. The present work evaluated some acrylamide mitigation strategies, employing a fractional factorial design experiment, and their influence on textural properties and bioaccesibility, by an in vitro simulated digestion assay. 2. Results and Discussion 2.1. Acrylamide Content Potatoes of the Atlantic variety were selected for crisp production, because of their low levels of glucose and fructose compared to other varieties, such as the Ranger Russet [15]. According to the storage period of potato tubers, two sampling points were considered: freshly harvested potatoes (summer of 2018) and four-month stored potatoes. The main variables described for acrylamide reduction in potato crisps are [7]: temperature control and time of frying process, blanching, and the thickness of slices. These variables were selected because they are easily applicable in the industry and do not represent extra costs in production. A fractional factorial design (24−1 ) was proposed, to evaluate the influence of frying process parameters in the total amount of acrylamide formed and its bioaccessibility. The levels selected for the variables were: temperature (T) of 150 and 180 ◦ C; frying time (t) of 5 and 10 min; blanching treatment (B)—no blanching treatment and 70 ◦ C for 5 min—and thickness of slice (w), at 1.5 and 2.0 mm. In this sense, eight experiments (in duplicate, a total of 16 experiments) were performed. Table 1 shows the matrix used and the acrylamide content for the experimental design. According to the obtained results, the concentration of acrylamide was higher in stored potatoes. This can be explained by the difference between the content of reducing sugars between samples, since a concentration of 23 mg kg−1 of reducing sugars was found in freshly harvested potatoes, while, in samples stored for 4 months, a concentration of 34 mg kg−1 was found. During storage, a phenomenon called cold sweetening occurs, consisting of a degradation of the potato starch caused by the low temperatures [16]. Even if appropriate storage conditions of potato tubers are followed, a slight increase of reducing sugars is expected, and will affect the acrylamide concentration [17]. 40 Molecules 2019, 24, 3827 Table 1. Matrix of the experimental fractional factorial design and the average of obtained responses. Exp T (◦ C) t (min) B (70 ◦ C, min) w (mm) 1 2 3 4 5 6 7 8 150(−1) 150(−1) 150(−1) 150(−1) 180(+1) 180(+1) 180(+1) 180(+1) 5(−1) 10(+1) 10(+1) 5(−1) 10(+1) 5(−1) 5(−1) 10(+1) 0(−1) 0(−1) 5(+1) 5(+1) 0(−1) 0(−1) 5(+1) 5(+1) 1.5(−1) 2.0(+1) 1.5(−1) 2.0(+1) 1.5(−1) 2.0(+1) 1.5(−1) 2.0(+1) Acrylamide Content (μg kg−1 ) ± sd Non-stored 4-month stored 973 ± 71 752 ± 63 825 ± 67 885 ± 62 2590 ± 243 795 ± 92 1138 ± 220 2250 ± 204 1257 ± 171 890 ± 52 1483 ± 58 1097 ± 205 4386 ± 270 1149 ± 82 1812 ± 70 3540 ± 254 A comparison of each variance with respect to the variance of the residual allows the determination of the the experimental F value (Fexp ), which is then compared with the Fcritical value (5.32, p = 0.05, 1,8). The F-test indicated that, in both cases (non-stored and stored tubers), the critical factors for acrylamide formation were (p = 0.05): temperature (T) and frying time (t). The contribution of potato thickness (w) was also observed for stored samples (Table 2). Table 2. Analysis of variance of the results obtained from the fractional factorial design, showing the factors significantly affecting acrylamide concentration. Factor T t B w Residual Non-Stored 4-month Stored Effect Variance Fexp Effect Variance Fexp 834.5 656.5 −3 −211 2,785,561 1,723,969 36 178,084 43,718 63.72 39.43 8.2 × 10−4 4.07 1540 1246 62.5 −565.5 9,486,400 6,210,064 15,625 1,279,161 56,593.5 216.99 142.05 0.36 29.26 Figure 1 shows the mean effect graph for the effect of evaluated control factors on acrylamide concentration. Independently of storage time, the mean effect profile is similar in both cases, demonstrating that the increase in acrylamide concentration is mainly a consequence of the increment of reducing sugars concentration, rather than the processing variables. Bertuzzi et al. also reported a strong influence of reducing sugar concentration on acrylamide formation in processed potato products [18]. A percentage of reducing sugars between 0.15%–0.20% has been suggested as an indicator of the suitability of potatoes for processing [19]. Despite the lower reducing sugar content found in potatoes in this study, acrylamide formation reached values over the 750 μg kg−1 limit established by the European Commission [6]. The results obtained are congruent with others previously described, in which an increment in acrylamide content in potatoes, in relation to temperature and frying time, was observed [18,20]. In general, the increase in acrylamide content follows a linear function over time, while the relationship with frying temperature is not linear, although temperatures over 175 ◦ C significantly increased acrylamide levels [1,18,20–22]. Any temperature–time combination proposed to mitigate acrylamide content should not affect the overall quality of the fried product, especially its texture. Although the other processing parameters (blanching and thickness) had a smaller contribution, their interaction was also significant (Figure 2). As acrylamide is formed on the potato surface, the size:volume ratio of the potato slice influences the acrylamide content. In general, thinner and smaller cut sizes result in increased acrylamide formation [22]. Also, the blanching of potato slides prior to the frying process has been recommended to reduce the acrylamide content, since this helps remove reducing sugars on the surface [8,23]. In this study, a blanching process was required for thinner potatoes, to decrease acrylamide formation. However, a lower concentration of acrylamide 41 Molecules 2019, 24, 3827 was observed in thicker potatoes without the blanching treatment. It seems that, in thicker slices, heat diffusion is slower when the slice has not been blanched and, consequently, a decrement of acrylamide content can be observed. So, the blanching treatment should not be suitable for potato slices with a thickness over 2.00 mm. This result is interesting for the industry because the application of blanching treatments at high temperatures and longer times represents an additional difficulty, since most industrial frying processes are continuous, and blanching could be unnecessary for certain types of crisps. 3000 Acrilamide concentration (μg kg-1) Non-stored 4-month stored 2500 2000 1500 1000 500 T(-1) T (+1) t (-1) t (+1) B(-1) B(+1) w(-1) w(+1) Factors and Levels Figure 1. Mean effect graph vs. acrylamide concentration (μg kg−1 ) of the experimental design for non-stored and 4-month stored potato tubers storage. Acrylamide concentration (μg kg-1) 3000 2500 w(+) 2000 w(+) 1500 w(-) 1000 w(-) 500 Non-stored 4-month stored 0 B(-1) B(+1) B(-1) B(+1) Factor and level Figure 2. Mean effect graph vs. acrylamide concentration (μg kg−1 ) for the interaction between blanching (B) and thickness (w) in non-stored and 4-month stored tubers. 42 Molecules 2019, 24, 3827 The combination of settings that generated the minimum acrylamide concentration was: temperature, 150 ◦ C (−1); time, 5 min (−1); no blanching treatment (−1); and a slice thickness of 2.0 mm (+1). Acrylamide content of 725 and 1030 mg kg−1 was determined at these conditions for non-stored and 4-month stored tubers, respectively. 2.2. Texture Analysis and Bioaccesibility Potato chips are consumed as indulgent foods, with a characteristic flavor and crispiness, the latter evaluated through textural parameters. In order to evaluate it, crisps were prepared according to the processing parameters detailed above. Figure 3 shows a representative profile of the force (N) vs. probe displacement (mm). The force displacement curves have a jagged appearance, with several fracture events, which are typical of crispy food [24]. These graphics have two well-differentiated regions, the first one starting from the first contact between the potato crisp and the probe, until the major force is achieved (associated with major structural breakdown and hardness). The second region starts from the major structural breakdown and continues until the end, where smaller force events take place. The parameters evaluated were the maximum force applied (N), related to hardness or firmness, and the gradient (N s−1 ), which is related to stiffness. 9.0 Non-stored 8.0 4-month stored 7.0 Force (N) 6.0 5.0 4.0 3.0 2.0 1.0 0.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Displacement (mm) Figure 3. Force displacement curves generated from the analysis of potato crisps obtained from frying non-stored and 4-month stored tubers. Frying conditions: temperature, 150 ◦ C; time, 5 min; no blanching treatment; and a slice thickness of 2.0 mm. The maximum force applied was 6.1 and 8.5 N, for non-stored and 4-month stored samples, respectively. Genovese et al. have reported values below 6 N for firmness [25]. The storage of the tubers promoted an increase in firmness. On the other hand, samples obtained from frying the non-stored potato slices presented the highest gradient (8.9 N s−1 ) compared to 4-month stored samples (7.2 N s−1 ), making them, therefore, the stiffest. Additionally, the number of total force peaks in each case can be evaluated: seven for non-stored tubers and 13 for 4-month stored tubers. Taking into account that values higher than six are considered high-sensory crispiness, both samples can be accepted as adequate snacks [26]. 43 Molecules 2019, 24, 3827 Once the effect of the control factors on acrylamide formation had been evaluated, it was important to know their effect on bioaccesibility. Values of 79.1% ± 1.5% and 76.9% ± 1.5% of bioaccesibility were obtained for the snacks using potatoes that were freshly harvested and stored for 4 months before frying, respectively. The results obtained in this study are in agreement with the predicted behavior of potato [10] and bakery products [12]. The main effect was observed at the end of the intestinal phase of the digestion process, and could be explained by the formation of Michael adducts, in which the acrylamide would be involved. During the gastric phase of the digestion process, pepsin hydrolyzes the proteins present in the potato, generating small peptide chains and some amino acid residues, such as cysteine and lysine, that have a nucleophilic character (-SH and -NH2 ). These peptides are able to interact with acrylamide and form adducts, which consequently causes an apparent reduction in acrylamide during the intestinal phase [12]. Taking into account the mentioned results, this can be proposed as an alternative method of reducing acrylamide concentration in the digestion process, through the formation of adducts with thiol compounds (i.e., glutathione) contained in other foods, such as spinach, avocados or asparagus [27,28] 3. Materials and Methods 3.1. Reagents Acrylamide, 2-naphthalenethiol, and the following enzymes: pepsin (≥250 U mg−1 solid), from porcine gastric mucosa, pancreatin (4 × USP), from porcine pancreas, α-amylase, from human saliva (500 U) and porcine bile salts, were purchased from Sigma–Aldrich (St. Louis, MO, USA). Potassium chloride, sodium bicarbonate and sodium chloride were purchased from MEYER (Mexico City, Mexico). Acetonitrile (HPLC grade) and acetic acid were obtained from J.T. Baker (Philisburg, NJ, USA). 3.2. Samples and Frying Conditions Potato (Atlantic variety) and vegetable oil (palm olein) were the raw materials used, provided by Fritos Totis (Tizayuca, Hidalgo, Mexico). Samples were collected in two periods, the first with potatos freshly harvested and the second after four months of storage, at 8 ◦ C and 95% relative humidity. Slices (with a thickness of 1.5 and 2.0 mm, and a diameter of around 35 mm) were cut using a slicing machine. Slices were rinsed immediately after cutting for 1 min in deionized water (resistivity of 18.2 MΩ cm), to eliminate some starch material adhered onto the surface, before frying. Forty grams of slides were deep-fried in an electrical fryer Blazer FE-3 (Mexico city, Mexico) 1600 W, 127 V and equipped with a bowl with 3 L of capacity, a static basket and a regulating thermometer. The main variables evaluated for acrylamide reduction in potato crisps were [6]: control of temperature and time of frying process, blanching, and slice thickness. These strategies were selected because they are easily applicable to the industry and do not represent extra costs in production. A fractional factorial design (24−1 ) was proposed, to evaluate the influence of frying process parameters in the total amount of acrylamide formed and its bioaccessibility. The factors selected were temperature (T), frying time (t), blanching treatment (B), and thickness of potato slices (w). Results obtained from the fractional factorial design were subjected to an analysis of variance study. The mean effect of each factor on acrylamide content and the variance was estimated through the Yates algorithm [29]. Experimental results were analyzed using MINITAB® version 17 software (Minitab Inc., State College, PA, USA). 3.3. Acrylamide Determination Acrylamide determination was carried out by chemical derivatization, using 2-naphthalenethiol as a derivatization reagent, followed by HPLC separation and quantification with fluorescence detection [30]. In brief, 3 g of potato crisps were homogenized with 30 mL of deionized water and then defatted with hexane. Then, the aqueous phase was centrifuged and filtered using a cellulose membrane (pore size, 0.45 μm). Afterwards, an aliquot of 7.5 mL was mixed with 1 mL of the solution 44 Molecules 2019, 24, 3827 2-naphthalenethiol (62 mM), in a solution of sodium hydroxide (0.1 M). The mixture was heated at 90 ◦ C for 45 min. The reaction was stopped with 1.5 mL of acetic acid (1%) and the solution was centrifuged at 700 g for 15 min. Acrylamide concentration of samples was calculated by standard additions, by spiking each sample with known concentrations of acrylamide. Chromatography separation was performed using an Agilent Technologies 1260 Infinity chromatography system (Agilent Technologies, Waldbronn, Germany). Samples were manually injected using a 20-μL loop, and a C-8 ZORBAX eclipse XDB column (5 μm; 150 × 4.6 mm internal diameter) from Agilent Technologies was used as the stationary phase. The mobile phase was acetic acid (1.0% v/v) and acetonitrile in a 50:50 ratio (v/v). The flow rate was 0.8 mL min−1 . 3.4. Texture Evaluation A TA.XTPlus Texture Analyser (Stable Micro Systems Ltd., Surrey, UK), equipped with a 5 kg load cell, was used for force/displacement measurements, with a stainless spherical probe (P/0.25S) of a 0.25 inch diameter. The samples were placed on a crisp fracture rig (HDP/CFS). The test settings were: test speed of 1 mm s−1 , trigger force of 0.406 N, travel distance of 3 mm. 3.5. In Vitro Digestion Bioaccessibility was estimated using an in vitro digestion model (oral, gastric and intestinal stages) and the simulated fluids—salivary (SFS), gastric (SFG) and intestinal (SFI)—were prepared according to an internationally agreed protocol (Table 3) [31]. Samples of potato crisps (5 g) were transferred to a conical tube and mixed with 5 mL SFS (containing 75 U α-amylase mL−1 ) for 3 min. After that, 5 mL of a pepsin solution (12.5 mg mL−1 in 0.1 M HCl) and 10 mL of SFG were added. The mixture was adjusted to pH 2.0 and incubated in a shaking water bath at 37 ◦ C for 2 h, with an agitation speed of 60 strokes per min. After incubation, the pH was adjusted to 7.5, and then bile salts (10 mg mL−1 ), dissolved in 20 mL of SFI together with a pancreatin solution (5 mL, 10 mg mL−1 in water), were added to the tube. The mixture was incubated at 37 ◦ C for 2 h, with shaking at an agitation speed of 60 strokes per min, to simulate the duodenal phase. Digestion products were placed in a dialysis bag (6–8000 molecular weight cut off; Sigma Aldrich) and dialyzed in 250 mL of sodium bicarbonate solution (pH = 7.5) for 12 h. Dialysis aliquots were removed and acrylamide dialyzed was determined by the method described above. Bioaccesibility was reported as the percentage of acrylamide in the dialyzed sample compared to the value in the original sample. Table 3. Composition of simulated salivary, gastric and intestinal electrolytes fluids. Compound KCl KH2 PO4 NaHCO3 NaCl MgCl2 ·6H2 O (NH4 )2 CO3 CaCl2 ·2H2 O Simulated Salivary Fluid (SSF) mmol L−1 Simulated Gastric Fluid (SGF) mmol L−1 Simulated Intestinal Fluid (SIF) mmol L−1 15.10 3.70 13.60 0.15 0.06 1.50 6.90 0.90 25.00 47.20 0.10 0.50 0.15 6.80 0.80 85.00 38.40 0.33 0.60 4. Conclusions The evaluation of potato crisps’ processing parameters was performed by a chemometric approach, through fractional factorial design. The factors with a higher contribution were temperature and frying time, and an increment on the acrylamide, with respect to the store time of raw material, was also observed. Crisps obtained in the most suitable conditions have adequate texture parameters, while bioaccesibility takes place mainly at the intestinal phase, through reaction with –NH2 and 45 Molecules 2019, 24, 3827 –SH containing compounds. 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Food Function 2014, 5, 1113–1124. [CrossRef] Sample Availability: Samples of the compounds are not available from the authors. © 2019 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/). 47 molecules Article Determination of N-Carbamylglutamate in Feeds and Animal Products by High Performance Liquid Chromatography Tandem Mass Spectrometry Yonghang Ma, Zhengcheng Zeng, Lingchang Kong, Yuanxin Chen and Pingli He * State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China * Correspondence: hepingli@cau.edu.cn; Tel.: +86-10-62733688 Academic Editor: Jose M. Miranda Received: 18 July 2019; Accepted: 30 August 2019; Published: 31 August 2019 Abstract: N-carbamylglutamate (NCG), a synthetic analogue of N-acetylglutamate, is an activator of blood ammonia conversion and endogenous arginine synthesis. Here, we established an accurate quantitative determination of NCG in feeds, animal tissues, and body fluids using the high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The sample pretreatment procedures included extraction with 0.5% of formic acid in water/methanol (80/20, v/v), and purification using an anionic solid phase extraction cartridge. Satisfactory separation of NCG was achieved in 20 min with the application of an Atlantis T3 column, and a confirmative detection of NCG was ensured by multiple reaction monitoring of positive ions. NCG spiked in feeds, tissues, and body fluids were evaluated in regard to linearity, sensitivity, recovery, and repeatability. Recoveries for different sample matrices were in the range of 88.12% to 110.21% with relative standard deviations (RSDs) less than 8.8%. Limits of quantification were within the range of 0.012 to 0.073 mg kg−1 and 0.047 to 0.077 μg mL−1 for solid and liquid samples, respectively. This study will provide a solid foundation for the evaluation of availability and metabolic mechanism of NCG in animals. Keywords: N-carbamylglutamate; feeds; animal products; milk; HPLC-MS/MS 1. Introduction N-carbamylglutamate (NCG) is a synthetic analogue of N-acetylglutamate (NAG) (Figure 1), which is the allosteric stimulator of carbamyl phosphate synthase-1 (CPS1). CPS1 is a key enzyme functioning in the urea cycle and endogenous arginine synthesis pathway [1]. Like NAG, NCG can activate CPS1 and lead the conversion of blood ammonia into the mitochondrial carbamoyl-phosphate, further stimulating the endogenous synthesis of arginine. Due to its good stability and safety [2,3], NCG was initially used for the clinical treating of hyperammonemia caused by the NAG synthetase deficiency [4,5], propionic aciduria and methylmalonic aciduria [6,7], and maple syrup urine disease [8]. NCG is also proven to be a novel, effective and low-cost substitute feed additive for arginine. In 2014, China’s ministry of agriculture approved NCG as a new feed additive to use in the livestock (new feed additive certificate no. 2014-01). The maximum addition limit of NCG in the compound feed is 800 g t−1 of sow, and 880 g t−1 of dairy cow, respectively. Compared with the sole supplementation of NAG or arginine, NCG can avoid being catabolized by the deacylase or amino acid metabolism enzymes, and does not cause nutritional antagonism against other amino acids, especially lysine, tryptophan, and histidine [1,9]. Previous studies have indicated that oral administration of NCG 50 mg per kg body weight two times a day, increases piglets’ plasma arginine concentration by 68%, and weight gain by 61% in 10 days [1]. More recently, several results have been reported that gilts with dietary supplementation of 0.05% and 0.1% of NCG had more pigs born alive, and significantly Molecules 2019, 24, 3172; doi:10.3390/molecules24173172 49 www.mdpi.com/journal/molecules Molecules 2019, 24, 3172 increased the live litter weight [10,11]. A similar effect of NCG on growth promotion and reproductive performance enhancement have also been found in other species of animals including the rat [12], cattle [13], sheep [14], goat [15], and yellow-feather broiler [16]. Figure 1. The chemical structure of N-carbamylglutamate. Although the physiological functions of NCG have been well elucidated, and NCG has long been shown to be non-toxic and free of side effects [2], its metabolic process in animals remains unclear. With the promising use of NCG in the livestock, it is necessary to develop an accurate measurement method for NCG in the feed and animal products. Based on the chemical structure of NCG, the infrared spectroscopy (IR), nuclear magnetic resonance (NMR) spectroscopy, and high performance liquid chromatography (HPLC) have been adopted in the analysis of the structure and content of the NCG additive (purity > 97%) [17]. However, due to the complexity of the feed and animal products matrices, few studies have been conducted to deal with the determination of NCG in the feeds [18], and to the best of our knowledge, there is no former report regarding the NCG residue detection in animal tissues or body fluids. Since NCG has poor absorption of the UV-Vis spectrum, using the HPLC based on the UV detection will result in low responsivity and sensitivity. Our group tried to determine the NCG using the beforeor past-column derivation, but it showed a multiple matrix interference leading to unsatisfactory resolution results, and the procedures were relatively complex. In recent years, the high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), due to its high selectivity and sensitivity, has become the preferred method to analyze the trace small molecular compounds in the complex substrates [19,20]. This study developed a HPLC-MS/MS method to qualitatively and quantitatively determine NCG in the feed, animal tissues, and body fluids with excellent accuracy and sensitivity. The proposed method can achieve a fast separation of NCG within a 20 min gradient elution, and the method validation was achieved by evaluating the linearity, sensitivity, recovery, as well as the accuracy and repeatability for NCG in the feeds and animal products. 2. Results and Discussion 2.1. Optimization of HPLC-MS/MS Conditions In order to improve the analytical sensitivity and selectivity, parameters of the mass spectrometry such as the ionization mode, source temperature, capillary voltage, nebulizer pressure, sheath gas flow, collision energy, and fragmentor voltage were optimized using the NCG standard solution. The result showed that the most abundant precursor ion of NCG was the pseudo-molecular ion [M + H]+ at m/z 191.0 (Figure 2A and Figure S1). The MS/MS spectrum of this precursor ion is shown in Figure 2B. The most abundant product ion (m/z 84.0) was used for quantitation while the two relatively abundant product ions (m/z 130.0, m/z 148.0) were selected for qualification (Figure 2C–E). With the aim to increase 50 Molecules 2019, 24, 3172 the signal responses of characteristic product ions and their stability, the parameters (fragmentor, collision energy) in the second mass analyzer were optimized. The optimal fragmentor of the precursor ion (m/z 191.0) is 50 V. The optimal collision energy of m/z 84.0, m/z 130.0, and m/z 148.0 are 15 V, 10 V, and 5 V, respectively. Figure 2. Cont. 51 Molecules 2019, 24, 3172 Figure 2. (A) Parent ion chromatogram of N-carbamylglutamate (NCG); (B) the tandem mass spectrometry (MS/MS) spectrum of the NCG precursor ion; (C–E) typical extracted mutiple reaction monitoring (MRM) ion chromatograms of the NCG standard conducted under optimal conditions. 52 Molecules 2019, 24, 3172 NCG is a compound with strong polarity. A Waters Atlantis T3 C18 column (4.6 × 250 mm, 5 μm) was compared with a Waters BEH C18 column (2.1 × 100 mm, 1.7 μm). The T3 column showed a better retention for polar compounds with reduced peak tailing thus better peak shapes and improved analytical results than the BEH column. In addition, considering that there are many free amino acids, fatty acids, and other small molecular metabolites in animal samples, the long analytical column with strong polarity is more suitable for the separation of NCG without a precolumnar derivation. Thus, the Atlantis T3 C18 column was chosen as the analysis column. Moreover, the mobile phase of HPLC is a critical factor influencing the analytical results and sensitivity of the detection. Compared with acetonitrile, using methanol as the organic mobile solvent can achieve better chromatographic separation of NCG. Due to the weak acidity of NCG, the addition of a small amount of formic acid could improve the peak shape and improve the sensitivity. Therefore, 0.1% of the formic acid aqueous solution and methanol were selected to be the mobile phases for the binary gradient pump system. Figure 2C showed the typical extracted MRM ion chromatogram of NCG conducted under optimal conditions. 2.2. Optimization of Extract Condition An effective pretreatment should serve the purpose that the impurity substance is excluded while the target analyte stays as much as possible. As NCG is soluble in both water and methanol, pilot experiments have been conducted to determine the optimal extraction solvent with the finest extraction result. For the feed samples, five extract solvents including water, water/methanol (80:20, v/v), water/methanol (50:50, v/v), water/methanol (20:80, v/v) and methanol were compared. The results show that the recoveries were water > water/methanol (80:20, v/v) > methanol > water/methanol (50:50, v/v) > water/methanol (20:80, v/v). However, using water as the extraction solvent was subjected to a greater matrix effect presumably due to the complex water-soluble additive in the feed. Thus, water/methanol (80:20, v/v) was selected as the extraction solvent. To better improve the extraction efficiency of NCG in the water/methanol (80/20, v/v) solvent, different concentration levels of formic acid in the water including 0, 0.1%, 0.5%, and 1% were compared. The results show that 0.5% of formic acid in water/methanol (80/20, v/v) manifested the best extraction result with a recovery of 99.1%. In addition, the high recovery (>100%) were shown in Figure 3C and D. We speculated that the matrix effect in the C and D extract may cause the enhancement of the NCG signal in MS, which leads to the recovery of NCG over 100%. Therefore, 0.5% of formic acid in water/methanol (80/20, v/v) was applied as the extraction solvent for NCG in the feeds (Figure 3). For the animal samples, the biggest interference came from the high lipid and protein content in the samples [21]. Similarly, different proportions of methanol and 0.5% of formic acid solution were also tested for the optimization of extraction agents. Moreover, the application of ice-cold methanol combined with the subsequent refrigerated centrifugation step can efficiently precipitate and remove most of the interference proteins present in milk, serum, and tissue samples [20,22]. Thus, ice-cold methanol (100%) was finalized as the optimal extraction solvent for NCG in the animal body fluids and tissues due to its precipitation effect of proteins and relatively good recovery (Figure 3). 2.3. Optimization of Purification Condition A subsequent purification experiment of the sample extracts was conducted using the solid-phase extraction (SPE) cartridges to further decrease the matrix interference and enhance sensitivity. NCG is a weak acid compound. A strong anion exchange SPE cartridge was used in virtue of its strong adsorption ability for the anionic compound. For the better retentivity of NCG, an appropriate amount of 5% ammonia solution was added to adjust the pH of the extract and make NCG anionic. The results show that the pH of the extract that ranged from 8 to 10 was optimal, the NCG retained above 95% on the SPE (Figure 4). Since the cartridge was a strong anion exchange column, considering the isoelectric point of the NCG (pI 3.02), a lower pH (<8) will not be able to sufficiently make the NCG anionic, resulting in lower retentivity. On the other hand, if the pH is too high (>10), other interfering 53 Molecules 2019, 24, 3172 compounds may be turned anionic and compete with NCG for the limited retention capacity of the cartridge, this will also cause lower retentivity of NCG in the column. In addition, the NCG content in the feeds were very high, a direct application of the sample extracts may cause oversaturation and lead to unsatisfactory recoveries of NCG due to limited adsorption capacity of the cartridges. Our study has indicated that a reduction of the NCG concentration in the sample extracts could significantly increase the NCG recoveries. To avoid oversaturation and improve the peak shape, feed sample extracts were diluted 10 times with water before loaded onto the cartridges. On the contrary, if the content of the NCG in the sample is too low, such as the biological sample, the loading volume should be increased before passing through the column. Figure 3. Recoveries of NCG from matrices using different solvents: (a) 0.5% of formic acid solution; (b) 0.5% of formic acid solution/methanol (80:20, v/v); (c) 0.5% of formic acid solution/methanol (50:50, v/v); (d) 0.5% of formic acid solution/methanol (20:80, v/v); (e) methanol. Figure 4. Dependence of the retention abilities of the NCG on the solid-phase extraction (SPE) cartridge with different pH of the loading solvent. 54 Molecules 2019, 24, 3172 2.4. Method Validation 2.4.1. Stability and Matrix Effect For the stability test, the NCG stock solution was diluted into 0.005, 0.1, 2 μg mL−1 to have six aliquots with concentrations around the linearity range. These aliquots were stored at −4 ◦ C and 20 ◦ C, respectively. Normally, the criterion for the stability is that degradation is equal to or less than 5%. The results show that the solution could be stored stably for at least three months and one week at the −4 ◦ C refrigerator and 20 ◦ C, respectively. Matrix effects come from various chemical and physical processes during ionization of the analytes in the ESI mode of mass spectrometry, which causes signal suppression or enhancement. The matrix effect was evaluated by comparing three NCG concentrations (0.005, 0.1, 2 μg mL−1 ) constructed in the solvent and fortified sample extract. The matrix effects were calculated by comparing the peak area of NCG in the solvent with the peak area of NCG in the fortified sample extract. The results show that the matrix effects ranged from 1.08 to 0.54 for the feed and biological samples. Especially for the biological samples, the matrix inhibition effects were obvious when a high concentration NCG was added. Therefore, the quantification of NCG must be performed using the matrix-matched calibrators. 2.4.2. Linearity, Precision, Accuracy, LOQ and LOD A series of experiments were carried out with the aim to validate the linearity, sensitivity, precision, and accuracy of the established method. The NCG stock solution was diluted into 0.0016, 0.08, 0.2, 0.5, 1, 2 μg mL−1 working solutions. Excellent linearity was obtained at the NCG concentrations ranging from 0.0016–2 μg mL−1 with a correlation coefficient of (R2 ) 0.999. To evaluate the sensitivity of this method, the limits of detection (LOD) and the limits of quantitation (LOQ) were tested. The LOD, defined as the analyte concentration at three times the signal to noise ratio (S/N), was determined for NCG in the feed, milk, serum, meat, liver, and kidney samples. The results indicated that the LOD values were within the range of 0.0035 to 0.022 mg kg−1 and 0.014 to 0.023 μg mL−1 for the solid and liquid samples, respectively (Tables 1 and 2). The LOQ, defined as the analyte concentration at 10 times the S/N, were in the range of 0.012 to 0.073 mg kg−1 and 0.047 to 0.077 μg mL−1 for the solid and liquid samples, respectively (Tables 1 and 2). Here, the compound feed, concentrated feed, and premixed feed are all called the feed. Since the three matrices are similar, the LOD and LOQ values for the three feeds were just slightly different. For convenient use, we have integrated the three similar results into one LOD and one LOQ. Table 1. Percentage recoveries and relative standard deviations (RSDs) of NCG in different feed matrices (n = 3). Matrices Compound feed Concentrated feed Premix a Added Concentration (%) Recoveries (%) LOD (mg kg−1 ) LOQ (mg kg−1 ) 0.01 0.05 0.1 0.05 0.2 1.0 0.5 2.0 10 96.85 (1.3) a 98.89 (2.9) 99.24 (5.0) 107.0 (3.5) 106.1 (7.1) 97.60 (8.8) 94.27 (6.8) 91.90 (3.9) 100.2 (3.6) 0.022 0.073 Figures in bracket represented the relative standard deviation (%). 55 Molecules 2019, 24, 3172 Table 2. Percentage recoveries and relative standard deviations (RSDs) of NCG in different animal samples (n = 3). Matrices Serum (μg mL−1 ) Milk (μg mL−1 ) Meat (mg kg−1 ) Kidney (mg kg−1 ) Liver (mg kg−1 ) a Added Concentration Recoveries (%) 0.1 1.0 10 0.1 1.0 10 0.05 0.1 0.05 0.1 0.05 0.1 94.25(3.8) a 110.2 (2.3) 96.34 (5.1) 88.12 (1.8) 102.5 (3.2) 89.58 (4.7) 93.84 (7.6) 101.6 (3.4) 89.14 (3.1) 93.65 (5.6) 94.54 (7.4) 97.36 (2.9) LOD (μg mL−1 ) LOQ (μg mL−1 ) 0.023 0.077 0.014 0.047 0.0038 0.013 0.0069 0.023 0.0035 0.012 Figures in bracket represented the relative standard deviation (%). The method precision and accuracy were evaluated by the recoveries of NCG in the spiked samples. Recoveries were determined for the feeds, milk, serum, meat, procine liver, and kidney, three replicates were used for each sample at different concentrations. The MRM ion chromatograms for a blank (free of NCG) feed sample and the compound feed sample spiked with NCG were demonstrated in Figure 5A,B and Figures S2–S19, respectively. The peak intensity ratio of the three product ions of NCG can be used to confirm the presence of NCG when compared with the peak intensity ratio obtained from the standard sample. The most abundant product ion of the mass spectrum was selected for the quantitative determination and evaluation of the recoveries for NCG. Calibration curves developed using an external standard were used to quantify NCG in the spiked samples. The spiked levels, spiked recoveries, and coefficients of variation were evaluated. According to the recommended dose of NCG in the feeds, NCG was spiked in the compound feed at 0.01%, 0.05%, and 0.1%, the premix feed at 0.05%, 0.2%, and 1%, and the concentrate feed at 0.5%, 2%, and 10%. Three replicates were analyzed for each concentration. A good consistency was found between the actual spiked amount of NCG and the estimated concentrations in three different feed matrices (Table 1). The recoveries were within the range of 91.90% to 107.0% with the coefficients of variation ranging from 1.3% to 8.8%. Similarly, the above established method was also applied to the detection of the NCG spiked animal products. The MRM ion chromatograms for the blank and spiked animal products samples were shown in Figure 5C–G and Figures S20–S49, respectively. NCG was spiked in meat, kidney, and liver at 0.05, and 0.1 mg kg −1 . For each sample concentration, three replicates were analyzed to evaluate the spiked levels, spike recoveries, and coefficients of variation. As presented in Table 2, reliable results were found for most test samples. The recoveries for NCG ranged from 89.14% to 101.56% and the coefficients of variation were less than 7.6%. The NCG were spiked in milk and serum at 0.1, 1, and 10 μg mL−1 . The recoveries ranged from 88.12% to 110.21% with the coefficients of variation ranging from 1.8% to 5.1% (Table 2). Both the intra-assay and inter-assay reproducibility of this method were evaluated. For instance, five repeats at an intermediate concentration (0.05%) were used to determine the recoveries of NCG spiked in the compound feed. According to the analytical results, the recoveries of NCG were within the range of 98.89% to 102.76% and the coefficients of variation were less than 2.9% and 3.8% for the intra-assay (within a day) and inter-assay (over a period of five consecutive days) measurements, respectively. For the animal samples, this method also showed satisfactory reproducibility. For 1 μg mL−1 of NCG spiked in the serum, the recoveries of NCG were within the range of 93.25% to 104.68% and the coefficients of variation were less than 4.7% and 5.4% for the intra-assay and inter-assay measurements, respectively. For 0.05 mg kg−1 of NCG spiked in the liver, 56 Molecules 2019, 24, 3172 the recoveries of NCG were within the range of 91.23% to 106.71% and the coefficients of variation were less than 3.9% and 6.7% for the intra-assay and inter-assay measurements, respectively. Figure 5. Quantitative product ion chromatograms of the NCG in different matrices: (A) Blank feed sample; (B) compound feed; (C) milk; (D) serum; (E) meat; (F) liver; (G) kidney. 2.5. The Analysis of Authentic Samples The method established in this study was applied to determine the NCG content in the authentic feed samples added with NCG additive. The results showed that NCG was detected in the compound feed, concentrated feed, and premix feed samples with the average content of 0.098%, 0.95%, and 9.53%, respectively. The variation between the two parallel samples was less than 10%, while NCG was not detected in the ordinary swine feed samples (<0.022 mg kg−1 ). According to the actual addition amount, the accuracy for the determination of the compound, concentrate, and premix feed was 98.0%, 95.0%, and 95.3%, respectively. The proposed method was also adopted for the determination of NCG in the authentic serum samples from pigs and milk sample from cows (each concentration has six replicates). The results showed that the concentration of NCG in the serum samples increased significantly with the increase of NCG in the diets (Table 3). Except for the control group, the NCG levels in the serum of four diets ranged from 0.11 to 1.31 μg mL−1 . Moreover, the multiple relationship between the serum NCG and dietary NCG was basically consistent. The NCG concentration in milk was about 57 Molecules 2019, 24, 3172 0.0647 μg mL−1 when the cow was fed 20 g of NCG per day. Therefore, the developed method proved both feasible and reliable for the quantitative determination of NCG in the routine analysis. Table 3. The results of NCG in the authentic samples (n = 6). Sample Diets Added (%) NCG Concentration (μg mL−1 ) Intra-Day Precision CV (%) Inter-Day Precision CV (%) 0 0.025 0.05 0.1 0.2 0 20 g/day ND a 0.112 0.253 0.561 1.31 ND 0.0647 6.8 2.4 7.8 12 11 8.7 5.6 11 5.1 7.4 Serum b Milk a Not detected, b Serum from piglets. 3. Materials and Methods 3.1. Materials and Reagents The NCG standard (purity >99.5%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The methanol and formic acid used were of a HPLC grade and were obtained from Fisher Scientific International (Hampton, NH, USA) and Dikma Technology (Richmond Hill, ON, Canada), respectively. Other chemicals used were all of the analytical grade. A Milli-Q system (Millipore Corporation, Bedford, MA, USA) was applied to provide ultrapure water for the preparation of all aqueous solutions. The NCG stock solution (1 mg mL−1 ) was prepared by dissolving the NCG standard in ultrapure water, and preserved at 4 ◦ C. The preparation of the NCG working solutions was done daily by diluting the stock solution to appropriate concentrations with ultrapure water. The extraction solvent, 0.5% of formic acid in water/methanol (80/20, v/v), was prepared by mixing 4 mL of formic acid with 796 mL of Milli-Q water, and then mixing with 200 mL of methanol. 3.2. Instruments and Apparatus The HPLC- MS/MS was performed on an Agilent 1200 UHPLC system coupled with an Agilent 6460 Triple Quadrupole Mass Spectrometer (Agilent Technologies, Fermont, CA, USA). An Ultrasonic Cleaner (Kunshan, China) was used to promote the sample dissolution and extraction. A low temperature high speed centrifuge (Eppendorf, Hamburg, German) was used to centrifuge the samples. 3.3. Sample Preparation Feed samples free of NCG were provided by the Ministry of Agricultural Feed Industry Centre (Beijing, China). The liver and kidney as blanks were from a porcine, and milk was from a cow. They were all purchased from local supermarkets. The serum of the pig as blanks were collected from the animal research station of China Agricultural University (Hebei, China). Prior to the extraction, the feed samples were crushed into about 60 mesh by a small mill. The meat, liver, and kidney tissues were homogenized in a homogenizer for five min. The milk and serum were pre-treated by centrifuging at 14,000 rpm for 10 min at 4 ◦ C, then the supernatant was collected. 3.4. Sample Extraction and Purification Extraction of the feed samples was carried out by adding 20 mL of the extraction solvent (0.5% of formic acid in water/methanol, 80/20, v/v) into the feeds (2 g for the compound feed, 0.2 g for the concentrated feed and premix). Usually, the recommended amounts of NCG in the pig compound feed, premixed feed, and concentrated feed were about 0.05%, 1% and 10%, respectively. Since the concentration of NCG in the premix or concentrated feed was too high, in order to avoid too much 58 Molecules 2019, 24, 3172 dilution before the HPLC-MS/MS analysis, the sample amount in the premix or concentrated feed was relatively reduced. After the vortex mixing and a 30 min ultrasonic bath, 10 min of centrifugation at 14,000 rpm at 4 ◦ C was completed and the supernatant was collected. For the fluid samples, 2 mL of pre-centrifuged milk or serum was pipetted into a Corning® 15 mL centrifuge tube, and 8 mL of an ice-cold extraction solvent was added. After the vortex mixing, the samples were centrifuged at 14,000 rpm for 10 min at 4 ◦ C and the supernatant collection was done. For the tissue analyses, 2 g of tissues samples (meat, kidney, or liver) were extracted by adding 20 mL of an ice-cold extraction solvent. After a thorough vortex mixing, the samples were placed on ice for 30 min and centrifuged for 10 min at 14,000 rpm at 4 ◦ C, then the supernatant collection was done. Before further purification, the above supernatant was diluted 10 times using water and adjusted by adding an appropriate amount of 5% of ammonia solution to pH 8–10. The strong anion exchange cartridge (Agilent SampliQ SAX, 200 mg, 3 mL, Agilent Technologies, Fermont, CA, USA) was firstly preconditioned with 3 mL of methanol following by 3 mL of water. Then, 3 mL of the diluted supernatant was loaded and slowly passed through the cartridge with a flow rate of approximately 1 mL min−1 . After rinsing with 3 mL 5% of the ammonia solution and 3 mL of methanol, elution was carried out with 3 mL 2% of formic acid in methanol. The eluate was then collected and gently evaporated under a nitrogen stream in a 50 ◦ C water bath. The residue was reconstituted with 1 mL 0.1% of an aqueous formic acid solution and filtered by a 0.1 μm Syringe Filter (Tianjin Fuji Science and Technology, China). 10 μL of the sample was injected by the autosampler for the HPLC-MS/MS analysis. 3.5. HPLC-MS/MS Conditions A Waters Atlantis T3 column (4.6 × 250 mm, 5 μm) was applied in the chromatographic separation of NCG. The column temperature was maintained at 35 ◦ C. The mobile phases consisted of solution A (0.1% of an aqueous formic acid solution) and B (methanol), and were delivered at a flow rate of 0.8 mL min−1 . The total run time was set at 20 min. A gradient elution program was developed as follows: 90% of solution A (initial), with 90–15% of solution A (from 6 to 6.1 min), 15–90% of solution A (from 12 to 12.1 min). Before the next injection, a 7.9 min equilibration was necessary. A calibration curve calculated from the NCG standard solution was used to quantify the NCG content in the spiked samples. The electron spray ionization source was operated under an optimized condition: Capillary voltage 3500 V, nebulizing gas temperature 350 ◦ C, nebulizing gas flow 5 L min−1 , sheath gas temperature 350 ◦ C, and sheath gas flow 7 L min−1 . Positive ions were monitored. The multiple reaction monitor (MRM) mode was applied for the quantitation analysis. Data were collected and processed on the Agilent MassHunter Workstation software (Version B.04.00). 3.6. The Method Validation The method validation was performed with the feeds, serum, meat, liver, and kidney samples. Extracts of the NCG-free samples were used for the preparation of matrix-matched calibration standards. The artificially prepared samples were spiked with the NCG standard solution of 0.01–10% in the feeds, 0.1–10 μg mL−1 in fluids, or 0.05–0.1mg kg−1 in tissues. The validity of the method including the stability, matrix effect, linearity, sensitivity, as well as the precision and accuracy of the method were evaluated in three replicates. 3.7. The Analysis of Authentic Samples One hundred kg of the commercially prepared compound feed, concentrated feed and premix were added with the NCG feed additive product (NCG content ≥ 97%) obtained from Ya Tai XingMu company (Beijing, China) to the concentrations of 0.1% for the compound feed, 1% for the concentrated feed and 10% for the premix, respectively. The animals breeding experiments were carried out at the animal research station of China Agricultural University (Hebei, China). All piglets used in this study were housed and handled 59 Molecules 2019, 24, 3172 according to the established guidelines of China Agricultural University, which were approved by the China Agricultural University Animal Care and Use Committee. Thirty weaned piglets (each 30–40 kg in weight) were randomly assigned to one of the five groups (n = 6 piglets per group). Five pig diets were prepared with NCG added 0%, 0.025%, 0.05%, 0.1%, and 0.2%, respectively. After 26 fed days, blood samples from the precaval veins were collected after fasting 12 h and centrifuged (3000 rpm) for 10 min. Then, the supernatant was transferred into new tubes and stored at −20 ◦ C until use. Milk samples were collected from the cow before and after feeding 20 g of NCG per day. Thirty weaned piglets (each 30–40 kg in weight) were randomly assigned to one of the five groups (n = 6 piglets per group). 4. Conclusions A fast, sensitive and reliable HPLC-MS/MS method was successfully developed and optimized for the qualitative and quantitative determination of NCG in the feeds, animal tissues, and body fluids. Based on the high selectivity and satisfactory separation result of the HPLC-MS/MS technique, this method can achieve confirmative identification and accurate determination of NCG in the complex matrices including feeds and animal products. Furthermore, the applicability of the established method has been validated using the real feed, serum and milk samples. Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/17/3172/s1. Author Contributions: Y.M. and Z.Z. designed and performed the experiments; L.K. and Y.C. analyzed the data; P.H. and Y.M. wrote the paper. Funding: The financial support from The National Key Research and Development Program of China (2018YFD0500402) is gratefully acknowledged. 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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/). 61 molecules Article Sequence Identification of Bioactive Peptides from Amaranth Seed Proteins (Amaranthus hypochondriacus spp.) Alexis Ayala-Niño 1 , Gabriela Mariana Rodríguez-Serrano 2 , Luis Guillermo González-Olivares 1, *, Elizabeth Contreras-López 1 , Patricia Regal-López 3 and Alberto Cepeda-Saez 3 1 2 3 * Chemistry Investigation Center, Universidad Autónoma del Estado de Hidalgo, Carretera Pachuca-Tulancingo km 4.5, Mineral de la Reforma Hidalgo C.P. 46067, Mexico Biotechnology Department, Universidad Autónoma Metropolitana, Unidad Iztapalapa, Mexico City C.P. 55355, Mexico Universidad de Santiago de Compostela, Campus Lugo, 15705 Santiago de Compostela, 27002 A Coruña, Spain Correspondence: lgonzalez@uaeh.edu.mx; Tel.: +52-771-717-2000 Received: 1 June 2019; Accepted: 3 August 2019; Published: 21 August 2019 Abstract: Amaranthus hypochondriacus spp. is a commonly grown cereal in Latin America, known for its high protein content. The objective of this study was to separate and identify bioactive peptides found in amaranth seeds through enzymatically-assisted hydrolysis using alcalase and flavourzyme. Hydrolysis was carried out for each enzyme separately and compared to two-step continuous process where both enzymes were combined. The biological activity of the resulting three hydrolysates was analyzed, finding, in general, higher bioactive potential of the hydrolysate obtained in a continuous process (combined enzymes). Its fractions were separated by RP-HPLC, and their bioactivity was analyzed. In particular, two fractions showed the highest biological activity as ACE inhibitors with IC50 at 0.158 and 0.134, thrombin inhibitors with IC50 of 167 and 155, and antioxidants in ABTS assay with SC50 at 1.375 and 0.992 mg/L, respectively. Further sequence analysis of the bioactive peptides was carried out using MALDI-TOF, which identified amino acid chains that have not been reported as bioactive so far. Bibliographic survey allowed identification of similarities between peptides reported in amaranth and other proteins. In conclusion, amaranth proteins are a potential source of peptides with multifunctional activity. Keywords: amaranth protein; flavourzyme; alcalase; bioactive peptides; hydrolysates 1. Introduction Many diseases that prevail nowadays could be tackled more efficiently, and even prevented, by combining a healthy diet with functional foods intake [1]. Among functional food components, we can find bioactive peptides, which are generally short sequences of amino acids encrypted in food proteins [2]. Bioactive peptides are released from proteins as a result of microorganisms’ metabolic activity during fermentation, by proteolytic enzymes, or finally by the action of gastrointestinal enzymes once proteins are ingested. Application of commercially available enzymes has become a simple and inexpensive way to access free amino acids and peptides from proteins [3]. Consequently, a vast number of studies using various proteinases target the preparation of hydrolysates from different protein sources, such as cereals and pseudocereals, in an ongoing effort to obtain highly active biopeptides [4–7]. One of the most important highly-consumed Mesoamerican original seeds, with a high content of proteins and an excellent amino acid balance, is amaranth. Molecules 2019, 24, 3033; doi:10.3390/molecules24173033 63 www.mdpi.com/journal/molecules Molecules 2019, 24, 3033 In Mexico, amaranth is consumed in a fresh form, offered by traditional confectionary and in typical dishes of Mexican cuisine [8]. Bioactive peptides found in amaranth exhibit various biological activities—such as anticholesterolemic, antihypertensive, antioxidant, and antithrombotic—and they are released mainly by in vitro digestion [9–12]. However, multiple bioactivities shown by the same peptide fraction of amaranth have never been studied. In order to find peptide sequences with different biological activities, our research work focused on the separation and identification of the peptide sequences from amaranth proteins released during hydrolysis with two commercial enzymes, alcalase and flavourzyme, in separate and continuous hydrolysis processes. The multiple biological activities exhibited by peptides released from amaranth proteins through enzymatically-assisted hydrolysis have not been reported until now. 2. Results 2.1. Free Amine Groups Analysis during Enzymatic Hydrolysis Once the hydrolitic enzymes were added to the protein sample, the progress of the reaction was monitored every 20 min by the measurement of free amine groups concentration released during the reaction course (Figure 1). It was observed that after 120 min of hydrolysis with alcalase (H1), the concentration of free amine groups equaled to 6170.53 ± 29.5 mg/L NH:− , whereas flavourzyme hydrolysis (H2) reached the highest concentration of free amine groups (5551.11 ± 33.83 mg/L NH:− ) after 90 min of reaction. It was concluded that, in terms of enzymatic efficiency, alcasase overdoes flavourzyme in the release of peptide fractions. Following the analysis of H1 and H2, the combined two-step hydrolysis (H3) was carried out, and a final free amine groups concentration of 7468.89 ± 34.79 mg/L NH:− was obtained after 40 min of reaction. This result proves that the continuous hydrolitic process (H3) was more efficient than using alcalase and flavourzyme separately. Figure 1. Free amine groups concentration during amaranth protein hydrolysis. * Different letters indicate statistically significant differences between treatments. 2.2. Hydrolysates Bioactivity Analysis Table 1 shows the assessment of the different biological activities exhibited by the hydrolysates H1, H2, and H3, determined during amaranth proteins hydrolysis. For thrombin inhibition essay, H1 hydrolysate showed the highest inhibitory potential (90%), followed by H2 hydrolysate which inhibited thrombin activity by approximately 80%. No significant difference was observed for H3 and H1 hydrolysates as thrombin inhibitors. When angiotensin-converting enzyme inhibition was evaluated, it was observed that H3 hydrolysate showed the highest inhibitory potential (58%), in comparison 64 Molecules 2019, 24, 3033 to H1 and H2 hydrolysates (49% and 39%, respectively). Finally, antioxidant activity was measured by three different methods based on the free radical scavenging principle, such as ABTS, DPPH, and FRAP. The highest antioxidant activity was observed by the H3 sample in DPPH and FRAP assays (388.94 μmol TE/100 g and 592.54 μmol Fe2 E/100 g, respectively), while H1 hydrolysate exhibited the highest antioxidant activity measured by the ABTS method (425.86 ± 0.66 mg TE/100 g). Noteworthy, the antioxidant potential of H3 hydrolysate increased fivefold in the DPPH assay and ninefold in the FRAP assay, compared to the control sample of unhydrolyzed amaranth proteins. Table 1. Bioactivity potential of amaranth protein hydrolysates after treatment with alcalase (H1), flavourzyme (H2), and in two-step combined hydrolysis (H3). Hydrolysis ACE Inhibition (%) Thrombin Inhibition (%) Amaranth Protein H1 H2 H3 10.58 ± 1.19 d 49.49 ± 1.47 b 39.77 ± 2.15 c 58.53 ± 2.58 a 11.90 ± 10.10 c 92.85 ± 3.36 a 80.95 ± 13.46 b 92.85 ± 3.36 a Antioxidant Activity DPPH (μmol Trolox E/100 g) ABTS (mg Trolox E/100 g) FRAP (μmol Fe2 E/100 g) 76.66 ± 1.60 d 340.17 ± 10.95 b 274.03 ± 10.84 c 388.94 ± 2.73 a 115.65 ± 10.30 d 425.86 ± 0.66 a 398.36 ± 3.62 c 404.90 ± 1.52 b 63.37 ± 5.72 c 241.70 ± 9.38 b 226.29 ± 11.20 b 592.54 ± 29.29 a Values are shown as mean ± standard deviation (n = 3); values in the same column with different superscript letters are significantly different (p < 0.05). 2.3. RP-HPLC Separation and Fraction Bioactivity Since the H3 hydrolysate (obtained by application of continuous enzymatic hydrolysis) showed the highest bioactivity, it was chosen for further analysis in order to separate fractions and identify peptide sequences. In total, 56 fractions were obtained through RP-HPLC separation (see Appendix A), of which only 14 could be identified with known proteins. Moreover, protein content was determined in samples collected after 27 min (Table 2). In the case of the 14 fractions identified with protein, their bioactivity was evaluated by measurement of the IC50 in the ACE and thrombin inhibition assays, and SC50 in the ABTS assay. Results are gathered in Table 2. Table 2. Bioactivities of amaranth protein fractions (Angiotensin I-Converting Enzyme and Thrombin inhibitory activity (IC50 , mg/L)) and antioxidant activity (ABTS radical scavenging; SC50 , mg/L). Fraction 2 3 9 18 19 22 23 27 28 32 34 39 40 45 ACE (IC50 ) cd 0.332 0.442 e NI 0.614 f 0.173 b 0.158 ab NI 0.808 c 0.346 d 0.192 b 0.317 cd NI 0.298 c 0.134 a Thrombin (IC50 ) ABTS (SC50 ) e i 4.204 NI NI 2.538 d NI 1.375 b 2.809 d 1.616 c 1.728 c 6.931 g 2.593 d 5.561 f 4.547 e 0.992 a 38.46 4.36 h 0.426 f 2.65 g 0.183 b 0.167 ab 0.349 e 0.402 f 0.135 a 0.298 d 0.247 c 0.247 c 0.26 cd 0.155 a Peptide Concentration (mg/L) 0.2125 0.8375 0.9062 0.7125 0.3750 0.4687 0.5625 0.3125 0.0937 0.4687 0.4937 0.4375 0.8375 0.8125 Values for ACE, thrombin, and ABTS (mg peptide/mL) are mean ± SD (n = 3); values in the same column with different superscript letters are significantly different (p < 0.05). NI: not identified. For the antihypertensive potential, the IC50 calculated for the test fractions ranged from 0.134 to 0.808 mg/mL (Table 2). These values stay in agreement with the results reported elsewhere for amaranth protein hydrolysates [10,13]. On the other hand, values obtained in this work were lower than those 65 Molecules 2019, 24, 3033 found for other vegetable protein sources [14], or higher in comparison to buffalo milk [15]. Antioxidant potential based on the ABTS assay and expressed as SC50 varied from 0.992 to 6.931 mg/mL, and was slightly higher than that obtained for amaranth hydrolysates by in vitro digestion [16]. This may be explained by the fact that the type of bioactive peptides released depends not only on the protein nature, but also on the enzymes used in the particular case of hydrolysis [17,18]. Concerning the thrombin IC50 values, the results obtained were in the range of 0.992 to 38.46 mg/L, which stay in agreement with the values reported for amaranth hydrolyzed by alcalase and pepsin [12]. Finally, no statistically important differences were found for thrombin inhibition potential between H3 hydrolysate (alcalase and flavourzyme) investigated in this study, and amaranth hydrolyzed by alcalase and pepsin as reported in Sabbione et al. [12], which was also obtained via sequential two-enzyme process. Based on the obtained results, fractions 22 and 45 were subjected to MALDI-TOF analysis (Figure 2), which allowed the identification of sequences with interesting multiple bioactivities. Figure 2. Fraction 22 and 45 MALDI-TOF mass Spectra. The structure of the aminoacidic chains and the corresponding protein source are presented in Table 3. Table 3. Peptides identified in the most active fractions from amaranth hydrolysate and their corresponding protein source. Fraction Mass m/z Calc MH+ Sequence 34 3 16 1375.6435 573.2252 874.4448 1375.6341 573.3516 874.4813 ITASANEPDENKS LVRW NIDMLRL Protein Fraction 22 66 Agglutinin Agglutinin Granule bound starch synthase I Molecules 2019, 24, 3033 Table 3. Cont. Fraction Mass m/z Calc MH+ Sequence Protein Fraction 22 12 5 3 13 794.3805 686.3414 573.2251 812.3607 794.4203 686.4081 573.3617 812.4265 27 14 14 4 29 26 24 19 13 6 3 2 1 1 853.4330 707.3505 707.3502 547.1921 861.3847 845.4098 841.4242 823.4281 686.3805 619.2805 531.1927 505.1907 503.1730 503.1731 853.4329 707.3212 707.3185 547.2997 861.4299 845.4254 841.3841 823.3696 686.4080 619.3566 531.3042 505.2447 503.2715 503.2823 RPVFEF DPKLTL IKEAL NVEVHKS Granule bound starch synthase I Granule bound starch synthase I Granule bound starch synthase I Cystatin Fraction 45 HVQLGHY SQIDTGS NWACTL VRWS CIHNIVY EGTESIPL PRYDQY MSNIDML DPKLTL IPSRF ARVW CQAAL EELL LGVAGS Agglutinin Agglutinin Agglutinin Agglutinin Granule bound starch synthase I Granule bound starch synthase I Granule bound starch synthase I Granule bound starch synthase I Granule bound starch synthase I Granule bound starch synthase I Granule bound starch synthase I Granule bound starch synthase I Granule bound starch synthase I Granule bound starch synthase I 3. Discussion Results obtained for the individual enzymatic hydrolysis were similar to those reported by Zhuang et al. [19] and Ma et al. [20], where it was observed that the enzymatic activity of alcalase and flavourzyme decreased after 90–120 min of the reaction course. The duration of enzymatic activity depends on the nature of the protein matrix and the amount of enzyme and substrate present in the medium. Additionally, other studies indicated a higher hydrolytic degree when only alcalase was used [21]. For H3 results, they are similar to those reported by Cumby et al. [22], who found that combined enzymatic hydrolysis increased peptide concentration when compared to hydrolysis performed solely with alcalase. It was also stated that hydrolysis with flavourzyme did not contribute to reach higher concentrations of free amine groups when alcalase was used in the first step of the combined enzymatic hydrolysis. On the other hand, the degree of hydrolysis depends on nature of protein and the specificity of the enzymes used [17,18]. Alcalase and flavourzyme have been used individually and in continuous hydrolytic processes applied to fragmentation of different food proteins in search of platelet inhibitory peptides [23,24]. In the case of amaranth proteins, the addition of a second enzyme different to flavourzyme, following hydrolysis with alcalase, has proven to enhance the inhibition potential of thrombin. This finding implies that the higher degree of hydrolysis, the greater bioactivity achieved [12,25]. In addition, Sabbione et al. [12] observed that the degree of hydrolysis of amaranth proteins, such as albumin and globulin, was an important factor in improved thrombin inhibition, reaching approximately 81%, which matches with the results obtained in present study (80% for H2, and 90% for H1 and H3). The increase in thrombin inhibition activity may be due to the fact that amaranth protein has peptide sequences capable of inhibiting fibrinogen. These peptides are released by the action of proteolytic enzymes. Furthermore, it was observed that not only was the size of the peptide important for thrombin inhibition, but also the amino acid sequence, which might be homologous to the fibrinopeptides A and B from human fibrinogen (GGGVR-GP and PPSAR-GH, respectively) [26]. Therefore, antithrombotic activity is affected by the competition for platelet receptors between casoplatelin and the γ-chain of human fibrinogen (HHLGGAKQAGDV) [27,28], which is implicated in one of the three steps in thrombosis cascade reactions. Additionally, alcalase has been reported as an enzyme capable of 67 Molecules 2019, 24, 3033 releasing antithrombotic peptides form proteins like peanut [18], but no reports have been available so far on hydrolytic potential of flavourzyme to release bioactive peptides from amaranth. In the case of antihypertensive activity, the results obtained in this study are similar to those reported by Ambigaipalan et al. [29]. They observed that the combination of two or more enzymes increased the inhibitory activity of angiotensin, converting the enzyme in date seed protein in a sequential enzymatic process. Many authors have reported that the variation in antihypertensive activity of the released peptides could be attributed to the differences in composition and hydrophobicity of the protein primary structure [30,31]. In this sense, hydrophobic residues of amino acids (leucine, valine, alanine, tryptophan, tyrosine, proline, and phenylalanine) bind at the ACE catalytic sites, acting as competitive inhibitors [32]. Nevertheless, milk proteins have been reported as a better source of bioactive peptides, especially those with antihypertensive activity reaching over 80% of ACE inhibition [33,34]. Furthermore, studies have been focused on peptides released from vegetable proteins, such as amaranth. In particular, 11S globulin was reported to show IC50 value in the ACE assay, ranging from 6.32 mM to 175 μM [9,35]. It has been known that the low molecular weight of peptides is a prerequisite for their antihypertensive activity [36]. In this study, peptides with the latter feature were identified using SDS-PAGE (data not shown), revealing typical weights of these biopeptides. In addition to antihypertensive activity, peptide size (peptide chain length less than 20 amino acids) has been related to antioxidant capacity, showing that smaller peptides have greater potential [37]. This observation could explain the higher antioxidant activity obtained for H3 hydrolysate, which contained a higher concentration of free amine groups. In conclusion, during the course of enzymatic hydrolysis H1, H2, and H3 carried out in this study, peptides with different biological properties were released—for example, those able to chelate reactive agents, or donate electrons or hydrogen [38]. Moreover, in several studies in which sequential hydrolysis of proteins with different enzymes was performed, it has been observed that the antioxidant activity remained the same or might be increased by the addition of a second enzyme [39,40]. Thus, the application of two hydrolytic enzymes allows the release of new bioactive peptides, which could affect the antioxidant capacity of the hydrolysates. Consequently, it has been observed that protein hydrolysates might present higher antioxidant properties as they increase their content of small peptides [41]. For RP-HPLC peptide separation, the results obtained in this work are similar to those reported by Moronta et al. [42], who hydrolyzed amaranth proteins with alcalase. It was found that peptides with a higher content of polar amino acids showed higher anti-inflammatory activity. Nonetheless, a fraction found in the non-polar region of the chromatographic analysis (45 min) exhibited higher potential in terms of antihypertensive, antithrombotic, and antioxidant activity than other polar fractions. In some studies, it has been demonstrated that peptides containing hydrophobic amino acids might enhance biological capacities, such as antihypertensive and antioxidant activities [43]. As observed in Table, fraction 22 contained the longest peptide sequence, identified as ITASANEPDENKS, with a molecular weight of approximately 1.44 kDa, being the highest molecular weight found in both fractions. In the case of the peptide NIDMLRL, the last part of its sequence (-LRL) has been identified in silico as an inhibitor of ACE in amaranth. This asseveration may be explained by the presence of leucine as hydrophobic amino acid interacting with the active site of ACE [44]. Additionally, the LVRW sequence was found in fraction 22. The three amino acid chain LVR was previously described as a bioactive peptide with antihypertensive activity in fig sap having an IC50 lower than 20 μM [45]. Additionally, W might be also a bonding amino acid in the active site of ECA. On the other hand, the IC50 of ACE inhibitory potential found for fraction 45 was lower than for fraction 22. Fraction 45 contained VRWS, and the dipeptide VR was described as an antihypertensive agent by itself, with an IC50 of 52.80 μM [46]. In addition, even though it was bound to other amino acids, such as Y or SP, it retained its bioactivity [47,48]. Similarly, the presence of Tyr in the C-terminal end should promote binding to ACE and thus enhance its inhibition [49]. It was proved for the tripeptide 68 Molecules 2019, 24, 3033 IVY [50], also present in the sequence CIHNIVY in fraction 45. Finally, in this fraction, various peptides containing Tyr residue were found. In previous works on antioxidant peptides, it was stated that peptides with a length between 5 and 16 amino acids showed antiradical activity [51]. The results obtained in the present study support these findings, since the potential of antioxidant peptides had a molecular weight in the range of 500–1400 Da and contained 4–13 amino acid residues (Table 3). One of the peptides described in fraction 22 was LVRW, which could play a role as an antioxidant agent. Haung et al. [52] determined that the presence of the RW amino acid sequence in the C-terminal end of the polypeptide chain might be accounted for by its high antiradical activity. A similar effect was observed for the peptide ARVW, were the antioxidant activity of Trp is mainly due to its indole group [53]. In fraction 45, the DPKLTL sequence was identified, which might have antioxidant capacity owning to the presence of the DPK fragment (previously described as an antioxidant peptide). Its bioactivity could be explained by the presence of aspartic acid, which has the ability to donate electrons and hydrogen. On the other hand, this fragment is bound to hydrophobic amino acids, namely Pro and Leu, which in turn could enhance the radical scavenging abilities of this peptide [54]. Additionally, the DPKLTL peptide might exhibit antithrombotic activity due to the presence of three amino DPK acid residues, previously known for inactivating thrombin in its active site [25]. According to the work of Wang et al. [55], peptides with antithrombotic bioactivity usually contain 3–20 amino acid residues. Table 3 shows that the longest peptide identified in fraction 22 had a 13-amino acid chain. Moreover, it has been reported that sequences containing Val and Tyr might possess antithrombotic activity [23]. These amino acids were mainly found among peptide sequences present in fraction 45. On the other hand, Pro-Arg bonding N-terminal end of polypeptide chains has been reported as a thrombin inhibitor in its active site [26]. This bond is present in the structure of PRYDQY; however, more exhaustive studies are necessary on antithrombotic peptides as thrombin contain three main structural domains (a catalytic site and two exosites (I and II)), and enzyme inhibition can take place to different extents at any of these sites [26,56]. 4. Materials and Methods 4.1. Sample and Treatments Raw Amaranth hypochondriacus spp. seeds were obtained from Xochimilco, Mexico City, in July 2016. Seeds were ground in a Chopin mill, segregated according to their molecules size, and only a fraction with molecule size between 200 and 800 μm was selected for the experiments. Protein extraction was performed following the methodology described by Martínez and Añón [57] with some modifications. First, flour was defatted with n-hexane (10% w/v) for 24 h. Then it was suspended in deionized water 10% (w/v), and pH 9 was adjusted with NaOH. The crude was incubated for 30 min at room temperature and centrifuged during 20 min at 10,000 rpm. The soluble protein fraction found in the supernatant was precipitated by pH 5 adjustment, using HCl. The sample was centrifuged 20 min at 10,000 rpm, and the pellet pH was adjusted to 7. Following the lyophilization, the protein extract was stored at 4 ◦ C until further use. This protein was called protein extract. 4.2. Enzymatic Hydrolysis The enzymatic hydrolysis was performed according to the method described by Tironi and Añón [58]. Briefly, 5 g of the protein extract was diluted in 100 mL of deionized water (milli Q 18.2 MΩ*cm, Manufacturer, Darmstadt, Germany). For alcalase hydrolysis (H1), prior to the reaction, the pH of the solution was adjusted to 10, and alcalase (≥2.4 U/g, Anson Units; Sigma-Aldrich (St. Loui, MO, USA) was added at concentration of 8 μL/100 mg of the sample. For flavourzyme hydrolysis (H2), the pH of the reaction media was adjusted to 7 and flavourzyme (≥500 U/g; Sigma Aldrich, St. Louis, MO, USA) was added at concentration of 5 μL/100 mg of the sample. Finally, when both enzymes were 69 Molecules 2019, 24, 3033 used in a two-step continuous hydrolytic process (H3), after 2 h of hydrolysis with alcalase, the enzyme was inactivated by heating to 85 ◦ C for 10 min. The solution was adjusted to pH 7 and hydrolysis with flavourzyme was carried out. For all three methods, hydrolysis was followed up during 4 h and progress of the reaction was monitored every 20 min. The aliquots were heated to 85 ◦ C during 10 min and frozen until further use. 4.3. Free Amine Groups Analysis by TNBS Test In order to determine free amine groups released during the enzymatic hydrolysis of the amaranth proteins, the 2,4,6-Trinitrobenzenesulfonic acid (TNBS) test was performed, with some modifications, according to the protocol described by Sashidhar et al. [59]. Briefly, 5% TNBS solution (Sigma-Aldrich, (St. Loui, MO, USA) was diluted in 0.21 M phosphate buffer (pH 8.2) to a final concentration of 1% (v/v). Two milliliters of the prepared substrate was added to 2 mL of phosphate buffer (0.21 M; pH 8.2), and 0.25 mL of the test sample. The mixture was incubated during 1 h at 50 ◦ C and the reaction was stopped by the addition of 2 mL of 0.1 N HCl. The absorbance was read at 340 nm. Results were plotted against a calibration curve prepared by different glycine concentrations (0, 0.05. 0.1, 0.15, 0.2, and 0.25 mg/mL) using the following equation (R = 0.9972): y = 0.004x + 0.134 (1) Results are expressed as milligrams of free amines per liter (mg/L NH:− ). 4.4. Antihypertensive Activity Inhibitory effect on angiotensin converting enzyme (ACE) was evaluated spectrophotometrically according to the method of Cushman et al. [60] using Hippuril-Histidyl-Leucine (St. Loui, MO, USA) as substrate. Briefly, 5 mM substrate solution was prepared in 0.1 M sodium borate buffer pH 8.3 containing 0.3 M sodium chloride. A 5 mM 100 μL aliquot of substrate solution was mixed with 40 μL of the test sample before adding 10 μL of angiotensin converting enzyme (EC 3.4.15.1, 5.1 U/mg; Sigma-Aldrich). The reaction mixture was incubated during 1 h 15 min at 37 ◦ C and the enzyme was inactivated with 1 mL of 0.1 M HCl. The hippuric acid formed in this reaction was extracted with ethyl acetate, concentrated under reduced pressure, and finally re-dissolved in distilled water. The absorbance was measured at 220 nm in a GENESYS spectrometer. The inhibitory activity of ACE was calculated using the formula: % inhibitory activity = (AbsC − AbsM)/(AbsC −AbsB) × 100 (2) where: AbsC: Hippuric acid formed during the reaction with ACE without inhibitor. AbsB: Hippuril-Histidyl-Leucine that did not react and was extracted with ethyl acetate. AbsM: Hippuric acid formed after the reaction with ACE in the presence of inhibitory substance. 4.5. Antithrombotic Activity To evaluate antithrombotic activity, the methodology developed by Zhang et al. [61] was followed, including modifications of sodium chloride concentration proposed by Pérez-Escalante et al. [56]. Absorbance at 405 nm was measured before adding the enzyme to a microplate reader (Power Wave XS UV-Biotek, software KC Junior, USA) and after 10 min of incubation with the enzyme at 37 ◦ C. The percentage of inhibition (% inhibition) was calculated following the equation: % inhibitory activity = [(C − CB) − (S − SB)]/(C − CB)*100 where: CB (control blank): The initial absorbance of the negative control of inhibition. 70 (3) Molecules 2019, 24, 3033 C (control): The absorbance of the negative control after 10 min of incubation with thrombin. SB (sample blank): The initial absorbance of the sample. S (sample): The absorbance of the sample after 10 min of incubation with thrombin. 4.6. Antioxidant Activity Antioxidant activity was evaluated by three different methods. 4.6.1. ABTS test Radical scavenging capacity was measured using the radical cation 2,2 -azino-bis 3-ethylbenzothiazoline-6-sulphonic acid (ABTS•+), which was produced by mixing 7 mM of ABTS•+ stock solution with 2.45 mM potassium persulfate in the dark at room temperature for 16 h prior to its usage. The ABTS•+ solution was diluted with deionized water, so that absorbance measured at 754 nm was of 0.70 ± 0.02. An aliquot of 20 μL of test sample was added to 980 μL of the diluted ABTS•+ solution, and after 7 min of incubation at room temperature, the absorbance readings were taken at 754 nm in a microplate reader (Power Wave XS UV-Biotek, soft-ware KC Junior, USA). Antioxidant capacity was expressed as milligrams Trolox equivalents per liter (mg TE/100 g) [62]. 4.6.2. DPPH Test Radical scavenging activity was measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical. An ethanolic solution (7.4 mg/100 mL) of the stable DPPH• radical was prepared. Then, 100 μL of the test sample was added to 500 μL of DPPH• solution, and it was left to sit at room temperature for 1 h. The solution was stirred and centrifuged at 3000 rpm for 10 min. Finally, absorbance of the supernatant was measured at 520 nm in a microplate reader. Antioxidant activity was expressed as micromole of Trolox equivalents per liter (TE μmol/100 g) [63]. 4.6.3. FRAP Test FRAP antioxidant activity was evaluated according to the Benzie and Strein method [64], in which 0.3 M sodium acetate buffer pH 3.6, TPTZ, 20 mM FeCl3, and 5M FeSO4 were elaborated. For the preparation of FRAP buffer, TPTZ and FeCl3 were mixed at 10:1 ratio (v:v). Briefly, 30 μL of test sample was mixed with 900 μL of FRAP solution and 90 μL of distilled water, and agitated. The mixture was incubated during 10 min at 37 ◦ C, after which the absorbance was measured at 593 nm in a microplate reader. Results were compared with a calibration curve constructed for FeSO4 standards at concentrations ranging from 0 to 1000 mM. Antioxidant activity was expressed as micromole equivalents of Fe (II) per 100 g (μmolEFeII/100 g). 4.7. Identification of Bioactive Peptides by RP-HPLC 4.7.1. Sample Preparation For sample preparation, 10 mg/mL of freeze-dried amaranth protein hydrolysates were prepared with phosphate buffer (pH 7.8). The crude mixture was stirred during 1 h at 37 ◦ C and followed by centrifugation at 10,000 rpm for 10 min at room temperature. The resulting supernatant with corresponding soluble fractions was separated and stored. 4.7.2. RP-HPLC Separation Peptides were separated by reversed-phase chromatography on a HPLC (Waters, USA) system equipped with a C8 column (250 mm × 4.6 mm × 5 mm; Waters) and photodiode array detector (Spectra System Thermo Scientific, USA) A gradient elution was applied from 100 to 0% A in 56 min at flow rate of 1 mL/min. A binary mobile phase consisted of solvent A: 0.065% trifluoroacetic acid [TFA] in water/acetonitrile [ACN] 98:2, and solvent B: 0.065% TFA in water/ACN 35:65. Detection was performed at 280 nm and temperature was set at 40 ◦ C. The injection volume was 200 μL. Fractions 71 Molecules 2019, 24, 3033 were manually collected every 1 min in Eppendorf tubes and protein content was evaluated by the Bradford assay. Finally, samples were lyophilized (Labconco DrySystem/freezone 4.5) and bioactivity analysis was carried out only on the lyophilized samples containing protein. 4.7.3. MALDI-TOF Spectrometry The collected fractions with the highest bioactivities were filtered through a Minisart RC4 filter (0.45 μm) and analyzed by a matrix-assisted laser desorption ionization (MALDI) mass spectrometer, equipped with a delayed extraction source and a 355 nm pulsed nitrogen laser. A MALDI scoutMTPTM was run in the linear mode. A 100-times diluted sample was mixed with 1 volume of 20 mg/mL of sinapinic acid in acetonitrile/water 50:50 (v/v). Finally, 0.5 μL of the mixture was deposited onto the MALDI target plate. All spectra were the result of signal averaging of 200 shots. The MALDI-TOF/TOF MS/MS was run in the positive refractor mode. The peptide sequencing was performed by processing the MS/MS spectra using Auto eXecute software. Mascot (Matrix Science Inc., Boston, MA, US) software was used to identify and characterize peptide structures. 4.8. Statistical Analysis All data were obtained in triplicate and expressed as mean ± standard deviation (SD). The data were analyzed by one-way analysis of variance (ANOVA) tests, and the differences among means were compared using the Tukey test with significance level set at p < 0.05, using the SPSS® System for WINTM version 15.0 (IBM®Armonk, New York, NY, USA). 5. Conclusions Amaranth protein hydrolysate, obtained through enzymatic reaction with alcalase and flavourzyme in a sequential two-step hydrolytic process, may be a source of bioactive peptides. Sequences were different from those obtained in more commonly performed enzymatic hydrolysis using pepsin and pancreatin for amaranth proteins digestion. The biological activities identified for some peptide sequences in this study have been proven in other food sources. Moreover, novel amino acid chains with possible multi-functional activities were identified. This is the first report of the multiple bioactivities of peptide fractions derived from hydrolyzed amaranth proteins. Additionally, this study shows that amaranth hydrolyzed with alcalase and flavourzyme could be used in the nutraceutical industry, as a value-added ingredient with multi-functional bioactive properties. Author Contributions: Conceptualization, L.G.G.-O. and A.A.-N.; methodology, L.G.G.-O., A.A.-N. and G.M.R.-S.; software, E.C.-L., P.R.-L. and A.C.-S.; validation, P.R.-L., A.C.-S. and L.G.G.-O.; formal analysis, L.G.G.-O., A.A.-N. and P.R.-L.; investigation, A.A.-N.; resources, A.C.-S. and L.G.G.-O.; data curation, E.C.-L. and A.C.-S.; writing—original draft preparation, A.A.-N. and L.G.G.-O.; writing—review and editing, A.A.-N. and L.G.G.-O.; visualization, L.G.G.-O.; supervision, L.G.G.-O. and G.M.R.-S.; project administration, L.G.G.-O.; funding acquisition, L.G.G.-O., E.C.-L. and G.M.R.-S. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. 72 Molecules 2019, 24, 3033 Appendix A Figure A1. 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[CrossRef] [PubMed] Sample Availability: Samples of the compounds are not available from the authors. © 2019 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/). 76 molecules Article Green Extraction of Phenolic Acids from Artemisia argyi Leaves by Tailor-Made Ternary Deep Eutectic Solvents Li Duan 1,† , Chenmeng Zhang 1,† , Chenjing Zhang 1 , Zijing Xue 2 , Yuguang Zheng 2, * and Long Guo 2, * 1 2 * † College of Chemistry and Material Science, Hebei Normal University, Shijiazhuang 050024, China School of Pharmacy, Hebei University of Chinese Medicine, Shijiazhuang 050200, China Correspondence: zyg314@163.com (Y.Z.); guo_long11@163.com (L.G.); Tel.: +86-0311-89926316 (Y.Z.); +86-0311-89926017 (L.G.) These authors contributed equally to this work. Academic Editor: Jose M. Miranda Received: 25 June 2019; Accepted: 3 August 2019; Published: 5 August 2019 Abstract: The Artemisia argyi leaf (AL) has been used as a traditional medicine and food supplement in China and other Asian countries for hundreds of years. Phytochemical studies disclosed that AL contains various bioactive constituents. Among bioactive constituents, phenolic acids have been recognized as the main active compounds in AL. To the best of our knowledge, no research has been focused on extraction method for the bioactive phenolic acids from AL. Nowadays, deep eutectic solvents (DESs) are emerging as a new type of green and sustainable solvent for efficient extraction of bioactive compounds from natural products. In the present study, an environmentally friendly extraction method based on DESs was established to extract bioactive phenolic acids from ALs. Diverse tailor-made solvents, including binary and ternary DESs, were explored for simultaneous extraction of four phenolic acids (3-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid) from AL. The results indicated that the ternary DES composed of a 2:1:2 molar ratio of choline chloride, malic acid, and urea showed enhanced extraction yields for phenolic acids compared with conventional organic solvents and other DESs. Subsequently, the extraction parameters for the four phenolic acids by selected tailor-made DESs, including liquid–solid ratios, water content (%) in the DESs, and extraction time, were optimized using response surface methodology and the optimal extraction conditions were: extraction time, 23.5 min; liquid–solid ratio, 57.5 mL/g (mL of DES/g dry weight of plant material); water content, 54%. The research indicated that DESs were efficient and sustainable green extraction solvents for extraction of bioactive phenolic acids from natural products. Compared to the conventional organic solvents, the DESs have a great potential as possible alternatives to those organic solvents in health-related areas such as food and pharmaceuticals. Keywords: deep eutectic solvents; Artemisia argyi leaves; phenolic acids; extraction; response surface methodology 1. Introduction The Artemisia argyi leaf (AL), which is widely distributed in China and other Asian countries, has been used as a traditional medicine or food supplement for hundreds of years [1]. As a traditional Chinese medicine, AL is reported to possess antioxidant, antibacterial, anti-inflammatory, anticancer, hemostatic, and analgesic activities and is commonly used for treatment of hemorrhage, pain, eczema, and menstruation-related symptoms [2–4]. AL is also consumed as a food ingredient because of its Molecules 2019, 24, 2842; doi:10.3390/molecules24152842 77 www.mdpi.com/journal/molecules Molecules 2019, 24, 2842 delicious flavor and distinctive smell. In China, AL is used as a common condiment and colorant for the traditional Chinese food “Qingtuan”. In Japan, AL is added into food as an additive to enhance the flavor and nutrition [5]. Although the AL has been used as an herbal medicine and food ingredient for a long time, studies on its bioactive compositions are still limited. Phytochemical studies disclosed that AL contains various bioactive constituents, mainly including volatile oils, phenolic acids, flavonoids, and terpenoids [6,7]. Among bioactive constituents, phenolic acids have been recognized as the main active compounds in AL [8,9]. Therefore, the content of the phenolic acids is an important index for quality analysis and normal applications of AL. To date, several bioactive phenolic acids, such as 3-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid, have been isolated from AL. A number of analytical methods, including high-performance liquid chromatography (HPLC) and high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS), have been used for qualitative and quantitative analysis of the main phenolic acids in AL [5,7]. However, the research focused on extraction methods for bioactive phenolic acids from AL is still limited. Nowadays, conventional organic solvents, such as alcohols, ethyl acetate, acetone, and chloroform, are widely used in the extraction of bioactive components from natural sources [10]. However, the consumption of large amounts of these volatile and hazardous organic solvents may contribute to environmental pollution and leave unacceptable solvent residues in extracts. Therefore, in analytical chemistry, green extraction methods which are environmentally friendly and sustainable for sample preparation have received more and more attention [11]. Since being introduced as a new type of green solvent, deep eutectic solvents (DESs) have rapidly gained great interest as sustainable alternatives to conventional organic solvents. DESs are prepared by simply mixing two or more naturally occurring, inexpensive, and biodegradable components together to obtain a eutectic mixture [12]. The availability, low cost, biodegradability, and environmental friendliness of the components make the DESs versatile alternatives to conventional organic solvents [13]. Due to their excellent properties, including biodegradability, low toxicity, solute stabilization, and low cost, DESs have been widely used in organic synthesis, separation processes, and biomedical applications [14,15]. Generally, DESs are prepared by simply mixing two or more naturally occurring, inexpensive, and biodegradable components together to obtain eutectic mixtures. As tunable solvents, diverse possible combinations of starting components have different targeted functionality, which means that we can increase the solubility and extraction efficiency of DESs for target compounds by selecting appropriate combinations of starting components. The tailor-made DESs have tremendous potential for efficient and simultaneous extraction and separation of compounds which have obvious differences in nature [16]. Recently, many reports have shown that tailor-made DESs were successfully employed in the extraction and separation of different kinds of bioactive compounds, such as phenolic acids, flavonoids, alkaloids, and saponins, from various plant materials [17–22]. Nonetheless, most of the research used tailor-made binary DESs as extraction solvents for bioactive compound extractions, the number of reports on the application of tailor-made ternary DESs for extraction is still limited, and the efficiency of DESs for extraction of bioactive phenolic acids from AL still remains unknown. In the present study, in order to evaluate DESs for the extraction of phenolic acids, several tailor-made binary and ternary DESs were used for simultaneous extraction of four bioactive phenolic acids (3-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid) from AL, and the extraction efficiency of tailor-made DESs was compared with that of conventional organic solvents. Moreover, the extraction parameters for phenolic acids by tailor-made DESs were systematically optimized using response surface methodology (RSM). 78 Molecules 2019, 24, 2842 2. Results and Discussion 2.1. Chromatographic Conditions and Method Validation In order to achieve a rapid and efficient analysis of the four phenolic acids (3-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid) in AL, different mobile phases (including water–methanol, water–acetonitrile, formic acid water–methanol, and formic acid water–acetonitrile), flow rates (0.7 mL/min, 0.8 mL/min, and 1.0 mL/min), as well as column temperatures (15 ◦ C, 20 ◦ C, 25 ◦ C, and 30 ◦ C, Supplementary Materials Figure S1) were examined and compared. As a result, the formic acid water–acetonitrile system at 15 ◦ C with a flow rate of 0.7 mL/min was finally selected for the suitable analysis duration, greater separation ability, and better peak shapes. The gradient elution was as follows: 0–5 min, 12% B; 5–15 min, 12–22% B; 15–25 min, 22% B; 25–35 min, 22–25% B; 35–40 min, 25–40% B. The typical HPLC chromatograms of the AL sample and four phenolic acids reference standards are shown in Figure 1. Figure 1. The typical HPLC chromatograms of (A) Artemisia argyi leaves sample (20 mg/mL) and (B) four phenolic acids reference standards. (1. 3-caffeoylquinic acid, 4.96 μg/mL; 2. 3,4-di-O-caffeoylquinic acid, 4.24 μg/mL; 3. 3,5-di-O-caffeoylquinic acid, 5.36 μg/mL; 4. 4,5-di-O-caffeoylquinic acid, 4.16 μg/mL). Method validation of quantitative analysis was performed. The linearity, limit of detections (LODs), limit of quantifications (LOQs), precision, repeatability, stability, and accuracy for the four phenolic acids were validated. Each calibration curve was performed with six different concentrations in triplicate. All calibration curves were of good linearity with high correlation coefficient (R2 > 0.9997) 79 Molecules 2019, 24, 2842 over the tested range. The LODs and LOQs of the four analytes were defined by the concentration that generated peaks with signal-to-noise values of 3 and 10 using standard solutions. The precision of the developed method was determined by the intra- and interday variations. For the intraday test, the sample was analyzed six times within the same day, while for the interday test, the sample was examined in duplicates for three consecutive days. The relative standard deviations (RSDs) of intraday and interday precisions were less than 1.61% and 2.14%, respectively. For the repeatability test, six replicates of the same sample were prepared and analyzed, and for the stability test, the same sample was stored at room temperature and analyzed by replicate injection analysis at 0, 2, 4, 8, 12, and 24 h. The repeatability presented as RSDs was less than 2.71%, and the stability was less than 2.06%. The recovery was used to evaluate the accuracy of the method. Known amounts of the four phenolic acids standard solutions were added into the same samples in sextuplicate, and then extracted and analyzed with the same procedures. The recovery of each analyte was calculated by the equation: Recovery (%) = (Detected amount − Original amount)/Spiked amount × 100%. The overall recoveries of the four analytes were in the range of 101.15–102.86% with RSDs less than 1.67%. The data of method validation are shown in Supplementary Materials Table S1. 2.2. Screening of DESs for the Extraction of Phenolic Acids from AL 2.2.1. Extraction of Phenolic Acids by Binary DESs DESs are composed of a mixture consisting of hydrogen bond acceptors (HBAs) with hydrogen bond donors (HBDs). The composition of DESs determines their physicochemical properties and consequently greatly influences extraction efficiency of natural compounds [23]. In the present study, six choline-chloride-based binary DESs, ChCl-Ma, ChCl-Ur, ChCl-Ga, ChCl-Pa, ChCl-Eg, and ChCl-Gl, were successfully synthesized (Table 1) and selected to test their extraction efficiency for phenolic acids. The high viscosity of most DESs at room temperature restricted their application due to a slow mass transfer. To overcome this problem, extraction conditions were adjusted to reduce the viscosity by increasing extraction temperature and adding a certain amount of water [24]. In the initial screening experiments, 75% DES solution in water (v/v) was employed, and the extraction conditions were as follows: extraction time, 30 min; extraction temperature, 50 ◦ C; liquid–solid ratio, 50 mL/g (mL of DES/g dry weight of plant material). Table 1. The binary DESs synthesized in this study. NO. Abbreviation Component 1 Component 2 Molar Ratio BD-1 BD-2 BD-3 BD-4 BD-5 BD-6 ChCl-Ma ChCl-Ur ChCl-Ga ChCl-Pa ChCl-Eg ChCl-Gl choline chloride choline chloride choline chloride choline chloride choline chloride choline chloride dl-malic acid urea glutaric acid propanedioic acid ethylene glycol glycerol 1:1 1:2 1:1 1:1 1:3 1:2 Extraction yields of the four phenolic acids (3-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid) with different binary DESs are shown in Figure 2. The initial screening results indicated that the extraction efficiency for phenolic acids was influenced by the types of DES solvents, and different types of DESs resulted in different extraction yields. In general, extraction yields of the four phenolic acids followed the order 3,5-di-O-caffeoylquinic acid > 3-caffeoylquinic acid > 4,5-di-O-caffeoylquinic acid > 3,4-di-O-caffeoylquinic acid. The phenolic acid, 3,5-di-O-caffeoylquinic acid, showed high extraction efficiency in ChCl-Eg with the concentration 9.35 ± 0.03 mg/g (mg/g dry weight of plant material), followed by ChCl-Ma (9.05 ± 0.05 mg/g) and ChCl-Pa (8.71 ± 0.03 mg/g). For 3-caffeoylquinic acid, ChCl-Ma (4.45 ± 0.03 mg/g) and ChCl-Ur (4.47 ± 0.03 mg/g) led to higher extraction yields. For 4,5-di-O-caffeoylquinic acid, ChCl-Ma (4.10 ± 0.02 mg/g), ChCl-Pa (3.90 ± 0.03 mg/g), and ChCl-Eg (3.72 ± 0.02 mg/g) exhibited higher extraction efficiency. For 80 Molecules 2019, 24, 2842 3,4-di-O-caffeoylquinic acid, ChCl-Ur (3.32 ± 0.03 mg/g) showed higher extraction efficiency, followed by ChCl-Ma (3.03 ± 0.02 mg/g) and ChCl-Pa (2.97 ± 0.03 mg/g).In conclusion, the extraction yields of the total phenolic acids were calculated, and it was clearly shown that ChCl-Ma (BD-1) was the best binary DES for extraction of four phenolic acids from AL with the extraction yields 20.64 ± 0.08 mg/g. Figure 2. Extraction yields of different binary DESs and different ratios of methanol for 3-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, and total four phenolic acids from Artemisia argyi leaves (n = 3). Numbers on horizontal axis are in accordance with the numbers in Table 1. Error bars indicate the SD (n = 3). Extraction yields which do not share the same letter are significantly different (p < 0.05). In order to comprehensively compare the extraction efficiency of DESs and conventional solvents for extraction of phenolic acids from AL, different ratios of methanol (25% MeOH, 50% MeOH, 75% MeOH, and 100% MeOH), efficient solvents commonly used in the extraction of bioactive compounds from natural products, were selected as reference solvents [7]. As shown in Figure 2, among the different ratios of MeOH, 75% MeOH displayed the highest extraction efficiency for the four phenolic acids. It was clear that all the six tailor-made binary DESs exhibited higher extraction efficiency for the four phenolic acids compared with 100% MeOH (16.56 ± 0.06 mg/g). However, compared to 75% MeOH (22.41 ± 0.03 mg/g), none of the six binary DESs exhibited a higher extraction yield. Based on the results above, ChCl-Ma (BD-1) was selected as the best binary DES for extraction of phenolic acids from AL, and we attempted to synthesize a series of ternary DESs based on ChCl-Ma to enhance extraction efficiency of phenolic acids in further tests. 2.2.2. Extraction of Phenolic Acids by Ternary DESs DESs can be synthesized from two or more components. According to the previous report, the ternary DESs forming with addition of glycerol to the binary DESs show lower melting points and viscosities [25]. Adding the third component to binary DESs may change the properties of ternary DESs and thus influence the extraction yields. In this study, based on the best binary DES (ChCl-Ma) selected above, several tailor-made ternary DESs were designed in order to further enhance the extraction yields of phenolic acids from AL. Five ternary DESs, including ChCl-Ma-Ur, ChCl-Ma-Ga, ChCl-Ma-Pa, ChCl-Ma-Eg, and ChCl-Ma-Gl, were successfully synthesized (Table 2) and further used to test their 81 Molecules 2019, 24, 2842 extraction efficiency for phenolic acids. The extraction yields of the four phenolic acids employing the tailor-made ternary DESs are listed in Figure 3. The results indicated that compared to the binary DESs, some tailor-made ternary DESs could change the extraction efficiency. Almost all of the tailor-made ternary DESs exhibited higher extraction efficiency for the four phenolic acids compared with the best binary DES (ChCl-Ma), except ChCl-Ma-Gl (TD-12 and TD-13). It could be speculated that the addition of the third component might reduce the viscosity of DESs, enhancing the hydrogen bond interactions between DESs and the target components, thus improving the extraction yields [26]. Table 2. The ternary DESs synthesized in this study. NO. Abbreviation Component 1 Component 2 Component 3 Molar Ratio Extraction Yield of Four Phenolic Acids (mg/g) TD-1 TD-2 TD-3 TD-4 TD-5 TD-6 TD-7 TD-8 TD-9 TD-10 TD-11 TD-12 TD-13 ChCl-Ma-Ur ChCl-Ma-Ur ChCl-Ma-Ur ChCl-Ma-Ga ChCl-Ma-Ga ChCl-Ma-Pa ChCl-Ma-Pa ChCl-Ma-Pa ChCl-Ma-Pa ChCl-Ma-Eg ChCl-Ma-Eg ChCl-Ma-Gl ChCl-Ma-Gl choline chloride choline chloride choline chloride choline chloride choline chloride choline chloride choline chloride choline chloride choline chloride choline chloride choline chloride choline chloride choline chloride DL-malic acid DL-malic acid DL-malic acid DL-malic acid DL-malic acid DL-malic acid DL-malic acid DL-malic acid DL-malic acid DL-malic acid DL-malic acid DL-malic acid DL-malic acid urea urea urea glutaric acid glutaric acid propanedioic acid propanedioic acid propanedioic acid propanedioic acid ethylene glycol ethylene glycol glycerol glycerol 2:1:1 2:2:1 2:1:2 2:1:1 2:2:1 2:2:1 2:1:1 1:1:1 2:1:2 1:2:0.5 2:2:1 1:2:0.5 2:2:1 21.92 ± 0.04 22.26 ± 0.04 22.43 ± 0.02 21.41 ± 0.04 21.89 ± 0.02 20.81 ± 0.06 21.30 ± 0.05 21.55 ± 0.04 22.08 ± 0.03 21.67 ± 0.04 22.12 ± 0.05 20.02 ± 0.02 20.09 ± 0.04 Figure 3. Extraction yields of different ternary DESs for 3-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, and total four phenolic acids from Artemisia argyi leaves (n = 3). Numbers on horizontal axis are in accordance with the numbers in Tables 1 and 2. Error bars indicate the SD (n = 3). Extraction yields which do not share the same letter are significantly different (p < 0.05). Several studies have revealed that different molar ratios of DES components result in different viscosities and surface tensions, thereby affecting the extraction efficiency of target components from natural biomass [27]. Thus, in the study, the effects of molar ratios of tailor-made ternary DESs were also investigated. As shown in Figure 3, the optimal molar ratio of ChCl-Ma-Ur was 2:1:2 (TD-3) 82 Molecules 2019, 24, 2842 and the extraction yields of the four phenolic acids were 22.43 ± 0.02 mg/g. The optimal molar ratio of ChCl-Ma-Ga was 2:2:1 (TD-5) and the extraction yields of the four phenolic acids were 21.89 ± 0.02 mg/g. The optimal molar ratio of ChCl-Ma-Pa was 2:1:2 (TD-9) and the extraction yields of the four phenolic acids were 22.08 ± 0.03 mg/g. The optimal molar ratio of ChCl-Ma-Eg was 2:2:1 (TD-11) and the extraction yields of the four phenolic acids were 22.12 ± 0.05 mg/g. The optimal molar ratio of ChCl-Ma-Gl was 2:2:1 (TD-13) and the extraction yields of the four phenolic acids were 20.09 ± 0.04 mg/g. None of the tailor-made ternary DESs with different molar ratios exhibited an extraction yield higher than 75% MeOH, the best conventional solvent. However, the tailor-made ternary DES, ChCl-Ma-Ur (2:1:2), produced similar extraction efficiency for the four phenolic acids compared to 75% MeOH (p > 0.05). Based on the results, the ChCl-Ma-Ur with a molar ratio of 2:1:2 (TD-3) was selected as the best ternary DES for extraction of phenolic acids from AL. 2.3. Optimization of the Extraction Parameters for Phenolic Acids The above extraction investigations showcased that the tailor-made ternary DES, ChCl-Ma-Ur (2:1:2), was selected as the best ternary DES for extraction of phenolic acids from AL. In order to obtain the optimal extraction efficiency for phenolic acids from AL, several numerical variables that could affect the extraction efficiencies were optimized by RSM, a valuable statistical technique to determine the optimal values of the independent variables and to enable the user to effectively investigate the effects of multiple factors [28,29]. Similar to previous studies [30], three variables of extraction time, liquid–solid ratios, and water content in DESs were evaluated using Box–Behnken design (BBD). After determining the range of extraction factors on the basis of preliminary single-factor test, the extraction time (A), liquid–solid ratios (B), and water content (C) were varied at three levels (−1, 0, +1) as follows: A, 8.0–40.0 min; B, 17.5–57.5 mL/g; C 20–70%. The total extraction amounts of the four phenolic acids were taken as the responses of the design experiments. The experimental orders, levels of variables, and response values are summarized in Table 3. Table 3. The experimental orders, levels of variables, and response values in Box–Behnken design. Run 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Factors Responses Extraction Time (A, min) Liquid–Solid Ratios (B, mL/g) Water Content (C, %) Total Extraction Amounts of Four Phenolic Acids (mg/g) 24.0 40.0 24.0 8.0 24.0 40.0 24.0 24.0 8.0 24.0 24.0 24.0 8.0 40.0 24.0 8.0 40.0 37.5 57.5 17.5 37.5 37.5 17.5 57.5 37.5 57.5 17.5 57.5 37.5 17.5 37.5 37.5 37.5 37.5 45 45 20 20 45 45 70 45 45 70 20 45 45 70 45 70 20 22.60 22.61 18.47 16.13 22.10 21.96 20.85 21.40 22.34 18.86 15.51 22.65 21.27 19.84 22.02 20.06 17.00 Experiments conducted according to the design resulted in a second-order polynomial equation for total extraction amounts (Y) expressed using coded variables (A, B, and C) as follows: Y = 22.16 − 0.20A + 0.095B + 1.56C − 0.11AB − 0.27AC + 1.24BC − 0.14A2 + 0.027B2 − 3.76C2 83 Molecules 2019, 24, 2842 The model was evaluated in terms of the square of correlation coefficient (R2 ) and the lack of fit by the analysis of variance (ANOVA) at the 95% confidence level (Table 4). The resulting R2 value was 0.9765, indicating that the experimental data were in relatively good agreement with predicted extraction yields. The lack-of-fit value, which evaluates the failure of the model to represent the data in the experimental domain points, was insignificant for the response with p-value of 0.3789 (p > 0.05). Table 4. The ANOVA results of the quadratic multiple regression model for phenolic acids. Source Sum of Squares df Mean Square F Value p-Value Prob > F Significance Model A B C AB AC BC A2 B2 C2 Residual Lack of Fit Pure Error R2 86.54 0.32 0.073 19.53 0.045 0.3 6.13 0.08 3.10 × 10−3 59.51 2.08 1.04 1.04 0.9765 9 1 1 1 1 1 1 1 1 1 7 3 4 9.62 0.32 0.073 19.53 0.045 0.3 6.13 0.08 3.10 × 10−3 59.51 0.3 0.35 0.26 32.38 1.08 0.24 65.78 0.15 1.02 20.64 0.27 0.01 200.42 <0.0001 0.3323 0.6359 <0.0001 0.7085 0.3468 0.0027 0.6188 0.9215 <0.0001 significant 1.34 0.3789 not significant Statistical analysis and 3D response plots (Figure 4) illustrated the significant variables affecting extraction yields of phenolic acids and the interaction effects between the variables. In the model, the water content (C) showed significant effects on the extraction efficiency of phenolic acids (p < 0.0001). Based on the adequate model, the calculated optimum conditions for the extraction of phenolic acids from AL were as follows: extraction time, 23.5 min; liquid–solid ratio, 57.5 mL/g; water content, 54%. Triplicate experiments were carried out under the optimal extraction conditions and mean values of experimental results were compared with the predicted values. Under the optimum conditions, the total extraction amounts of the four phenolic acids were 22.80 mg/g, which were closed to the predicted values of 22.79 mg/g. The results obtained through confirmation experiments indicated the model is adequate for predicting the expected optimization. Figure 4. Response surface plots of the model for extraction of phenolic acids from Artemisia argyi leaves. (A. extraction time, min; B. liquid–solid ratios, mL/g; C. water content, %). 3. Materials and Methods 3.1. Materials and Reagents AL samples were purchased from a Chinese herbal medicine market (Anguo, China). The samples were dried in the shade and stored in the desiccator. The AL samples were authenticated by Prof. Yuguang Zheng from Department of Pharmacognosy, Hebei University of Chinese Medicine and the voucher specimens were deposited in Hebei University of Chinese Medicine, Shijiazhuang, China. Four phenolic acids reference compounds, 3-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid, were purchased from Chengdu Must Bio-technology Co., Ltd. (Chengdu, China). The purities of the four reference compounds were 84 Molecules 2019, 24, 2842 determined to be higher than 98% by high-performance liquid chromatography diode array detection analysis. The structures of the four phenolic acids are shown in Figure 5. Figure 5. Chemical structures of 3-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid. 3,4-di-O-caffeoylquinic acid, Chemical compounds for DES preparation including choline chloride (ChCl), DL-malic acid (Ma), urea (Ur), glutaric acid (Ga), propanedioic acid (Pa), ethylene glycol (Eg), glycerol (Gl) were obtained from Aladdin Reagent Company (Shanghai, China). Acetonitrile, methanol, and formic acid (chromatographic grade) were purchased from Merck (Darmstadt, Germany). Deionized water was prepared by a Milli-Q water purification system (Millipore, Billerica, MA, USA). Other reagents and chemicals used in this work were of analytical grade. 3.2. HPLC Analysis HPLC analysis was performed on an Agilent 1260 HPLC system equipped with a quaternary pump, a degasser, an autosampler, a thermostated column compartment, and a diode array detector (Agilent Technologies, Palo Alto, CA, USA). Chromatographic separation was achieved on an Agilent SB C18 column (4.6 × 250 mm, 5 μm). The mobile phase was composed of 0.1% formic acid water (A) and acetonitrile (B) with a gradient elution as follows: 0–5 min, 12% B; 5–15 min, 12–22% B; 15–25 min, 22% B; 25–35 min, 22–25% B; 35–40 min, 25–40% B. The detection wavelength was 330 nm. The flow rate was set at 0.7 mL/min and the column temperature was set at 15 ◦ C. 3.3. Preparation of DESs The DESs were synthesized according to the previous studies [31]. Briefly, different DESs were obtained by simply mixing hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs) together in a proper molar ratio with constant stirring at 80 ◦ C until a clear and homogeneous liquid formed. 3.4. Extraction of Phenolic Acids From AL The AL samples were powdered and screened through 40 mesh sieves. An accurately weighed powder (20 mg) was extracted with 1 mL of different solvents (different DESs, 25% MeOH, 50% MeOH, 75% MeOH, and 100% MeOH) in a 2 mL centrifuge tube by ultrasonic cleaner (KQ5200B, 200W, 40 kHz, Kunshan, China) for 30 min. After extraction, the extracted solution was centrifuged at 13,000 rpm/min 85 Molecules 2019, 24, 2842 for 10 min. Then, 0.1 mL of supernatant was sampled and diluted 10-fold with methanol. The diluted solution (2 μL) was injected into the HPLC instrument for analysis. Three replicates of each sample were prepared and analyzed (n = 3). 3.5. DESs Tailoring In order to determine the effect of the DES compositions on the extraction of the four phenolic acids from AL, six ChCl-based binary DESs, including ChCl-Ma, ChCl-Ur, ChCl-Ga, ChCl-Pa, ChCl-Eg, and ChCl-Gl, were successfully synthesized in initial screening. Then, the extraction efficiency of the different binary DESs for the four phenolic acids was investigated and the optimal binary DES was selected. Next, different efficient components were added into the optimal binary DES obtained in initial screening to design different ternary DESs. Five tailored ternary DESs with different molar ratios, including ChCl-Ma-Ur, ChCl-Ma-Ga, ChCl-Ma-Pa, ChCl-Ma-Eg, and ChCl-Ma-Gl, were synthesized and the extraction efficiencies for the four phenolic acid compounds were tested. 3.6. Optimization of the DES Extraction Parameters for Phenolic Acids The optimal extraction parameters for the phenolic acids from AL were obtained using RSM. RSM was performed using the Design-Expert Ver. 8.0.6 (Stat-Ease Inc., Minneapolis, MN, USA). After determining the range of extraction variables on the basis of preliminary single-factor test, Box–Behnken design (BBD) was used to find the optimal values for three independent variables: extraction time (A), liquid–solid ratios (B), and water content (%) in DESs (C). The total extraction amounts of the four phenolic acids were taken as the response of the design experiments. Regression analysis was performed according to the experimental data. Subsequently, additional confirmation experiments were conducted to confirm the validity of the statistical experimental strategies. 4. Conclusions In this work, a green and efficient extraction method using tailor-made DESs as extraction solvents was established for extraction of bioactive phenolic acids from AL. Six binary DESs and five ternary DESs were successfully synthesized and used to extract four phenolic acids from AL. The results indicated that the tailor-made DESs were efficient solvents for the extraction of phenolic acids. The ternary DES, ChCl-Ma-Ur with a molar ratio of 2:1:2, proved to be the most efficient solvent for phenolic acid extraction from AL. Moreover, the optimal extraction conditions for phenolic acids by ChCl-Ma-Ur (2:1:2) were determined using RSM and the optimal extraction conditions are extraction time, 23.5 min; liquid–solid ratio, 57.5 mL/g; and water content, 54%. The present study suggests that the DES-based extraction method is efficient and sustainable for extraction of bioactive phenolic acids from AL. The DESs are truly designed and efficient solvents which could be used as green solvents for the extraction of bioactive compounds from natural products and have a great potential as possible alternatives to those organic solvents in health-related areas such as food and pharmaceuticals. Supplementary Materials: The Supplementary Materials are available online. Author Contributions: Conceptualization, L.D. and L.G.; Data curation, C.Z. (Chenmeng Zhang); Investigation, L.D. and C.Z. (Chenmeng Zhang); Methodology, C.Z. (Chenjing Zhang); Project administration, Y.Z.; Resources, Z.X.; Writing—original draft, L.D. and L.G.; Writing—review & editing, Y.Z. and L.G. Funding: This research was funded by National Natural Science Foundation of China (81803697, 81803685), Natural Science Foundation of Hebei Province (H2018205185, H2018423032), Research Foundation of Hebei Province Education Department (BJ2019006, QN2017093), Research Foundation of Hebei Provincial Administration of Traditional Chinese Medicine (2018108), and Innovation Team of Hebei Province Modern Agricultural Industry Technology System (HBCT2018060205). Conflicts of Interest: The authors declare no conflict of interest. 86 Molecules 2019, 24, 2842 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Abad, M.J.; Bedoya, L.M.; Apaza, L.; Bermejo, P. The artemisia, Genus: A review of bioactive essential oils. 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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/). 88 molecules Article Biochemical, Micronutrient and Physicochemical Properties of the Dried Red Seaweeds Gracilaria edulis and Gracilaria corticata Thomas Rosemary 1 , Abimannan Arulkumar 1,2 , Sadayan Paramasivam 1, *, Alicia Mondragon-Portocarrero 3 and Jose Manuel Miranda 3 1 2 3 * Department of Oceanography and Coastal Area Studies, School of Marine Sciences, Alagappa University, Thondi Campus, Thondi-623 409, India; rosemarythomas@gmail.com (T.R.); aruul3@gmail.com (A.A.) Department of Biotechnology, Achariya Arts and Science College, Villianur, Puducherry-60 110, India Departamento de Química Analítica, Nutrición y Bromatología, Facultade de Veterinaria, Universidade de Santiago de Compostela, Pabellón 4, Planta Baja, 27002 Lugo, Spain; aliciamondragon@yahoo.com (A.M.-P.); josemanuel.miranda@usc.es (J.M.M.) Correspondence: drparamsan@gmail.com; Tel.: +91-9047733300; Fax: +91-4565-225525 Academic Editor: Gavino Sanna Received: 25 April 2019; Accepted: 11 June 2019; Published: 14 June 2019 Abstract: The present study sought to evaluate the nutritional composition and physicochemical properties of two dried commercially interesting edible red seaweeds, Gracilaria corticata and G. edulis. Proximate composition of the dried seaweeds revealed a higher content in carbohydrates (8.30 g/100 g), total crude protein (22.84 g/100 g) and lipid content (7.07 g/100 g) in G. corticata than in G. edulis. Fatty acids profile showed that G. corticata samples contain higher concentrations of saturated fatty acids, such as palmitic and stearic acids, and polyunsaturated ones such as α-linolenic and docosahexaenoic acids. Contrariwise, G. edulis contained higher amounts of monounsaturated oleic acid. Total amino acid content was 76.60 mg/g in G. corticata and 65.42 mg/g in G. edulis, being the essential amino acid content higher in G. edulis (35.55 mg/g) than in G. corticata (22.76 mg/g). Chlorophyll a was found in significantly higher amounts in G. edulis (17.14 μg/g) than G. corticata, whereas carotenoid content was significantly higher in G. corticata (12.98 μg/g) than in G. edulis. With respect to physical properties, both water- and oil-holding capacities were similar in both seaweeds, whereas swelling capacity was higher in G. edulis. In view of the results, the present study suggests that G. corticata and G. edulis contains important nutrients for human health and are possible natural functional foods. Keywords: water holding capacity; seaweeds; Gracilaria; carbohydrates; fatty acids; vitamins 1. Introduction Seaweeds are very important natural resources from the oceans that are employed as human foods and animal feeds in their whole form, and as sources of polysaccharides (mainly alginates, carrageenans and agar), carotenoids, lipids, vitamins, minerals, dietary fiber, proline and amino acids for use in food and pharmaceutical industry [1]. Seaweeds have been included for a long time in the traditional diet of East Asian countries such as Japan, Korea and China; more recently, their presence in all forms in the diet of Western countries has been progressively increasing [2]. Seaweeds are considered healthy foods because, despite their low caloric content, they are rich in important nutrients such as protein, essential amino acids, vitamins, minerals and some bioactive compounds [1]. Seaweeds are also an excellent source of both soluble and insoluble dietary fiber. Among red algae, the genus Gracilaria contains a broad diversity of valuable contents for human Molecules 2019, 24, 2225; doi:10.3390/molecules24122225 89 www.mdpi.com/journal/molecules Molecules 2019, 24, 2225 nutrition and are one of the world’s most cultivated and valuable marine seaweed [3]. Its lipid content is low (1–5% dry weight, DW) [1], but it contains docosahexaenoic acid (DHA) which is recognized as the most important n-3 polyunsaturated fatty acid (PUFA) to reduce the risk of cardiovascular diseases [4,5]. In particular, n-3 PUFAs act as excellent antioxidants, strengthening the cell membrane, repairing damaged cells and tissues, improving heart function and fighting against cancer [6]. n-3 PUFAs were also found to prevent the growth of atherosclerotic plaque that affects blood clotting and blood pressure and improve the immune function, while n-6 PUFAs decrease low-density lipoprotein cholesterol and may also decrease high-density lipoprotein, cholesterol which reduces heart disease risk [7]. With respect to their protein content, the most abundant amino acids in Gracilaria species are aspartic acid, alanine, glutamic acid and glutamine. These amino acids provide the typical flavor of algae and accumulate in response to stress conditions [8]. Gracilaria is also a good source of both soluble and insoluble dietary fiber, so it can be employed as a potential alternative to cereal-based fiber in Western countries [1]. Soluble dietary fiber helps to increase viscosity and reduce glycemic response and plasma cholesterol in humans [1]. Insoluble dietary fiber improves the bulking effect caused by water absorption in feces and thus contributes to weight management, improvement of cardiovascular and gastrointestinal functions and cancer prevention [1]. Polysaccharides isolated from red seaweeds show potent antibacterial, antiviral, antioxidant, anticoagulant and anti-inflammatory activity [9]. Seaweeds such as Gracilaria can concentrate minerals from seawater and reach a mineral content 10–20 times higher than that of terrestrial plants [10]. Consequently, they are a valuable source of minerals, with important human nutrition functions [11,12]. Chlorophyll, an important pigment constituent present in algae, has positive effects on inflammation, oxidation and wound healing [13]. Chlorophyll acts directly as a reducer of free radicals and has the potential to protect lymphocytes against oxidative DNA damage by free radicals [14]. Moreover, a large number of potentially bioactive compounds such as phenols, polyphenols, terpenes, steroids, halogenated ketones and alkanes, fucoxanthin, polyphloroglucinol and bromophenols have been isolated [15–17]. However, the nutrient profile of seaweeds such as Gracilaria is influenced by different factors such as seaweed species, habitat, maturity stage, season, water temperature and the sampling conditions and method employed in the determinations [1,2]. Gracilaria edulis and G. corticata is abundantly available in almost all seasons in Palk Bay, on the southeast coast of India, rather than other Gracilaria sp. Both G. edulis and G. corticata are commercially important and commonly edible seaweeds in India. These two algae exhibited in a previous work high biological activities (proximate composition, antioxidant, antibacterial, and biopreservative effects in seafoods during preservation and extended shelf life than other Gracilaria species in a previous work [18]. Thus, the present study sought to evaluate and compare the chemical composition (proximate composition, lipid profile, amino acids, vitamins and pigments such as chlorophyll and carotenoids) and physicochemical properties of both, Gracilaria corticata and Gracilaria edulis from the Thondi coast of Palk Bay, southeast India. 2. Results Proximate, polysaccharide content and fatty acids profile of both G. corticata and G. edulis in a DW basis are shown in Table 1. 90 Molecules 2019, 24, 2225 Table 1. Proximate composition (g/100 g dry weight seaweed) G. corticata and G. edulis. Parameter G. corticata G. edulis Moisture Protein Fat Carbohydrates Ash Polysaccharides 8.40 ± 0.65 b 22.84 ± 0.87 b 7.07 ± 0.33 a 8.30 ± 1.89 a 8.10 ± 0.49 49.64 ± 3.89 a 10.40 ± 0.69 a 25.29 ± 0.67 a 4.76 ± 0.73 b 4.71 ± 0.60 b 7.36 ± 0.39 38.02 ± 4.32 b Values are mean ± standard deviation, n = 3. significantly different. a–b values with different superscripts within the same line were The crude polysaccharide content found for G. corticata and G. edulis was 49.64 g/100 g and 38.02 g/100 g, respectively. The moisture content (in dried seaweeds) of G. corticata and G. edulis was 8.40 g/100 g and 10.40 g/100 g, respectively. With respect to proximate composition, important differences were obtained for the two seaweeds investigated. Carbohydrates and fat content were significantly higher in G. corticata, whereas protein content was significantly higher in the case of G. edulis. With respect to fatty acids profile, total fatty acid content, expressed as g fatty acids methyl esters (FAME)/100 g total fat, of G. corticata and G. edulis was 5.49 ± 0.30 g/100 g and 3.92 ± 0.13 g/100 g, respectively (Table 2). The main saturated fatty acids (SFAs) found in both G. corticata and G. edulis were palmitic acid (C16:0), margaric acid (C17:0) and stearic acid (C18:0). With respect to PUFAs, linoleic acid (C18:2n-6), α-linolenic acid (C18:3n-3), stearidonic acid (C18:4n-3) and DHA (C22:6n-3) were found in both seaweeds. In the case of monounsaturated fatty acids (MUFAs), only oleic acid (C18:1) was detected in relevant amounts in both G. corticata and G. edulis. Margaric, linoleic and stearidonic acids were found in similar amounts in G. corticata and G. edulis. Palmitic, stearic, α-linolenic acid were found in higher amounts in G. corticata than in G. edulis. Overall, in G. corticata, SFAs accounted 49.4% of total fatty acids, MUFAs accounted a 3.3% and PUFAs accounted a 47.3%, whereas in the case of G. edulis, SFAs accounted 43.9% of total fatty acids, MUFAs accounted a 27% of total fatty acids, and PUFAs accounted a 29%. Table 2. Fatty acids profile (g fatty acid methyl esters/100 g total fat) of G. corticata and G. edulis.  Parameter G. corticata G. edulis Palmitic acid Margaric acid Stearic acid Oleic acid Linoleic acid α-Linolenic acid Stearidonic acid Docosohexaenoic acid  FA 1.22 ± 0.04 0.16 ± 0.25 1.31 ± 0.03 a 0.18 ± 0.12 b 0.63 ± 0.04 1.26 ± 0.04 a 0.21 ± 0.01 0.48 ± 0.14 a 5.49 ± 0.30 a 0.63 ± 0.09 b 0.15 ± 0.10 0.93 ± 0.05 b 1.05 ± 0.05 a 0.65 ± 0.18 0.14 ± 0.04 b 0.22 ± 0.05 0.12 ± 0.18 b 3.92 ± 0.13 b a FA = Total fatty acids. Values are mean ± standard deviation, n = 3. the same line were significantly different. a–b values with different superscripts within The protein of G. corticata and G. edulis is shown in Table 3. The total amino acid content was higher in G. corticata (76.60 ± 5.14 mg/g), than in G. edulis (65.42 ± 3.58 mg/g). These values are comparable to their corresponding crude protein content of 22.84 ± 0.87 and 25.29 ± 0.67 g/100 g, respectively, indicating that the amount of non-protein nitrogenous materials in these red seaweeds is low. Nine essential amino acids (EAAs), and 11 non-essential amino acids (NEAAs), were found in both G. corticata and G. edulis. Total EAAs where significantly higher in G. edulis (35.55 ± 1.75 mg/g) than in G. corticata (22.76 ± 1.81 mg/g), whereas total NEAAs where higher in G. corticata (36.14 ± 3.33 mg/g) than in G. edulis (29.86 ± 1.83 mg/g). The EAAs/total amino acid ratio suggests that more than 50% of the amino acids found in G. edulis are EAAs. The results also indicate a good ratio of 91 Molecules 2019, 24, 2225 essential amino acids to non-essential amino acids in G. corticata (0.62 ± 0.54 mg/g) and G. edulis (1.19 ± 0.95 mg/g). It was noted that a much higher concentration of the essential amino acid threonine (20.57 ± 0.62 mg/g) was found in G. edulis than in G. corticata. Contrariwise, alanine content was much higher in G. corticata (21.11 ± 0.54 mg/g) than in G. edulis (1.46 ± 0.18 mg/g). Aspartic acid content was similar in both seaweeds. The mineral content of G. corticata and G. edulis is shown in Table 4. G. corticata showed a higher content of Mg (463.23 ± 8.87 mg/kg) and Fe (1072.48 ± 20.97 mg/kg) than G. edulis. Moreover, G. edulis was found to possess more of trace elements like Zn (42.73 ± 2.12 mg/kg) and Cu (14.61 ± 0.46 mg/kg) than G. corticata. In view of the present results, both G. corticata and G. edulis contain an adequate amount of minerals, which suggests that these seaweeds could act as important sources of mineral supplements which are essential for human nutrition. Table 3. Protein composition of G. corticata and G. edulis. Amino Acid G. corticata G. edulis Aspartic acid Glutamic acid Asparagine Serine Glutamine Glycine Theronine * Arginine * Alanine Cysteine Tyrosine * Histidine Valine * Methionine * Isoleucine * Phenylalanine * Leucine * Lysine * Proline Tryptophan Total amino acids Total EAAs Total NEAAs EAAs/Total AAs EAAs/NEAAs 14.37 ± 0.78 2.54 ± 0.06 1.45 ± 0.05 b 2.23 ± 0.18 b 2.01 ± 0.7 b 4.71 ± 0.18 a 1.32 ± 0.09 b 3.41 ± 0.30 21.11 ± 0.54 a 1.49 ± 0.30 1.25 ± 0.15 b 2.46 ± 0.27 a 0.16 ± 0.01 8.73 ± 0.31 a 2.53 ± 0.16 a 1.42 ± 0.17 b 1.58 ± 0.35 a 2.37 ± 0.27 a 0.47 ± 0.20 1.00 ± 0.07 a 76.60 ± 5.14 a 22.76 ± 1.81 b 36.14 ± 3.33 a 0.29 ± 0.35 b 0.62 ± 0.54 b 12.67 ± 0.64 2.77 ± 0.15 1.89 ± 0.15 a 2.73 ± 0.13 a 2.42 ± 0.29 a 3.42 ± 0.27 b 20.57 ± 0.62 a 3.33 ± 0.17 1.46 ± 0.18 b 1.27 ± 0.06 2.50 ± 0.24 b 0.18 ± 0.02 a 0.15 ± 0.02 4.98 ± 0.48 b 1.22 ± 0.07 b 2.20 ± 0.10 a 0.38 ± 0.02 b 0.22 ± 0.03 b 0.46 ± 0.18 0.59 ± 0.16 b 65.42 ± 3.58 b 35.55 ± 1.75 a 29.86 ± 1.83 b 0.54 ± 0.48 a 1.19 ± 0.95 a Values are mean ± standard deviation, n = 3 expressed as mg/g seaweed in a dry weight basis. * EAAs: Essential amino acids; NEAAs: Non-essential amino acids. a–b values with different superscripts within the same line are significantly different. Table 4. Mineral content (mg/kg) in G. corticata and G. edulis. Minerals G. corticata G. edulis Zn Cu Mg Fe 31.52 ± 0.69 b 11.42 ± 0.72 b 463.23 ± 8.87 a 1072.48 ± 20.97 a 42.73 ± 2.12 a 14.61 ± 0.46 a 89.56 ± 0.77 b 557.36 ± 0.57 b Values are mean ± standard deviation, n = 3 on dry weight basis. Macro minerals: Zinc (Zn), copper (Cu), magnesium (Mg) and iron (Fe). a–b values with different superscripts within the same line are significantly different. For both G. corticata and G. edulis, the presence of water-soluble vitamins (vitamin B1 , vitamin B2 , vitamin B3 , vitamin B6 , vitamin B9 and vitamin C) and fat-soluble vitamins (vitamin A and vitamin E) 92 Molecules 2019, 24, 2225 was found, as shown in Table 5. G. corticata had a higher vitamin A (2.67 ± 0.31 mg/g vs. 2.14 ± 0.17 mg/g) and vitamin B9 contents (1.00 ± 0.07 mg/g vs. 0.45 ± 0.06 mg/g) than G. edulis, whereas G. edulis showed a significantly higher content of vitamin B2 (1.54 ± 0.39 mg/g vs. 0.05 ± 0.01 mg/g) and vitamin B6 (4.77 ± 0.23 mg/g vs. 3.79 ± 0.30 mg/g) than G. corticata. Table 5. Vitamin composition (mg/g), chlorophyll and carotenoid content (μg/g) of G. corticata and G. edulis. Name of Vitamins/Pigments G. corticata G. edulis Vitamin B1 Vitamin B2 Vitamin B3 Vitamin B6 Vitamin B9 Vitamin C Vitamin A Vitamin E chlorophyll a chlorophyll b carotenoid 0.38 ± 0.02 0.05 ± 0.01 b 1.54 ± 0.39 3.79 ± 0.30 b 1.00 ± 0.07 a 14.66 ± 0.23 2.67 ± 0.30 a 1.40 ± 0.10 8.96 ± 0.39 b 7.74 ± 0.33 12.82 ± 0.50 a 0.36 ± 0.02 1.54 ± 0.07 a 1.10 ± 0.29 4.77 ± 0.23 a 0.45 ± 0.06 b 13.41 ± 0.57 2.07 ± 0.06 b 1.49 ± 0.10 17.14 ± 0.55 a 8.44 ± 0.63 2.99 ± 0.56 b Values are mean ± standard deviation, n = 3 on dry weight basis. same line are significantly different. a–b values with different superscripts within the The methanolic extracts of G. corticata and G. edulis at 1 mg/mL concentration indicate the presence of three major compounds, chlorophyll a, chlorophyll b and carotenoids, in G. corticata (Retention factor (Rf) value = 0.97, 0.92 and 0.95, respectively) and G. edulis (Rf = 0.96, 0.96 0.84, respectively). G. corticata and G. edulis contained 8.96 ± 0.39 μg/g and 17.14 ± 0.55 μg/g of chlorophyll a and 7.74 ± 0.33 μg/g and 8.44 ± 0.63 μg/g of chlorophyll b, respectively. With respect to the carotenoid content, it was higher for G. corticata (12.82 ± 0.50 μg/g) than for G. edulis (2.99 ± 0.56 μg/g). Table 6 shows the swelling capacity (SWC), water-holding capacity (WHC) and oil-holding capacity (OHC) of G. corticata and G. edulis. In general, as temperature varied, the SWC and WHC of G. corticata and G. edulis powder varied, due to an increase in the solubility of the dietary fiber and the presence of protein in G. corticata and G. edulis. However, it also reaches significant differences for the case of SWC, whereas no statistical differences were obtained for WHC or OHC. The SWC of G. edulis were higher than G. corticata at both 25 ◦ C and 37 ◦ C (8.66 ± 0.53 mL/g vs. 7.90 ± 0.32 mL/g, and 7.70 ± 0.60 mL/g vs. 5.70 ± 0.65 mL/g, respectively). Table 6. Swelling capacity (SWC), water holding capacity (WHC) and oil holding capacity (OHC) of G. corticata and G. edulis. Seaweeds G. corticata G. edulis SWC (mL/g) 25 ◦ C WHC (g/g) 37 ◦ C 7.90 ± 0.32 8.66 ± 0.53 aA bA 5.70 ± 0.65 7.70 ± 0.6 aB bB OHC (g/g) 25 ◦ C 37 ◦ C 25 ◦ C 37 ◦ C 4.03 ± 0.39 4.09 ± 0.32 3.96 ± 0.58 3.64 ± 0.40 2.06 ± 0.24 1.87 ± 0.28 1.84 ± 0.40 1.91 ± 0.18 Values are mean ± standard deviation, n = 3 on dry weight basis. a–b values with different superscripts within the same column are significantly different between seaweeds. A–B values with different superscripts within the same line shows significant differences between temperatures. With respect to the WHC of G. corticata and G. edulis, values of 4.03 ± 0.39 and 4.09 ± 0.28 g/g, respectively, were obtained at 25 ◦ C, reduced to 3.96 ± 0.58 g/g in G. corticata and 3.64 ± 0.18 g/g in G. edulis at 37 ◦ C. In this study, both G. corticata and G. edulis exhibited similar OHC values (about 2 g/g) at both 25 ◦ C and 37 ◦ C. 93 Molecules 2019, 24, 2225 3. Discussion With respect to proximate content, the moisture of G. corticata and G. edulis was lower than most results obtained for Gracilaria sp. in general, such as the 12.15 g/100 g obtained for G. acerosa [6] the 19.2 g/100 g for G. edulis [8], and the 12.86 g/100 g for G. edulis [1], but higher than the 5.32 g/100 g obtained for G. changii [19]. In this work, G. edulis showed a higher ash content in a DW basis than G. corticata. Similarly, it was reported an ash content of 8.70 g/100 g in G. edulis [8], whereas other authors reported a higher ash content (40.30 g/100 g) in G. changii [19] than those found in the present work. A high ash content shows the presence of appreciable amounts of diverse minerals found in both seaweeds. A similar observation was for G. changii [19] in which were found an ash content of 6.40 g/100 g. Interestingly, total dietary fiber is known to have physiological properties for the prevention and treatment of cancer, obesity and diabetes [20,21]. Therefore, G. corticata and G. edulis may have the potential to be used as a source of dietary fiber in the nutraceutical industries. Other authors found much lower crude protein contents in Gracilaria spp. than those found in the present work. Thus, it was reported a crude protein content of 6.68 g/100 g for G. edulis [8], 0.61 g/100 g for G. acerosa [6], 12.57 g/100 g in G. changii [19] or 19.70 g/100 g for G. cervicornis [22]. Moreover, the high protein content of G. corticata and G. edulis indicates that these seaweeds may be considered as potential marine plant sources of protein [22]. Proteins from seaweeds can have antibacterial, antioxidant, immunostimulating, antithrombotic and anti-inflammatory activities. Consequently, they can be used for prevention and treatment of hypertension, diabetes and hepatitis among other positive effects in the organism [20]. The total carbohydrate content of both G. corticata and G. edulis was markedly lower than that reported [8] for G. edulis (10.2 g/100 g) or the 29.44 g/100 g reported for G. changii [19]. However, other authors found lower carbohydrate content in Gracilaria species, such as G. acerosa, for which was reported a carbohydrate content of 1.05 g/100 g [6]. The wide variation in the carbohydrate content observed in red and brown seaweed species might be due to the influence of different factors like salinity, temperature and sunlight intensity [2]. Moreover, carbohydrate content is also influenced by biomass, which reveals the link between growth and carbohydrate content [23]. In general, seaweeds have a low fat content [23]; that makes seaweeds low-calorie foods and in the present work both seaweeds contained fat amounts of 7.07 g/100 g DW seaweed (G. corticata) and 4.71 g/100 g DW seaweed (G. edulis). These results are lower than those obtained by other authors [8], whose reported a crude lipid content for G. edulis of 8.30 g/100 g but significantly that the 0.3% reported for G. changi [19], or the 1.7–3.6% reported for G. fisheri and G. tenuistipitata [5]. Thus, Gracilaria content in fat can widely vary depending on the species and source. Polysaccharides are polymers composed of at least 10 monosaccharides linked by glycosidic bonds [9]. Recently, seaweed polysaccharides have been given large attention by the scientific community due to their outstanding bioactivities and correspondingly low toxicity [9]. They have been shown to have other beneficial health effects, including their prebiotic effect and antioxidant or anti-inflammatory activity [20]. The polysaccharide content obtained in the present work was higher than the polysaccharide extracted from Gracilaria species in previous works, such as 29.08 g/100 g [24], 27.20 g/100 g [25], 21.40 g/100 g [26], and 32.80 g/100 g [27]. Contrariwise, it was also reported a higher polysaccharide content in G. debilis [28], in the range 52–67 g/100 g. A previous work [29] reported that the polysaccharide yield from Gracilaria species varies due to seasonal variations, physiochemical factors, environmental conditions and extraction methods. Additionally, the variations in the polysaccharide content of Gracilaria can vary depending on atmospheric temperature at the time of extraction [26]. Hence the present study significantly indicates that the crude polysaccharides present in G. corticata and G. edulis may exert varied biological activity [25]. With respect to the fatty acids composition, those of seaweeds often differ from those of terrestrial plants whereby seaweeds have a higher proportion of PUFAs than terrestrial vegetables. Red seaweeds are particularly rich in SFAs and PUFAs which have nutritional applications that lead to their extensive use in food, feed, cosmetic, biotechnological and pharmaceutical applications [30,31]. Variation in 94 Molecules 2019, 24, 2225 fatty acid content may also be due to the season of collection as well as other abiotic factors such as nutrition, salinity, light and temperature [8,20]. In the present work, total fatty acids were significantly lower than those obtained by other authors [8], who found 11.41 g/100 g in G. edulis. According to this work [8], the most abundant fatty acids in both seaweeds were palmitic, stearic and α-linoleic acid acids. The same fatty acids were also found abundant in G. changii [20]. However, our results were significantly lower than those obtained in G. changii for DHA content, in which DHA were found as the most abundant fatty acids, with a 48.36% of total fatty acids. The results of the present study revealed that both seaweeds are rich in SFAs and especially in PUFAs, which provide important health benefits. With respect to most commonly found n-3 PUFA, eicosapentaenoic acid (EPA) and DHA, it is common that their contents vary dramatically from Gracilaria spp. and even into the same species [32]. No EPA presence were found for the seaweeds tested in the present work. The presence of this n-3 fatty acid in Gracilaria spp. is inconstant, because it was found in G. gracilis [20], but it was not detected in G. changii [19] or G. edulis [8]. Fatty acids overall profile obtained in this work were significantly different than 57.5% SFAs, 18.3% MUFAs and 18.4% PUFAs reported for Gracilaria sp. [3] or the 7.5% SFAs, 38.3% MUFAs and 51.2% PUFAs 18.4% reported for Gracilaria changii [19]. The protein composition found in this work for G. corticata and G. edulis was lower than those found in a previous work [20], which reported an amino acid content of 91.90 mg/g in G. changii. The EAAs/total amino acid ratio was higher than those previously reported [6,19,26]. Aspartic acid content, that is important for the organoleptic point of view because it was reported that it is responsible for the special flavor and taste of seaweeds [33], was in similar contents in both seaweeds. Seaweeds are one of the richest sources of minerals and trace elements, because the cell-wall polysaccharides and proteins of seaweed contain sulfate, anionic carboxyl and phosphate groups which act as binding sites for metal retention [34]. With respect to the mineral content, G. corticata showed a higher content of Mg (463.23 mg/kg) and Fe (1072.48 mg/kg) than G. edulis. Moreover, G. edulis was found to possess more of trace elements like Zn (42.73 mg/kg) and Cu (14.61 mg/kg) than G. corticata. Both seaweeds had a higher or similar content of minerals like Zn, Cu, Mg and Fe when compared with the content of G. acerosa [6], G. edulis [8], G. fisheri and G. tenuistipidatata [5] or G. changii [19], with the exception of Mg in G. edulis which were lower than those found for other previous works as G. changii [19]. The ability of seaweeds to accumulate metals will depend on a variety of factors such as location, exposure, salinity, temperature, pH, light, nitrogen content, season, plant age, metabolic processes or the affinity of the plant for each element among others [35]. In view of the present results, both G. corticata and G. edulis contain an adequate amount of minerals, which suggests that these seaweeds could act as important sources of mineral supplements which are essential for human nutrition. For both G. corticata and G. edulis, the presence of water-soluble vitamins (vitamin B1 , vitamin B2 , vitamin B3 , vitamin B6 , vitamin B9 and vitamin C) and fat-soluble vitamins (vitamin A and vitamin E) was found. G. corticata had a higher vitamin A (2.67 mg/g vs. 2.14 mg/g) and vitamin B9 contents (1.00 mg/g vs. 0.45 mg/g) than G. edulis, whereas G. edulis showed a significantly higher content of vitamin B2 (1.54 mg/g vs. 0.05 mg/g) and vitamin B6 (4.77 mg/g vs. 3.79 mg/g) than G. corticata. With respect to previously published works, the vitamin content reported for Gracilaria species is widely different between the different authors [1,6,8]. Perhaps the more remarkable difference in vitamin content is that in the present work both G. corticata and G. edulis showed a significantly higher vitamin A content (2.67 and 2.07, respectively) than those previously reported for G. acerosa [6] or for G. edulis [1]. Another important difference was found for the case of vitamin C that showed a higher content than those previously reported [6,8] for G. edulis or G. acerosa, respectively. The variation in vitamin content may be due to some environmental factors such as salinity, atmospheric temperature, seasonality and methods of preservation and processing [6]. G. corticata and G. edulis contained 8.96 μg/g and 17.14 μg/g of chlorophyll a and 7.74 μg/g and 8.44 μg/g of chlorophyll b, respectively. In a previous work describing the composition of several 95 Molecules 2019, 24, 2225 seaweeds [36] it was reported that chlorophyll a and b in red seaweeds ranged from 68 to 162 μg/g and from 25 to 46 μg/g, respectively. Specifically, for Gracilaria spp., it was reported high chlorophyll a of 577.89 μg/g and low chlorophyll b of 1.11 μg/g in G. changii [19]. With respect to carotenoid content, a higher total carotenoid content was reported for G. changii than in the present study (74.22 μg/g) [19]. Carotenoids such as β-carotene, lutein, zeaxanthin and antheraxanthin have been identified in red seaweed, including Gracilaria species. Further, seaweed carotenoids, especially β-carotene, are preferred by the market of natural products, because they are a mixture of cis and trans isomers, which may possess anticancer activity [37]. The SWC, WHC and OHC properties of seaweeds are generally related to their content and type of polysaccharides as well as protein which links to the cell wall of polysaccharide [5]. Previous works described that variations in temperature can widely vary physicochemical properties of seaweeds, due to increase in the solubility of the dietary fiber and the presence of protein [5,8]. However, in our work, only were found significant variations in the case of SWC. Previous works [8] reported a SWC of 20 mL/g in G. edulis, higher than those found in the present study. Similarly, a SWC at 37 ◦ C of 7.68 mL/g in G. changii was reported [19], whereas for G. acerosa [6] an SWC at 37 ◦ C of 5 mL/g was reported. With respect to the WHC of G. corticata and G. edulis, a similar observation was also made in a previous work [8], that reported a WHC for G. edulis of 3.08 g/g. Other authors found better WHC than in the present work for other Gracilaria spp., such as G. fisheri, for which a WHC of 5.53 g/g was reported [5], and G. changii [26], for which WHC values of 6.15 g/g at 24 ◦ C and 9.93 g/g at 37 ◦ C were reported. Both SWC and WHC of seaweeds might be attributed due to different protein content and increases in the number and nature of the water binding sites on the protein molecules [38]. OHC is another functional property of food ingredients used in formulated foods for consumption. Ingredients with high OHC values allow the stabilization of food emulsions and high-fat food products [19]. For other Gracilaria spp., it was reported [8] that G. edulis showed an OHC of 1.64 g/g, which is very similar to the OHC values of the present study. Moreover, for G. changii [19] OHCs of 3.11 g/g at 24 ◦ C and 1.17 g/g at 37 ◦ C were reported. The low oil absorption capacity of red seaweeds is generally related to the hydrophilic nature of the changed polysaccharides (agar, carrageenan, fucans and alginates) of soluble dietary fiber [39]. The results of the present study for physicochemical properties confirmed that G. corticata and G. edulis could be considered as a source of food ingredients including proteins, dietary and soluble fiber [39]. 4. Materials and Methods 4.1. Sample Collection Samples of the commercially important and commonly edible red seaweeds G. edulis and G. corticata were collected by hand from the Thondi Coast (Latitude: 9◦ 44 N and Longitude: 79◦ 00 E), Palk Bay, on the southeast coast of India. Freshly collected seaweeds were washed thoroughly in seawater and transported to the laboratory immediately. Epiphytes, sediment particles and other debris were removed by washing thoroughly using potable water and thoroughly washed with distilled water immediately after washing with potable water. Seaweeds were identified using a standard manual [40]. The voucher specimen was deposited in museum at Department of Oceanography and Coastal Area Studies, School of Marine Sciences, Alagappa University. Seaweeds were shade dried for 5 days at constant temperature of 25 ◦ C ± 2 ◦ C. Dried seaweed samples were powdered using a mechanical blender and stored at room temperature in an airtight container (Tarsons, Kolkatta, India) for further analysis within a maximum period of one week. Further the remaining powder sample stored at frozen condition (−20 ◦ C) for future use. 4.2. Proximate Composition The proximate composition of each Gracilaria species was determined in all cases following Association of Official Analytical Chemists (AOAC) methods [41]. AOAC methods were employed 96 Molecules 2019, 24, 2225 to determine in G. corticata and G. edulis the ash content by heating at 550 ◦ C for 24 h in a muffle furnace (AOAC, 930.05), moisture content by heating at 105 ◦ C for 24 h (AOAC, 934.01), total fat by Soxhlet extraction with petroleum ether (AOAC 991.36) and protein by the Kjeldahl method (N × 6.25) (AOAC 981.10) [41]. The total carbohydrate content of G. corticata and G. edulis was determined by the phenol–sulfuric acid method [38]. All measurements were performed in triplicate for each seaweed and expressed as g/100 g seaweeds in a dry weight matter. 4.3. Isolation of Polysaccharides Polysaccharides were separated from G. corticata and G. edulis using 2 g samples of each seaweed according as previously described [42]. Powdered G. corticata and G. edulis were dissolved and homogenized with distilled water under constant stirring for 2 h at 100 ◦ C. The residues obtained were then removed by centrifuging the sample at 6300 g for 10 min using a CPR 30 plus centrifuge (Remi Lab World, Mumbai, India). The obtained supernatant was then precipitated by addition of an ethanol-in-water solution (1:3 v/v), followed by subsequent washing with 30 mL of acetone. The precipitated polysaccharides were then collected and subsequently air-dried, re-dissolved in distilled water and washed with acetone. Afterwards, the collected polysaccharides were stored at −20 ◦ C in a deep freezer (Blue Star, Mumbai, India) for further use. Isolation were performed in triplicate for each seaweed and results are expressed as g/100 g seaweeds in a dry weight basis. 4.4. Extraction of Crude Lipid and Determination of Fatty Acids Content Portions of 500 mg each of powdered G. corticata and G. edulis were mixed with 5 mL of a chloroform:methanol solution (2:1 v/v), tightly covered with aluminum foil and kept at room temperature for 24 h. After this period, solutions were filtered through 11 μm Whatman No. 1 filter paper, and the filtered extract was placed in a pre-weighed and oven-dried beaker. The beaker was weighed with lipids, and the difference in weight was taken as total lipid content and expressed as a percentage [43]. Afterwards, an aliquot of the total lipids of each sample was used to determine the fatty acids content, based on a method published [6]. For this purpose, 0.45 g was introduced into a 10 mL volumetric flask, dissolved in hexane containing 50 mg of butylated hydroxytoluene per L and diluted to 10 mL with the same solvent. Afterwards, 2 mL of the solution was transferred into a quartz tube and evaporated by means of a nitrogen flow. Further, 1.5 mL of a 20 g/L solution of sodium hydroxide in methanol, covered with nitrogen, was added, capped tightly with a polytetrafluoroethylene-lined cap, mixed and heated in a water bath for 7 min. After the water bath, samples were cooled at room temperature, and 2 mL of boron trichloride-methanol solution was added; then, they were blanketed with nitrogen, capped tightly, mixed and heated in a water bath for 30 min. After this period, samples were cooled to 40–50 ◦ C, and 1 mL of trimethylpentane was added; then, they were capped and shaken vigorously for at least 30 s. Immediately, 5 mL of saturated sodium chloride solution was added, then the samples were covered with nitrogen, capped and vortexed or shaken thoroughly for at least 15 s. The upper layer was allowed to become clear and then transferred to a separate tube. In the separate tube, the methanol layer was shaken once more with 1 mL of trimethylpentane and combined with trimethylpentane extracts. The organic solvent was then removed, and FAME were subjected to gas chromatography (GC), performed on a Perkin Elmer Clarus 580 gas chromatograph (Perkin Elmer, Gaithersburg, MD, USA) equipped with a flame ionization detector and an HP-5 capillary column (30 m × 0.25 mm). Initial temperature was maintained at 70 ◦ C, then increased to 250 ◦ C (10 ◦ C/min); the injection temperature employed was 225 ◦ C. Helium was used as carrier gas, with a flow rate of 1 μL/min. FAME peaks were identified by comparison of their retention times and quantified by comparison with individual calibration curves performed with a standard FAME mix (Supelco, Sigma-Aldrich, St Louis, MO, USA). Tricosanoic acid (C23; Sigma-Aldrich) was used as internal standard. Fatty acid composition was determined in triplicate for each seaweed and was expressed as g FAME/100 g total fat. 97 Molecules 2019, 24, 2225 4.5. Protein Composition Protein composition of G. corticata and G. edulis was determined based on a reversed-phase high performance liquid chromatography (HPLC) analysis method [6]. Two grams each of powdered seaweeds was mixed with phosphate buffer (pH 7.0) and centrifuged at 1200 g (Remi Lab World) for 20 min at 4 ◦ C. The supernatant was collected, and protein content was precipitated by adding 10% v/v trichloroacetic acid (TCA). The protein pellet was resuspended in 1 N NaOH and hydrolyzed by heating the solution with 6 N HCl in a boiling water bath for 24 h. After incubation, the supernatant was collected by centrifuging the sample at 3500 g for 15 min. The supernatant was then filtered and neutralized by the addition of 1 N NaOH. The filtered supernatant was diluted to 1:100 (v/v) with deionized water. The sample was subjected to reversed-phase HPLC analysis (Lachrome Hitachi, Tokyo, Japan) with UV and fluorescence detectors. One μl of sample was injected into a Denali C18 5-mm column (4.6 mm × 150 mm) at 23 ◦ C with detection at 254 nm. The mobile phase used was 20 mM sodium acetate/triethylamine (0.018% v/v) in phase A. The pH was adjusted to 7.2 using 1–2% acetic acid. In phase B, 20% of 100 mM sodium acetate (pH 7.2) with 1–2% acetic acid was used; 40% acetonitrile was used as the mobile phase. The protein composition was determined in triplicate for each seaweed and expressed as mg of amino acid/g of seaweed in a DW basis. 4.6. Determination of Mineral Content Zn, Cu, Mg and Fe analysis of G. corticata and G. edulis was performed according to the European Standards, with minor modifications according to the method previously described [17]. One gram of homogenized seaweed sample was added to mixed reagent at a ratio of 5:2:1 (nitric acid:perchloric acid:sulfuric acid). Mineralization was performed on a hot plate at 50 ◦ C for 30 min. After the end of digestion, 10 mL of 2 N HCL was added; digested solvents were filtered and made up to 25 mL with distilled water and stored at room temperature for further analysis [44]. Zn, Cu, Mg and Fe were determined by atomic absorption spectroscopy (AAS; Anton Paar-AAS, Graz, Austria). The metal standards were prepared and run to check the precision of the instrument throughout the analysis. Quality assurance and quality control protocols set by the US Environmental Protection Agency [45] for metal analysis were used. Quality assurance testing relied on the control of blanks and yield for the chemical procedure. Mineral content was determined in triplicate for each seaweed and expressed as mg/kg of DW seaweed. 4.7. Determination of Vitamin Content The vitamin content of G. corticata and G. edulis was determined by the method previously described [20]; vitamin A or retinol (328 nm), vitamin B1 or thiamine monohydrate (420 nm), vitamin B2 or riboflavin (254 nm), vitamin B6 or pyridoxine HCl (254 nm), vitamin C or ascorbic acid, vitamin E or tocopheryl acetate (520 nm) and folic acid (550 nm) were determined by HPLC methods, and the results were compared with the respective standards retinyl acetate, thiamine monohydrate, riboflavin, pyridoxine HCl, ascorbic acid, tocopheryl, and folic acid (Sigma Aldrich). Vitamin content was determined in triplicate for each seaweed and was expressed as mg/g of DW seaweed. 4.8. Determination of Chlorophyll a, b and Carotenoids Thin-layer chromatography (TLC) was used to screen chlorophyll a and b and carotenoid content in the G. corticata and G. edulis extracts [8]. The mobile phase contained methanol and chloroform (1:9). The sample (approximately 1 mg/mL) was spotted onto the TLC plates and air-dried. The spots were identified under long-wave and short-wave UV light, and also in an iodine chamber. The Rf value, which is the distance moved by the solute relative to the distance moved by the solvent, was calculated to find chlorophyll a and b and carotenoid content. Afterwards, for chlorophyll a and b determination, 1 g each of G. corticata and G. edulis powder was extracted with 96% CH3 OH, and the supernatant was collected by centrifugation at 1000 g for 1 min. 98 Molecules 2019, 24, 2225 After filtration, the supernatant was again filtered with Whatman No. 1 filter paper, centrifuged at 2300 g (Remi Lab World) for 10 min, and the absorbance of the collected supernatant was measured using a UV–Vis spectrophotometer [8]. The chlorophyll a and b contents were calculated using the following formulas in triplicate for each seaweed and expressed as μg/g of DW: Chlorophyll a = 15.65 (A666 ) − 7.340 (A653 ) (1) Chlorophyll b = 27.05 (A653 ) − 11.21 (A666 ) (2) where A666 = absorbance at 666 nm; A653 = absorbance at 653 nm. Carotenoid content was determined according to [46]. One gram each of G. corticata and G. edulis powder was extracted with 5 mL of acetone and incubated in the dark for 45 min, and the supernatant was collected by centrifugation at 10,000 g for 5 min. The supernatant was then stored in a refrigerator, and the extraction repeated with acetone until it became colourless. The supernatant was pooled and made up to 10 mL with acetone, and the absorbance of the collected supernatant was measured at 450 nm using a UV–Vis spectrophotometer (Shimadzu, Kyoto, Japan). Carotenoid content was calculated using the following formula in triplicate for each seaweed and expressed as μg/g of DW: Carotenoid content = A450/2500 (3) where 2500 is the extinction coefficient. 4.9. Physicochemical Properties In order to determine the physicochemical properties of the G. corticata and G. edulis powder, the SWC, WHC and OHC were determined for each powder. The SWC of G. corticata and G. edulis was assessed based on a method previously described [38]. Briefly, 500 mg of G. corticata and G. edulis were taken and mixed with 20 mL of distilled water and stirred vigorously. The influence of temperature on SWC was determined by maintaining the tubes at 25 ◦ C and 37 ◦ C overnight. The SWC of G. corticata and G. edulis was calculated using the following formula and expressed as ml of swollen sample per g of DW: SWC = Initial volume of water (mL) − Volume of water after incubation (mL) (4) The WHC of G. corticata and G. edulis was assessed by a modified method [38]. Briefly, 500 mg each of G. corticata and G. edulis was put into two sets of centrifuge tubes, and 20 mL of deionized water was added. The tubes were kept separately in an incubator shaker for 24 h at 25 and 37 ◦ C. The supernatant was discarded after centrifuging the tubes at 12,000 g for 30 min (Remi Lab World). The wet weight of G. corticata and G. edulis was noted. The samples were dehydrated by keeping them in an oven at 160 ◦ C for 2 h, and the dry weight of the sample was noted. The WHC was determined in triplicate for each seaweed and calculated using the following formula and expressed as the weight in g of water held by 1 g of DW sample: WHC = Wet weight of the sample (g) - Dry weight of the sample (g) (5) The OHC of G. corticata and G. edulis was assessed according to previous methods [20,38]. About 3 g of dried G. corticata and G. edulis was taken in a tube and mixed with 10.5 g of corn oil. The tubes were placed in a shaker at room temperature for 30 min. The oil supernatant was collected by centrifugation at 3000 g for 30 min (Remi Lab World). The OHC was determined in triplicate for each seaweed and was determined using the following formula and expressed as the number of g of oil held by 1 g of DW sample: OHC = Initial volume of oil (g) − Volume of oil after incubation (g) 99 (6) Molecules 2019, 24, 2225 4.10. Statistical Analysis All results were expressed as mean ± SD. Paired sample t-test was used to compare composition values between G. edulis and G. corticata. One-way analysis of variance (ANOVA) and Duncan’s test were used to compare the effects of temperature on the physicochemical properties. All determinations were performed using SPPS version 14 (SPSS Science, Chicago, IL, USA). A positive significant variation was defined at the significance level of p < 0.05. 5. Conclusions The polysaccharide content of the investigated dried red seaweeds (G. corticata and G. edulis) were found to be rich sources of polysaccharides. Because of the potential as prebiotics, antioxidant and anti-inflammatory compounds, carbohydrates from seaweeds has are compounds in high demand by consumers today. The physicochemical properties and proximate composition revealed that both G. corticata and G. edulis have appreciable levels of ash, protein, carbohydrate, fatty acid, essential and non-essential amino acid and vitamin content. It is suggested that both the seaweeds tested have great potential as potential food supplements and may be used in the food industry as a source of ingredients with an appreciable amount of nutritional value. Since both red seaweeds were found to be a good source of essential nutrients, their commercial value can be enhanced by marketing them as value-added products. However, depending on their composition, G. edulis and G. corticata have important differences that make it more adequate for certain cases. G. edulis showed higher concentration of essential amino acids, chlorophyll, vitamin B2 and Zn. Thus, it could be a good nutrient for low-protein diets or people whose need to reduce their oxidative status, because of its content in chlorophyll and Zn. Contrariwise, G. corticata showed higher PUFA content, carotenoids and minerals as Fe and Mg. Thus G. corticata is more adequate than G. edulis for people who need to reinforce their intake of such nutrients. Author Contributions: Conceptualization, S.P. and A.A.; methodology, T.R. and A.A.; software, A.A. and A.M.-P.; validation, A.A.; formal analysis, T.R. and A.A.; investigation, A.A. and S.P.; resources, S.P.; data curation, J.M.M.; writing—original draft preparation, J.M.M.; writing—review and editing, A.M.-P.; visualization, S.P. and J.M.M.; supervision, S.P.; project administration, S.P.; funding acquisition, S.P. Funding: This research received external funding by RUSA–2. 0 grant sanctioned vide Letter No. F. 24–51/2014–U, Policy (TNMulti–Gen), Department of Education, Government of India and by FEADER (The European Agricultural Fund for Rural Development (EAFRD)), grant number 2018/001B and ED431C 2018/05. Acknowledgments: The authors thank the authorities of Alagappa University for providing necessary facilities. Conflicts of Interest: The authors declare no conflict of interest. References 1. 2. 3. 4. 5. Debbarama, J.; Rao, B.M.; Murthy, L.N.; Mathew, S.; Venkateshwarlu, G.; Ravishankar, C. Nutritional profiling of the edible seaweeds Gracilaria edulis, Ulva lactuca and Sargassum sp. Indian J. Fish. 2016, 63, 81–87. [CrossRef] Torres, P.; Santos, J.P.; Chow, F.; dos Santos, D.Y. A comprehensive review of traditional uses, bioactivity potential, and chemical diversity of the genus Gracilaria (Gracilariales, Rhodophyta). Algal Res. 2019, 37, 288–306. 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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/). 102 molecules Article Concentration of EPA and DHA from Refined Salmon Oil by Optimizing the Urea–Fatty Acid Adduction Reaction Conditions Using Response Surface Methodology Gretel Dovale-Rosabal 1 , Alicia Rodríguez 1, *, Elyzabeth Contreras 1 , Jaime Ortiz-Viedma 1, *, Marlys Muñoz 1 , Marcos Trigo 2 , Santiago P. Aubourg 2, * and Alejandra Espinosa 3 1 2 3 * Department of Food Science and Chemical Technology, Faculty of Chemical and Pharmaceutical Sciences, Santos Dumont 964, University of Chile, Santiago 8380000, Chile; gretel.dovale@ug.uchile.cl (G.D.-R); ely_tcn@hotmail.com (E.C.); marlys_dayane@hotmail.com (M.M.) Department of Food Technology, Marine Research Institute (CSIC), Eduardo Cabello, 6, 36208 Vigo, Spain; mtrigo@iim.csic.es Department of Medical Technology, School of Medicine, University of Chile, Santiago 8380000, Chile; bespinosa@med.uchile.cl Correspondence: arodrigm@uchile.cl (A.R.); jaortiz@uchile.cl (J.O.-V.); saubourg@iim.csic.es (S.P.A.) Academic Editor: Jose M. Miranda Received: 19 March 2019; Accepted: 20 April 2019; Published: 26 April 2019 Abstract: This research focused on obtaining eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3) (EPA+DHA) concentrates from refined commercial salmon oil (RCSO). Independent variables of the complexation process were optimized by means of the application of response surface methodology (RSM) in order to obtain the maximum content of such fatty acids (FAs). As a result of employing the optimized conditions for all the variables (6.0, urea:FA content ratio; −18.0 ◦ C, crystallization temperature; 14.80 h, crystallization time; 500 rpm, stirring speed), high contents of EPA and DHA could be obtained from RCSO, achieving increases of 4.1 and 7.9 times in the concentrate, with values of 31.20 and 49.31 g/100 g total FA, respectively. Furthermore, a 5.8-time increase was observed for the EPA + DHA content, which increased from 13.78 to 80.51 g/100 g total FA. It is concluded that RCSO can be transformed into a profitable source of EPA and DHA (EPA+DHA), thus leading to a product with higher commercial value. Keywords: refined commercial salmon oil; n-3 long-chain polyunsaturated fatty acids (n-3 LCPUFAs) concentration; EPA; DHA; EPA+DHA; total FA yield; process variable maximization; response surface methodology (RSM); multiple response optimization; desirability function 1. Introduction In recent years, it has been recognized that the consumption of eicosapentaenoic acid (EPA) is associated with a low prevalence of coronary, circulatory, and inflammatory diseases [1–4]. Furthermore, docosahexaenoic acid (DHA) has been associated with fetal development, the prevention of neurodegenerative diseases, and the correct functioning of the nervous system and visual organs in the fetus [5–10]. According to the Food and Agriculture Organization/World Health Organization (FAO/WHO) [1], the recommended intake of EPA+DHA is at least 250 mg/day for adult males and non-pregnant/non-lactating adult females. Interestingly, the optimal brain development of children would need a 150 mg/day diet of such fatty acids. EPA+DHA concentrates may be produced by various methods, such as supercritical fluid chromatography, supercritical fluid fractionation, molecular distillation, silver complexation, enzymatic methods, and urea Molecules 2019, 24, 1642; doi:10.3390/molecules24091642 103 www.mdpi.com/journal/molecules Molecules 2019, 24, 1642 complexation [11,12]. Among them, complexation with the urea can be considered as the most efficient method, since polyunsaturated fatty acids (PUFAs) may be separated from saturated and monounsaturated ones by means of an economic process at low temperature [13–18]. The present research was focused on the employment of refined commercial salmon oil (RCSO) as a profitable source of EPA+DHA concentrates, which in time could lead to a product of higher commercial value. For it, independent variables of the urea adduction reaction conditions (urea:FA content ratio, crystallization time and temperature, and crystallization stirring speed) were optimized by response surface methodology (RSM) in order to achieve the maximum content of EPA, DHA and EPA+DHA. Additionally, the quality of the starting salmon oil was determined and evaluated. 2. Results 2.1. Characterization of the Initial Refined Commercial Salmon Oil Results for the composition of RCSO reported that the most abundant fatty acids were 18:1 9c (29.61%), 18:2 9c, 12c (16.69%), and 16:0 (13.74 %) followed by EPA (7.53%) and DHA (6.25%) (g/100 g total FA). The total value of saturated fatty acids (SFAs) was 21.28%, which was composed mainly of palmitic, stearic, and myristic acids (Table 1). Special interest is the confirmation of the absence of phytanic acid within the lipid composition of RCSO. This fatty acid has been linked to neurological disorders in some people, but it is also associated with the prevention of metabolic syndrome or type 2 diabetes [19]. Table 1. Composition of fatty acids in (RCSO) and the optimized concentrate from RCSO (g/100 g total FA) *. FA or FA Groups RCSO RCSO Optimum 12:0 14:0 15:0 16:0 16:1 9t 16:1 7c 16:1 9c 16:1 11c 16:1 13c 17:0 17:1 10c 18:0 18:1 9c 18:1 11c 18:2 9t, 12t 18:2 9c, 12c 18:2 9c, 15c 18:3 6c, 9c, 12c 20:0 18:3 9c, 12c, 15c 20:1 5c 20:1 8c 20:1 11c 18:4 6c, 9c, 12c, 15c 20:2 11c, 14c 20:3 8c, 11c, 14c 20:3 11c, 14c, 17c 20:4 8c, 11c, 14c, 17c 22:1 13c 20:5 5c, 8c, 11c, 14c, 17c 24:1 15c 0.07 3.19 0.20 13.74 0.15 Nd 4.66 Nd Nd 0.13 0.56 3.69 29.61 3.69 Nd 16.69 Nd 0.22 0.26 3.25 Nd Nd 1.60 Nd 0.79 0.30 0.12 0.40 0.21 7.53 Nd Nd 0.12 0.09 Nd Nd Nd 0.51 Nd Nd 0.10 1.28 0.28 0.59 0.04 Nd 7.48 Nd 1.09 Nd 2.60 Nd Nd Nd Nd 0.05 1.16 0.03 1.37 Nd 31.20 Nd 104 Molecules 2019, 24, 1642 Table 1. Cont. 22:5 7c, 10c, 13c, 16c, 19c 22:6 4c, 7c, 10c, 13c, 16c, 19c Total SFAs Total MUFAs Total PUFAs Total n-3PUFAs Total n-3LCPUFAs EPA+DHA 2.69 6.25 21.28 40.48 38.24 20.45 18.08 13.78 2.70 49.31 0.59 2.42 96.99 87.21 85.82 80.51 * Abbreviations employed: DHA (docosahexaenoic acid), EPA (eicosapentaenoic acid), FA (fatty acid), SFAs (saturated fatty acids), MUFAs (monounsaturated fatty acids), RCSO (refined commercial salmon oil), n-3LCPUFAs (n-3 long chain polyunsaturated fatty acids), Nd (not detected). Values obtained for the oxidative stability of RCSO were: peroxide value (PV) = 5.23 ± 0.05 meq. active oxygen kg−1 oil; p-anisidine value (pAV) = 6.84 ± 0.46; and total oxidation value (TOTOX) = 17.30 ± 0.40 and free fatty acids (FFA) = 0.30 ± 0.01 g oleic acid/100 g oil. Previous studies performed on refined salmon oil samples [18] indicated average values of PV= 3.54 ± 0.16 meq active oxygen kg−1 oil; p-anisidine value (pAV) = 5.14 ± 1.02 and FFA= 0.23 ± 0.00 g oleic acid/100 g oil. and at and 12.22 ± 1.34, respectively. 2.2. Effect of Process Variables on Total FA Yield, EPA Contents, and DHA Contents of RCSO Concentrate 2.2.1. Refined Commercial Salmon Oil Concentrate According to the experimental design, showed in Table 2, 28 assays were performed to obtain different refined commercial salmon oil concentrates. This table reports the experimental values obtained for the different response variables: R1 (total FA yield; g FA in the non-urea complexing fraction/100 g initial saponified oil FA), R2 (EPA content; g/100 g total FA), and R3 (DHA content; g/100 g total FA) of RCSO concentrate. Table 2 also includes the predicted values for the described corresponding variables, whereas the experimental values were replaced by the application of the model (R1’, R2’, and R3’ values, respectively). As a result, all the independent variables (A: urea/FA content ratio, w/w; B: crystallization temperature, ◦ C; C: crystallization time, h; D: stirring speed, rpm) significantly affected (p < 0.05) the response variables during the urea complexation process. On the other hand, the enrichment of EPA and DHA in concentrates varied inversely according to total FA yield, obtaining correlation coefficient values (r) of −0.7756 and −0.7185, respectively. Table 2. Values obtained for the experimental and predicted response variables of RCSO concentrate by central composite rotatable design 24 + star based on the response surface methodology 1 . 1 2 3 4 5 6 7 8 9 10 11 12 Response Variables ** Process Variables * Run Experimental Values Predicted Values A B C D R1 R2 R3 R1 ’ R2 ’ R3 ’ 1.5 4.5 1.5 4.5 1.5 4.5 1.5 4.5 1.5 4.5 1.5 4.5 −15 −15 15 15 −15 −15 15 15 −15 −15 15 15 14.3 14.3 14.3 14.3 36.8 36.8 36.8 36.8 14.3 14.3 14.3 14.3 200 200 200 200 200 200 200 200 600 600 600 600 40.44 12.64 48.71 17.45 36.25 10.44 4.22 18.12 41.74 12.77 78.45 16.11 10.47 28.41 9.22 25.09 10.46 24.42 7.96 24.87 10.23 30.20 9.02 25.16 11.04 44.38 9.52 32.69 11.13 55.91 8.18 30.47 10.89 46.43 9.41 34.34 33.77 12.23 40.35 18.82 29.02 21.55 20.22 12.76 44.89 12.14 68.43 21.40 13.77 28.09 9.52 23.85 13.22 27.55 8.98 23.30 14.52 28.84 10.27 24.60 14.00 45.89 10.23 26.94 15.24 47.14 11.47 28.18 14.71 46.61 10.94 27.65 105 Molecules 2019, 24, 1642 Table 2. Cont. 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 1.5 4.5 1.5 4.5 0 6 3 3 3 3 3 3 3 3 3 3 −15 −15 15 15 0 0 −30 30 0 0 0 0 0 0 0 0 36.8 36.8 36.8 36.8 25.5 25.5 25.5 25.5 3.05 48.0 25.5 25.5 25.5 25.5 25.5 25.5 600 600 600 600 400 400 400 400 400 400 0 800 400 400 400 400 45.49 9.86 58.94 12.64 57.38 15.86 12.21 33.82 12.71 16.31 19.11 16.47 20.77 20.74 17.20 22.74 9.91 27.56 7.84 23.05 6.33 25.45 29.26 13.52 20.41 23.01 22.62 26.07 22.43 21.40 24.00 18.23 10.06 48.55 7.91 28.93 6.39 30.43 44.37 14.65 22.67 28.93 25.72 33.39 25.82 25.20 29.15 20.23 40.14 7.17 48.30 15.33 67.84 13.34 19.61 34.36 25.65 14.83 13.39 27.09 20.24 20.24 20.24 20.24 13.97 28.30 9.73 24.05 -0.77 27.88 24.94 16.45 21.24 20.15 19.95 21.44 20.69 20.69 20.69 20.69 15.96 47.85 12.18 28.89 0.94 49.55 36.61 13.88 24.00 26.49 24.53 25.95 25.24 25.24 25.24 25.24 R2 Adjusted Equations Total FA yield = 57.60 − 19.47A + 0.26B − 0.87C + 0.08D + 2.26AA+ 0.01BB + 0.21AC − 0.02AD − 0.02BC + 0.001BD EPA = −0.90 + 9.53A − 0.14B − 0.79AA DHA = −1.19 + 8.10A + 0.17B − 0.19AB EPA+DHA = 3.52 + 12.88A − 0.52B 0.72(1) 0.84(2) 0.81(3) 0.80(4) 1 Central composite design: 24 + star, which studies the effects of 4 factors in 28 runs based on the RSM. * Independent variables: A (urea/FA content ratio, w/w), B (crystallization temperature, ◦ C), C (crystallization time, h), and D (stirring speed, rpm).** Response variables: R1 (total FA yield, g FA in the non-urea complexing fraction/100 g initial saponified oil FA), R2 (EPA content, g/100 g total FA), and R3 (DHA content, g/100 g total FA). Predicted response variables: R1 ’, R2 ’, and R3 ’. 2.2.2. Effect of Process Variables on EPA, DHA, and EPA+DHA Content and Total FA Yield: Pareto Charts and RSM Analysis Pareto charts (Figure 1) were obtained for the different dependent variables as a function of the concentrate processing variables from RCSO; furthermore, the linear, quadratic, and interaction terms in the second-order polynomial were used to generate a three-dimensional response surface graph. Panel (A) indicates that the total FA yield (R1) of concentrates was dependent (p < 0.05) on the linear terms of the urea:FA content ratio (A), crystallization temperature (B), crystallization time (C), stirring speed (D), the quadratic terms AA and BB, and the interactions terms AD, BD, BC, and AC. Figure 1 (Panel B) shows the response surface of the urea complexation process for the total FA yield. The total FA yield decreased when the urea:FA content ratio increased and the crystallization temperature decreased. A similar result was found for the total FA yield effect in concentrate obtained from a by-product of rainbow trout processing where the total FA yield presented a minimum value in the response surface analysis when considering high urea:FA content ratios, at low crystallization temperature levels and stirring speeds, and at intermediate levels of crystallization time [17]. In the case of the EPA content (Figure 1, Panel C), the Pareto charts showed that certain linear terms—the urea:FA content ratio (A), crystallization temperature (B), and the quadratic urea:FA content ratio (AA)—provided a significant effect (p < 0.05). However, linear terms such as the crystallization time (C) and stirring speed (D) did not produce significant changes (p > 0.05). Figure 1 (Panel D) exhibits the response surface of the urea complexation process for EPA content. It was found that the EPA content increased with the urea:FA content ratio while it decreased as the crystallization temperature increased. For the DHA content (Figure 1, Panel E), the Pareto charts reported that the linear terms of the urea:FA content ratio (A), crystallization temperature (B), and the interaction between the urea:FA content ratio and the crystallization temperature (AB) revealed a significant effect (p < 0.05). The DHA content increased when the urea:FA content ratio increased, while it decreased as the crystallization temperature increased (Figure 1, Panel F). Finally, concerning the EPA+DHA 106 Molecules 2019, 24, 1642 content, the urea:FA content ratio showed a positive effect, whereas the crystallization temperature had a negative effect (p < 0.05) (Figure 1, Panel G). These results agree with those obtained by authors concerning the employment of the Asian catfish (Pangasius bocourti) and by-product of rainbow trout oil, which reported an inverse relationship between the urea:FA content ratio and the crystallization temperature on the urea concentrate process [16,17]. When the urea:FA content ratio increased and the crystallization temperature decreased as result, high values were obtained for DHA in the non-urea complexing fraction, as well as a great retention of saturated and monounsaturated FA in the urea crystal adducts. In this case, similar results for the EPA content were found. In all the cases, the EPA, DHA, and EPA+DHA contents did not significantly varied depending on the stirring speed (p > 0.05). 3DQHO% 3DQHO$ $8UHDIDWW\DFLGV $$ $' %&U\VWDOOL]DWLRQWHPS %' '6WLUULQJVSHHG %& $& &&U\VWDOOL]DWLRQWLPH %% 7RWDO)$<LHOG                      8UHD)$FRQWHQWUDWLRZZ 6WDQGDUGL]HGHIIHFW 3DQHO' 3DQHO&   (3$JJWRWDO)$ $8UHDIDWW\DFLGV %&U\VWDOOL]DWLRQWHPS $$ '6WLUULQJVSHHG &&U\VWDOOL]DWLRQWLPH         &U\VWDOOL]DWLRQWHPSƒ& (B) (A)                              &U\VWDOOL]DWLRQWHPSž& 8UHD)$FRQWHQWUDWLRZZ    6WDQGDUGL]HGHIIHFW              (C) (D) 3DQHO) 3DQHO(              '+$JJWRWDO)$ $8UHDIDWW\DFLGV %&U\VWDOOL]DWLRQWHPS $% &&U\VWDOOL]DWLRQWLPH '6WLUULQJVSHHG                6WDQGDUGL]HGHIIHFW    8UHD)$FRQWHQWUDWLRZZ (E) (F) $8UHDIDWW\DFLGV %&U\VWDOOL]DWLRQWHPS '6WLUULQJVSHHG &&U\VWDOOL]DWLRQWLPH    (3$'+$JJWRWDO)$          &U\VWDOL]DWLRQWHPSž& 3DQHO+ 3DQHO*               8UHD)$FRQWHQWUDWLRZZ 6WDQGDUGL]HGHIIHFW                  &U\VWDOOL]DWLRQWHPSž& (H) (G) Figure 1. Pareto charts and response surfaces for the effects of different process variables: Panels in total FA yield (%, panels A,B), EPA content (g/100 g total FA, panels C,D), DHA content (g/100 g total FA, panels E,F), EPA+DHA content (g/100 g total FA, panels G,H). A: urea/FA contents ratio, w/w; B: crystallization temperature, ◦ C; C: crystallization time, h; and D: stirring speed, rpm). 107 Molecules 2019, 24, 1642 2.2.3. Models Obtained for the Concentration of EPA, DHA, and EPA+DHA Equations obtained for the experimental process variables of the response surface model calculated by multiple regression are shown in Table 2. According to the equations obtained, all the response variables were found to be dependent on the same process variables expressed in the Pareto analysis (Figure 1). The four RCSO concentrated models had an adjusted R2 by degrees of freedom of 72.0% for total FA yield (Equation (1)), 84.0% for EPA (Equation (2)), 81.0% for DHA (Equation (3)), and 80.0% for EPA+DHA (Equation (4)). Such values reported that the models adequately represented the variability of the results. Since the p-values obtained for lack-of-fit in the ANOVA study (0.014, 0.35, 0.19, 0.28; Equation (1) to Equation (4), respectively) was greater or equal to 0.05, except for Equation (1), the model appears to be adequate for the observed data at the 95.0% confidence level. 2.2.4. Independent Variables and Multiple Response Optimization Table 3 (part a) shows the optimization of the independent variables for the response variables (EPA, DHA, and EPA+DHA) of the urea complexation process. The optimum values of dependent variables for EPA, DHA, and EPA+DHA content were 33.01, 76.81, and 98.85 (g/100 g total FA), respectively. In all the cases, a tendency toward the same values for the urea:FA content ratio and crystallization temperature was observed, so that the optimum EPA, DHA, and EPA+DHA contents were obtained. Table 3. Process variables * optimization and multiple response optimization of the response variables. Part a) Optimization of the Process Variables Dependent Variables EPA DHA EPA+DHA Process Variables A 5.99 6.00 6.00 B −29.79 −29.98 −29.95 C 3.05 48.05 47.79 D 599.00 108.30 271.36 Stationary Point Optimum Value ** Maximum Maximum Maximum 33.01 76.81 98.85 Part b) Multiple Response Optimization of the Response Variables Dependent Variables EPA DHA EPA+DHA Maximum desirability Stationary Point Process Variables A B C D 5.84 −17.69 14.83 453.36 Maximum Predicted Value ** 30.7162.94 90.07 1.0 Part c) Experimental Validation of the Multiple Response Optimization of the Dependent Variables Dependent Variables EPA DHA EPA+DHA Process Variables A B C D 6.00 −18 14.80 500 Stationary Point Experimental Value ** Maximum 31.20 49.31 80.51 * Process variables (A, B, C, and D) as expressed in Table 2. ** Values expressed as g/100 g total FA. Table 3b shows the levels of factors that maximized the EPA, DHA, and EPA+DHA contents (g/100 g total FA) by means of multiple response optimization and the stationary point that predicted a maximum of 30.71, 62.94, and 90.07 (g/100 g total FA) in EPA, DHA, and EPA+DHA, respectively. Maximum desirability score of 1 (range, 0–1) was attained. A maximum predicted value could be obtained, provided that the following process conditions were applied: 5.84 (urea: FA content ratio), −17.69 ◦ C (crystallization temperature), 14.83 h (crystallization time), and 453.00 rpm (stirring speed). 108 Molecules 2019, 24, 1642 Figure 2 (panels A, B) shows the contours and estimated response surface of the urea:FA content ratio and crystallization temperature of the combination of factors levels to maximize the desirability function for RCSO concentrate. It is observed that the highest desirability values were reached by taking into account the high values of the urea:FA content ratio and the low crystallization temperature values. These results were similar to those previously reported for rainbow trout belly oil [17], where a maximum desirability score of 0.91 was obtained in the multiple response optimization of EPA, DHA and EPA+DHA contents. Furthermore, the predictive values of 32.50, 37.00, and 67.70 (g/100 g total FA) in the by-product concentrate of rainbow trout oil, respectively, were obtained. As a result, the following process conditions were applied in such study: 4.21 (urea:FA contents ratio), −15.00 ◦ C (crystallization temperature), 24.0 h (crystallization time), and 1000 rpm (stirring speed). &U\VWDOOL]DWLRQWHPSž& 3DQHO%                         'HVLUDELOLW\            8UHD)$FRQWHQWUDWLRZZ (B) (A) Figure 2. Combination of factors to maximize the desirability function for RCSO concentrate: response surface (A) and contour surface (B). 2.2.5. Validation of the Optimized Process and Characterization of the EPA+DHA Content Obtained Table 3c shows the validation of multiple response optimization after experimentally performing the process conditions. For EPA content, comparison of the predicted value and the value obtained experimentally showed that both values were similar (i.e., 30.71 and 31.20 g/100 g total FA, respectively); however, the experimental value of DHA content was substantially different from the predictive value (49.31 and 62.94 g/100 g total FA, respectively). Experimental values performed on rainbow trout belly oil [17] were shown to be similar to the predicted values obtained in the current study for EPA and DHA (36.10 and 47.70 g/100 g of total FA, respectively). In the experimental validation, an 80.51 g/100 g total FA value was obtained for the EPA+DHA content, which agrees with the value revealed by other authors who obtained a stationary point of 89.38% for the EPA+DHA content [14]. 2.3. Composition of FA in the RCSO Optimized Concentrate after Validation Table 1 shows the FA composition of the RCSO compared to the composition of FA from the validated optimum concentrate. Optimization process validation was performed experimentally by combining the factors levels in which the optimal EPA+DHA content was attained (Table 3b,c). In these experiments, the effect of the urea complexation process on the composition of FA and FA groups in the optimized concentrate (g/100 g FA) after validation can be observed. When compared to the initial RCSO, there was a marked increase in the concentration of total PUFAs (96.99%) and a substantial decrease in the concentration of SFAs (0.59%) and MUFAs (2.42%). Furthermore, the predominant FAs in the optimum concentrate oil were EPA, DHA, and EPA+DHA (31.20, 49.31, and 80.51 g/100 g total FA, respectively). The urea complexation under the optimization process conditions has revealed a high efficiency, since a marked increase of total PUFAs from 38.24 to 96.99 g/100 g total FA could be observed; this increase was 1.21 times higher than that reported by Pando et al. [18]. Additionally, the total EPA+DHA final content was 2.5 times higher than that reported for refined salmon oil concentrate without the optimization process [18]. 109 Molecules 2019, 24, 1642 3. Discussion In the present research, the distribution of SFAs in RSCO showed that this FA group was mainly composed of palmitic, stearic, and myristic acids. Similar values have been established for the SFAs group from crude commercial salmon oil [18]; interestingly, refined commercial salmon oil and Asian catfish oil also showed these three FA as the major ones, but a higher presence of stearic acid compared with myristic acid was detected in our study [16,18]. Among the MUFAs, the most abundant in the present study were 18:1 9c, 16:1 9c, 18:1 11c, and 20:1 11c. The total n-3 long chain polyunsaturated fatty acids (n-3 PUFAs) content was 1.14 times higher than the content of total n-6 PUFAs, showing values higher than those previously reported in different kinds of oils obtained crude commercial salmon, and rainbow trout (Oncorhynchus mykiss) [18,20]. The analysis of process variables shows a high recovery performance of RCSO concentrate when the urea:FA content ratio has the lowest value, and the value considered for the crystallization temperature is the highest. The results show a higher content of EPA and DHA when the total FA yield is lower, indicating that this experiment eliminated most of the SFAs and MUFAs from the starting oil, only leaving a small fraction of such acids in the urea non-complexed fraction. Similar values have been observed by other authors [17,21], who reported major yields with higher stirring speed, corroborating a significant effect of the stirring speed on the total FA yield in the urea complexation process (p < 0.05). Current results show an inverse relationship between the urea:FA content ratio and the crystallization temperature on the DHA content, which is a conclusion that has already been than those reported by several authors, whose employed different oils from marine origin such as seal blubber oil, tuna oil, Asian catfish oil, and a by-product of rainbow trout [13,14,16,17]. In this case, results similar for EPA content were found. As an explanation for this, it could be argued that the urea:FA content ratio has a significant positive effect (p < 0.05) on the concentration of EPA and DHA, probably as a result of increasing the concentration of urea with respect to that of FFA; consequently, this would lead to an increase of the number of adducts formed between the flat structures of SFA and urea molecules, this favoring the formation of hexagonal complexes and crystals [13], and leading to an increased concentration of EPA and DHA in the non-urea complexing solution. Previous studies on tuna oil concentrates have revealed that the regression models for total FA yield and total EPA+DHA content were highly significant with satisfactory R2 coefficients [14,22]. The present data proved that the urea complexation under the optimization process conditions has shown to be highly efficient, since an increase in the total PUFAs content from 38.24 to 96.99 g/100 g total FA could be reached, which was 1.2 times higher than that reported by Pando et al. [18]. Additionally, the total EPA+DHA final content was 2.5 times higher than that reported for refined salmon oil concentrate without applying an optimization process [18]. On the other hand, the oxidative and hydrolytic stability parameters (PV, pAV, TOTOX, and FFA) indicate that the quality of the oil used in this study was included within the acceptable quality limits for edible fats and oils of marine origin according to the Chilean Food Sanitary Regulations. Government institutions and trade associations, such as the Council for Responsible Nutrition (CRN) and the Global Organization for EPA and DHA Omega-3s (GOED), have set strict guidelines for marine oil quality and safety parameters. According to the previously described recommended values [23,24], the starting RCSO can be considered included in the 98% of fish oil products compliant with the PV limit of 10 meq. active oxygen kg−1 oil set by British Pharmacopeia Fish Oil Type I, European Union (EU) Pharmacopeia Fish Oil Type I, and Australian government guidelines. Furthermore, salmon oil samples were compliant with the pAV limit of 15 set by British and EU Pharmacopeia Fish Oil Type II, as well as the pAV limit of 20 set by the GOED, Canada Natural Health Product Directorate (NHP), United States Pharmacopeia, and Codex Alimentarius Commission, Food and Agriculture Organization of the United Nations (CODEX/FAO). Thus, the primary and secondary lipid oxidation levels of RCSO are not exceeding the regulatory thresholds in the testing peroxide values and p-anisidine values. Interestingly, such lipid oxidation scores can be considered similar to those previously obtained for refined salmon oil samples [18] and salmon oil [24]. On the other hand, the FA composition of the RCSO showed that most abundant FAs were C18:1 9c, 18:2 9c, 12c, and C16:0, followed by EPA and DHA. The value of the total SFAs was 110 Molecules 2019, 24, 1642 found to be 21.28% in RCSO. Concerning PUFAs, the most abundant in RCSO concentrates were DHA, EPA, and 18:2 9c, 12c. 4. Materials and Methods 4.1. Materials and Chemicals Refined commercial salmon oil was provided by Fiordo Austral S.A. (Puerto Montt, Chile). Fatty acid methyl ester (FAME) standards, fatty acid (FA) standards, and C23:0-methyl ester (2COT N-23M-A29-4 NU-CHECK-PREP-INC) were obtained from NU-CHECK-PREP, INC (Elysian, MN, USA). All the solvents and chemicals used (including urea, ethanol, α-tocopherol, and n-hexane) were of analytical grade (Merck, Santiago, Chile). 4.2. Characterization of Refined Commercial Salmon Oil Initial RCSO characterization was carried out by chemical analyses. For it, the following standard Association of Official Analytical Chemists (AOAC) and official methods [25] were carried out: peroxide value (PV; method Cd 8b-90:1-2), p-anisidine value (pAV; method Cd 18-19:1-2), and total oxidation value (TOTOX; method Cg 3-91) and free fatty acids (FFA) contents (method Ca 5a-40:1). 4.3. FA Composition of the RCSO and n-3 LCPUFA Concentrates For analyzing the FA composition of the RCSO and the different LCPUFA concentrates, a methylation process was performed to obtain FAMEs. For it, a two-step process was performed, according to previous research [18,26]. FAME analysis was carried out on an HP 5890 series II GLC with a flame ionization detector (FID) with the injection system split. A fused silica capillary column (100 m length × 0.25 mm × 0.2 μm film thickness) coated with SPTM-2560 (Supelco, Bellefonte, PA, USA) was used [18,26–29]. DataApex ClarityTM software (DataApex Ltd., Prague, Czech Republic) for chromatogram analysis was applied. The reference standard NU-CHEK GLC463 was used to identify the FA profiles. The concentration of the different FAME was determined from the calibration curves by assessment of the peak/area ratio. The quantification of all the individual FAs (g/100 g total FA) was achieved by employing C23:0 methyl ester as the internal standard according to the (AOCS Official Method (Ce 1j-7, 2009) [26]. 4.4. n-3 LCPUFA Concentrates from RCSO The procedure included salmon oil saponification, which was in agreement with other authors [17,18,30]. Concentrate from RCSO was prepared by FFA collection, the formation of urea FFA inclusion complexes, and the extraction of free n-3 LCPUFA. For it, the urea complexation method was carried out by 28 experimental runs with different urea:FA content ratios (0 to 6 w/w), crystallization temperatures (−30 to 30 ◦ C), crystallization times (0 to 48 h), and stirring speeds (0 to 800 rpm). The FFAs were mixed with urea and 95 % ethanol, and the mixture was subsequently stirred and heated at 60 ◦ C with magnetic stirring. Then, it was cooled with constant stirring to different conditions of temperature and time as described in the experimental design. The crystals formed were separated from the liquid phase by filtration with a Whatman No.1 paper. The non-urea complexing fraction was diluted with 100 mL of distilled water by each 10 g, acidified to pH 4.5 with 6 N HCl, and washed in a separating funnel with hexane (400 mL). The hexane phase was filtered with anhydrous sodium sulfate in a Whatman No.1 paper, and the solvent was partially removed using a rotatory evaporator at 40 ◦ C under vacuum [16,17]. The resulting n-3 LCPUFA concentrates were stored at −80 ◦ C with 0.5% of α-tocopherol under nitrogen atmosphere until use for further analysis. 4.5. Experimental Design and Optimization Procedure A rotational central composite design 24 + star, with 4 factors and 5 levels, was carried out, with 28 experimental runs that included 4 repetitions of the central point based on the RSM. The following 111 Molecules 2019, 24, 1642 conditions for the independent variables were considered (Table 1): urea:FA content ratio (variable A: 0 to 6 w/w), crystallization temperature (variable B: −30 to 30 ◦ C), crystallization time (variable C: 3.05 to 48.0 h), and stirring speed (variable D: 0 to 800 rpm). On the basis of the non-urea complexing fraction, the following response variables (R variables) of the experiment design were chosen: total FA yield (variable R1: g FA in the non-urea complexing fraction/100 g initial RCSO), EPA content (variable R2: g/100 g total FA in concentrate), and DHA content (variable R3: g/100 g total FA in concentrate). Four replicates were carried out at the central point of the experimental design in order to evaluate the experimental error. All experiments were performed randomly to minimize the effect of unexplained variability in responses resulting from extraneous factors [31]. In order to obtain response surfaces, multiple regression equations were fitted to the responses obtained by discarding non-significant terms (p > 0.05) to obtain response surfaces. To maximize the desirability function a multiple response optimization was performed to optimize several responses simultaneously, which maximized the desirability function scores that ranged between 0 and 1 [32]. The RSM was used to optimize and maximize the response variables, and a quadratic polynomial regression model was assumed for predicting individual Y variables. The model proposed for each Y value was as according to Equation (5): 4 4 3  4    βi Xi + βii Xi2 + βij Xi X j + ε, (5) Yi = β0 + i=1 i=1 i=1 j=i+1 where β0 , βi , and βii represent the intercept, linear, and quadratic coefficients, respectively; βij corresponds to the interaction coefficient terms for the interaction of variables i and j; Xi represents the independent variables; and E denotes to the random error [31,32]. 4.6. Statistical Analysis Multiple regression analysis, ANOVA. canonical, and ridge maximum of data in the response surface regression (RSREG) procedure was used. The estimated response surface and contours of the estimated response surface were developed using the fitted quadratic polynomial equations obtained from the response surface regression (RSREG) analysis, holding the independent variables with the least effect on the response at a constant value and changing the levels of the other two variables [31,32]. A multiple-response optimization was performed to assess the combination of experimental factors that simultaneously optimize several responses; as a result, maximization of the desirability function was obtained, this function ranging from 0 to 1 [32]. Analyses were performed in triplicate considering the standard deviation of each sample. The lack-of-fit test was carried out by comparison of the variability of the current model residuals with the variability between observations at replicate settings of the factors [31,32]. Statgraphics Centurion XV.II (Manugistics Inc., Rockville, USA) was used. 5. Conclusions The physical–chemical analyses of refined commercial salmon oil indicate that it is a good quality raw material that complies with the characteristics that are typical of oils of marine origin. A high concentration of EPA and DHA was obtained from RCSO, achieving an increase of up to 4.1 and 7.9 times in the concentrate, with values of 31.20 and 49.31 g/100 g total FA, respectively. Interestingly a 5.8-time increase was observed for the EPA+DHA content, from 13.78 to 80.51 g/100 g total FA. Therefore, it has been proved that it is possible to maximize the EPA and/or DHA content by the optimization of the variables in the urea inclusion process. The results of this study could serve as a basis for the food and nutraceutical industry, whose processes with clean technologies can develop new functional foods enriched with EPA and DHA. 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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/). 114 molecules Review Potential Use of Marine Seaweeds as Prebiotics: A Review Aroa Lopez-Santamarina, Jose Manuel Miranda *, Alicia del Carmen Mondragon, Alexandre Lamas, Alejandra Cardelle-Cobas, Carlos Manuel Franco and Alberto Cepeda Laboratorio de Higiene Inspección y Control de Alimentos, Departamento de Química Analítica, Nutrición y Bromatología, Universidade de Santiago de Compostela, 27002 Lugo, Spain; a_lo_san@hotmail.com (A.L.-S.); aliciamondragon@yahoo.com (A.d.C.M.); alexandre.lamas@usc.es (A.L.); alejandra.cardelle@usc.es (A.C.-C.); carlos.franco@usc.es (C.M.F.); alberto.cepeda@usc.es (A.C.) * Correspondence: josemanuel.miranda@usc.es; Tel.: +34-982-252-231 (ext. 22407); Fax: +34-982-254-592 Academic Editors: Sylvia Colliec-Jouault and Jesus Simal-Gandara Received: 16 December 2019; Accepted: 21 February 2020; Published: 24 February 2020 Abstract: Human gut microbiota plays an important role in several metabolic processes and human diseases. Various dietary factors, including complex carbohydrates, such as polysaccharides, provide abundant nutrients and substrates for microbial metabolism in the gut, affecting the members and their functionality. Nowadays, the main sources of complex carbohydrates destined for human consumption are terrestrial plants. However, fresh water is an increasingly scarce commodity and world agricultural productivity is in a persistent decline, thus demanding the exploration of other sources of complex carbohydrates. As an interesting option, marine seaweeds show rapid growth and do not require arable land, fresh water or fertilizers. The present review offers an objective perspective of the current knowledge surrounding the impacts of seaweeds and their derived polysaccharides on the human microbiome and the profound need for more in-depth investigations into this topic. Animal experiments and in vitro colonic-simulating trials investigating the effects of seaweed ingestion on human gut microbiota are discussed. Keywords: prebiotic; polysaccharides; seaweed; Rhodophyceae; Phaeophyceae; Chlorophyceae 1. Introduction Marine seaweeds have been consumed whole by East Asian populations for centuries, if not millennia, appearing in traditional recipe books in many countries [1]. Additionally, their human consumption in Western countries has been increasing in the latest decades because of their association with improved human health. Some benefits of their consumption include a lower incidence of cancers, decreased blood pressure and blood sugar, and antiviral, anti-inflammatory, immunomodulatory or neuroprotective activities [1,2]. A mechanistic link proposed to explain the prevention of such diseases by seaweed consumption implicates the presence of diverse health-promoting bioactive compounds in seaweeds, including sulphated polysaccharides, polyphenols, pigments (chlorophylls, fucoxanthins, phycobilins), carotenoids, omega-3 fatty acids or mycosporine-like amino acids [1,3,4]. In some cases, these compounds are not produced by terrestrial plants or are not consumed in adequate quantities as part of a typical Western diet. Nowadays, with a continuously expanding world population, fresh water is an increasingly scarce commodity, and the world’s desertification processes continue. About 45% of the world’s land surface is considered drylands, while 12 million hectares of land are degraded yearly through lack of water and related processes [5]. According to the Food and Agriculture Organization of the United Nations [6], agricultural productivity is persistently declining at over 1% per year. It is thus reasonable to expect that in the next decades there will be a need for increasing algae production to replace or supplement Molecules 2020, 25, 1004; doi:10.3390/molecules25041004 115 www.mdpi.com/journal/molecules Molecules 2020, 25, 1004 the intake of plant foods of terrestrial origin. Seaweeds have numerous advantages over terrestrial plants, such as rapid growth rates, and they do not require arable land, fresh water or contaminating fertilizers [7]. Additionally, an increase in seaweed cultivation would provide environmental services as an added benefit [5]. Marine seaweeds constitute approximately 25,000–30,000 different species, with a great diversity of forms and sizes [8]. The taxonomic groups, which reflect their pigmentation, include red algae (Rhodophyceae), brown algae (Phaeophyceae) and green algae [8]. Although they are still produced on a very modest scale relative to global food production, their worldwide cultivation has increased rapidly in the last decades, reaching a current yearly production of about 29 million tonnes [8]. Asian countries dominate world production, with 99% of the total, while most other maritime countries produce little or none [5]. Polysaccharides account for the majority of seaweed biomass (up to 76% of dry weight in some species; [8] and together with oligosaccharides have been the key focus of many studies of seaweed-derived compounds. Phenolic compounds and proteins from seaweeds have also attracted interest as potential functional ingredients [8]. Besides their consumption as an entire food, seaweeds or their polysaccharides are considered valuable additives in the food industry because of their rheological properties as gelling and thickening agents [2]. Additionally, seaweeds were largely employed in the formulation of animal feed [9], as well as in the formulation of cosmetics, drugs and fertilizers [10]. Regarding specific benefits on human health, seaweeds have demonstrated to exert preventive effects against several non-transmissible diseases such as cardiovascular diseases [11,12], antihypertensive [13], anti-obesity effects [14] and anti-diabetic effects [15,16], anti-cancer [17,18] or antioxidant activities [19]. Regarding cardiovascular diseases, there are large contributing risk factors that overlap and intertwine, contributing overall to the onset and growth of the disease [11]. Between them, it can be cited a cascade of mechanisms including vascular inflammation, oxidative stress, hypercoagulability and activation of the sympathetic and renin-angiotensin systems. Although in some cases the exact mechanisms through seaweeds can prevent cardiovascular diseases are not always fully understood, it was demonstrated that seaweed consumption can prevent cardiovascular diseases [11,12]. Functional oligosaccharides from seaweeds are associated with a variety of biological processes linked to hypoglycaemic and hypolipidaemic activities, although the concrete mechanisms have not been well studied [15]. With respect to hypertension, various compounds from seaweeds, such as protein-derived bioactive peptides and phlorotannins, can prevent hypertension by inhibition of angiotensin-I converting enzyme activity [13]. The potential anti-obesity activity derived from seaweeds consumption may involve a large variety of mechanisms and alterations in lipid metabolism, suppression of inflammation, suppression of adipocyte differentiation and delay in gastric emptying [14]. Between then, an important anti-obesity activity of seaweeds is the inhibition of peroxisome proliferator-activated receptor γ (PPARγ) expression and activation of the adenosine monophosphate-activated protein kinase (AMPK) phosphorylation [13]. Other important anti-obesity mechanism of seaweeds is related with inhibition of lipases, especially pancreatic lipase, that is one of the main therapeutic targets of anti-obesity drugs [14] and was recently demonstrated for various seaweeds species [20]. Additionally, anti-obesity mechanisms related with concrete seaweed components, such as phlorotannins, that target the inhibition of adipocyte differentiation or fucosterol, that decreases the expression of the adipocyte marker proteins PPARγ and CCAAT/enhancer-binding protein alpha were reported. Seaweeds can also prevent obesity by means of the modification of the relative quantities of phyla in the gut microbiota (GM), and polysaccharides from seaweeds can reduce obesity also by repairing the intestinal barrier and reducing inflammation [21]. Although abundant signaling pathways have been found to be involved in the process of glucose metabolism and anti-diabetic effects [15,16], among them, the IRS1/PI3K/JNK/AKT/GLUT4 pathways are important mechanisms in insulin signal transduction. Some seaweed components were shown in animal models to ameliorate the hepatic insulin resistance by regulating the cited signaling pathways [15,16,22]. In addition, seaweeds significantly increased the abundance and diversity of gut 116 Molecules 2020, 25, 1004 microbiota in animal models and showed the ability to increase the population of beneficial microbiota and maintain the homeostasis of the GM [15,22]. With respect to the antioxidant activity of seaweeds, this protective affects depends mainly on phlorotannins, secondary metabolites that exert their great antioxidant activity via the scavenging of reactive oxygen species [19]. Secondary metabolites of seaweeds are also responsible for the anti-cancer activity, whose includes different mechanisms such as repairing the intestinal barrier by intensifying the expression of the tight junction proteins via increasing the phosphorylation of MAPK and ERKT/2 genes [18], and activating the caspase cascades. Other potential mechanisms are reducing the expression of cyclin-dependent kinases and matrix metalloprotease family [17] and inducing decreased levels of pro-apoptotic metabolic signals [19]. 2. Polysaccharides from Marine Seaweeds Indigestible dietary polysaccharides attract attention as functional food ingredients with health benefits [7]. Most carbohydrates entering the colon are fermented in the proximal colon, which is considered a saccharolytic environment. As digesta moves through the distal colon, carbohydrate availability decreases, and proteins and amino acids become the main metabolic energy sources for bacteria in this region. The main end-products of saccharolytic fermentation are short chain fatty acids (SCFA), which contribute towards the host’s daily energy requirements. On the contrary, the end-products of proteolytic fermentation include metabolites such as phenolic compounds, and nitrogenous ones like amines and ammonia, some of which are carcinogens. This means that the GM exerts a key contribution to the human energy balance and nutrition, by extending the host metabolic capacity to indigestible polysaccharides. In addition, intestinal microorganisms contribute to develop and maintain the host immune system, defending the host from colonization by opportunistic pathogens [2]. The effects of polysaccharides on the GM are generally evaluated by the contents of SCFAs, the composition and the abundance of beneficial intestinal bacteria [20]. Polysaccharides in today’s human diet originate primarily from terrestrial plant cell walls, while other sources, such as seaweeds, are less represented [8]. Studies indicate that polysaccharides and oligosaccharides derived from seaweeds can modulate intestinal metabolism, including fermentation, inhibit pathogen adhesion and evasion, and potentially treat inflammatory bowel disease [8,23]. Some seaweed polysaccharides also demonstrate anticoagulant [24], antitumor [25], anti-inflammatory [26], antiviral, antihyperlipidemic [27] or antioxidant activity [28]. Other research has focused on their use as prebiotics to aid in limiting the occurrence of non-transmissible chronic diseases common in Western countries, such as obesity, diabetes, cardiovascular diseases or some types of cancer [2]. Nevertheless, many seaweed fibres are high-molecular-weight polymers that need to be transformed into oligosaccharides to increase their fermentability by the GM [2]. Depending on the taxonomic classification of algae, polysaccharides can vary greatly in their composition [8]. Seaweeds feature an integrated network of biopolymers in their cell walls, mainly formed by polysaccharides associated with other compounds, such as proteins, proteoglycans, polyphenols and some mineral elements, like calcium and potassium [8]. It is because of this complexity that for most seaweed polysaccharides, the exact structures, constituents and chemistry are not fully known [29]. Depending on the algal taxa, both structural and storage polysaccharides may vary. Structural polysaccharides are the most abundant, and their composition can be influenced by the seaweed species [29], as well as environmental factors, such as salinity, water temperature and sunlight intensity [30]. Some of the structural polysaccharides are carboxylated or sulphated, which can affect their fermentability [2]. Green seaweeds contain mostly sulphated structural polysaccharides, like ulvans (the most abundant, representing 8–29% of dry weight) and sulphated galactans, xylans and mannans. These polymers are composed mainly of rhamnose, xylose, glucose, glucuronic acid and sulphates, with smaller amounts of mannose, arabinose and galactose [31,32]. These polysaccharides are not fully fermented by the human GM [33,34]. Conversely, the main carbohydrate in storage is starch. 117 Molecules 2020, 25, 1004 Contrariwise, brown seaweeds contain mainly cellulose, alginic acids, fucoidans and sargassans as structural polysaccharides, while the storage polysaccharides are alginates [35] (the most abundant at 17–45% of dry weight), fucoidans and laminarins [2,8,36]. Finally, red seaweeds contain agars, carrageenans, xylans, sulphated galactans and porphyrins as main structural polysaccharides, while the main storage polysaccharide is starch [21,37]. Through a long time of co-evolution between GM and host, the intestinal microbes have evolved diverse strategies for degrading polysaccharides from terrestrial plants [38]. However, because the consumption of seaweeds polysaccharides was not common over human evolution, the human GM did not acquire the same efficacy to degrade seaweed polysaccharides. Thus, although humans possess the enzymes necessaries to degrade some algal polysaccharides, such as starches, they are unable to digest the most complex polysaccharides [2,38]. An elegant work carried out by Hehemann et al. [39], showed that specific genes coding for enzymes with potential capacity to degrade seaweed polysaccharides, as porphyranases or agarases, can be transferred from a member of marine Bacteroidetes, Zobellia galactanivorans to the GM bacterium Bacteroides plebeius in Japanese individuals. As a consequence, GM of those subjects acquires the ability to degrade porphyran and agarose, as compared to the GM from North American individuals, who are incapable of degrading it [39]. De Jesus Raposo et al. [40] suggested that most seaweed polysaccharides can be regarded as dietary fibre, as they are resistant to digestion by enzymes present in the human gastrointestinal tract, reaching the distal gut. In this allocation, polysaccharides are fermented and become food for the commensal bacteria, stimulating their growth. For this reason, great efforts have been placed on developing efficient methods for seaweed polysaccharides extraction, purification and structural characteristics elucidation in order to improve their bioavailability, especially for insoluble fibre [2]. It was previously shown that variations in the chemical structure of a prebiotic can impact its selective fermentation by bacteria [41]. For this reason, there were published large works regarding the investigation of the potential prebiotic effect of single polysaccharides, often contained in seaweeds, but employing pure standards [42–44]. However, it should be considered that seaweeds contain other components than also can affect GM. Consequently, trials employing whole seaweeds are required to investigate their real effect in human GM. Furthermore, although several works investigated the effects of seaweeds in livestock gut microbiota, they are more oriented to study the effects of seaweeds in animal production and welfare. An elegant review was recently published where they can be checked [32]. 3. Other Bioactive Compounds from Marine Seaweeds In addition to polysaccharides, seaweeds also contain other bioactive compounds, called secondary metabolites much of them with antioxidant activity [45]. Among these, polyoletides (such as phlorotannins), isoprenoids (such as terpenes, carotenoids and steroids), alkaloids and shkimates (such as flavonoids) are the main groups of secondary metabolites found in algae [46]. Compared with other macroalgae, red seaweeds are richer sources of these secondary metabolites [47]. The human health benefits afforded by these bioactive compounds include anti-inflammatory, antioxidant, anticoagulant, antiviral, antimicrobial, antidiabetic, antitumor, antihypertensive, antiallergic and immunomodulatory activities [45–48]. Exceptionally, phlorotannins or polyphenols are recognized as structural classes of polyketides, found primarily in brown algae. These compounds can also reach the large intestine where GM can convert them into beneficial bioactive metabolites. Phlorotannins are highly hydrophilic components formed by polymerization of monomeric units of phloroglucinol (1,3,5-trihydroxybenzene). There are six main groups: fucols, floretoles, fucofloretols, fuhalols, isofuhalols and eckols, and all display strong antioxidant properties and act against oxidative stress [46]. Certain polyphenols are used as prophylactics against problems such as cardiovascular diseases, cancers, arthritis and autoimmune disorders [47]. In addition, some phlorotannins have been shown to decrease blood glucose levels after carbohydrate-rich meals. This action is achieved by interfering with the enzymes amylase and sucrase that intervene in the digestion and assimilation of these carbohydrates. In addition to their 118 Molecules 2020, 25, 1004 effects on the metabolic functions of the host, phlorotannins also appear to have some antibacterial activities [47], which may explain the low production of AGCC derived from seaweeds in which they are an important part [7]. However, like other phenolic compounds, bacterial growth inhibition occurs selectively in microbial populations, including some pathogens, and its antibacterial effect is minor in commensal bacteria [7]. Because of this selective inhibition of bacterial pathogens, large whole seaweeds and seaweeds ethanolic extracts has been used to extend the shelf life of fresh fishery foods, such as Fucus spiralis [48,49], Bifurcaria bifurcata [50], Cytoseira compressa [51] or Gracilaria verrucosa [52] Another of its potentially therapeutic functions is that extracts with high phlorotannins content have demonstrated a potent inhibitory action on the growth of cancerous cell lines [53,54]. Bromophenols present in marine algae have attracted much attention in the field of antimicrobial agents [55]. Previous studies indicate that marine bromophenols possess promising antibacterial [56,57] and antiviral activities [58]. In addition, symphyocladin G, a new bromophenol adduct derived from the red seaweed Symphyocladia latiuscula, is found to have antifungal activity against Candida albicans [59]. Several bromophenols isolated from the red alga Odonthalia corymbifera, are promising candidates for antifungal agents in crop protection [56]. These properties are not exclusive to red algae because compounds, such as bis(2,3-dibromo-4,5-dihydroxybenzyl) ether, isolated from brown algae Leathesia nana, showed cytotoxic activity against some cancer cells [54], and exhibited antibacterial activity against several strains of Gram-positive and Gram-negative bacteria [56]. With respect to terpenes, there are about 200 different diterpenoids, of which some have important cytotoxic and antiviral, antimicrobial and antiparasitic activities (such as against Leishmania) [60,61]. These compounds are found in red and brown algae. Flavonoids and their glycosides are present in green, brown and red algae. These compounds possess antioxidant properties and have demonstrated action against arteriosclerosis and cancer [45]. Within this group, fucoxanthin, β-carotene and violaxanthin stand out. Besides its strong anticancer activity, fucoxanthin has promise in preventing obesity [62]. The correlation between a carotenoid-rich diet and a low risk of cardiovascular and ophthalmological diseases has been supported by recent research with different types of carotenoids in cellular systems and human intervention studies [63]. Specifically, flavonoids from Enteromorpha prolifera influenced the GM balance in diabetic mice, increasing the presence of Alistipes, Lachnospiraceae and Odoribacter genera [63]. Alistipes spp. is one of the most abundant bacterial genera in the mouse intestine and is capable of fermenting glucose and lactic acid to produce propionic, acetic and succinic acid, which modulate the release of intestinal hormones, thereby influencing the release of insulin and appetite. It is perhaps for this reason that E. prolifera is traditionally used in China as a natural herb to treat diseases associated with inflammation [63]. It has recently been observed that a polysaccharide of E. prolifera could be used as a novel agent to treat obesity and hyperlipidaemia [62]. Other important secondary metabolites contained in seaweeds and responsible of important beneficial effect in human health are peptides, such as lectins [48]. Lectins primarily show antiviral, antibacterial, and antifungal activities. Specially one type of lectin, griffithsin, showed important antiviral activity and is nowadays considered a promise antiviral agent, with great potential concerning the prevention of sexually transmitted infections [48], including HIV [64]. Other important peptides are renin inhibitor tridecapeptide [65] and dipeptide [66], which demonstrated hypotensive effect dipeptide. Phycoerythrin [43] and kahalalide F [67] are other important peptidic compounds isolated from seaweeds than showed antitumor effect. Besides the so-called secondary metabolites, seaweeds contain other minor nutrients of immense importance for human health. Phycobiliproteins, responsible for the characteristic bright pink appearance of red algae, are classified into phycoerythrin (red) and phycocyanin (blue). These pigments are used commercially in food, nutraceuticals, and for their therapeutic properties, mainly antimicrobial, antioxidant, anti-inflammatory, neuroprotective, hepatoprotective, immuno-modulatory and anticancer effects [67–72]. Such compounds may improve the efficacy of standard anticancer drugs, decrease their side effects, and act as photosensitisers for the treatment of tumour cells [30]. 119 Molecules 2020, 25, 1004 4. Effects of Seaweed Polysaccharides on Human Health Compounds with prebiotic activity, such as oligosaccharides, lactulose, fructo-oligosaccharides (FOS), inulin, galacto-oligosaccharides and arabinoxylano-saccharides are used as functional ingredients in the food industry [10]. While most of the above compounds are now derived from terrestrial plants, some studies have shown that polysaccharides and oligosaccharides derived from marine algae can also modulate intestinal metabolism, including fermentation, inhibit adhesion and invasion of pathogens, and treat inflammatory bowel disease [23,73]. Furthermore, these compounds have demonstrated anticoagulant, antioxidant, immunomodulatory, antitumor and antiviral activities [10]. Being much less degradable by enzymes from the human upper gastrointestinal tract than their terrestrial plant counterparts, polysaccharides from marine algae reach a greater proportion in the descending colon. For this reason, some authors [42,74] have found that polysaccharides from marine algae, such as alginate, agarose oligosaccharides and κ-carrageenan oligosaccharides, have a higher prebiotic activity than FOS in vitro. Specifically, sulphated polysaccharides from marine algae show anticoagulant, antiviral, antitumor, anti-inflammatory, antibacterial, immunological, antioxidant and many other biological and physiological activities [8,75]. Sulphated polysaccharides include fucoidans (l-fucose and sulphated ester groups) from brown seaweeds, agars and carrageenans (sulphated galactans) from red seaweeds, and ulvans (sulphated glucuronoxylorhamnan) and other sulphated glycans from green seaweeds [8]. The consumption of these sulphated polysaccharides can block the adhesion of leukocytes to the epithelium of blood vessels, preventing the migration of these cells to the site of inflammation [76]. These polysaccharides often stimulate the growth and activity of beneficial bacteria by acting as substrates for fermentation in the large intestine, leading to the production of SCFA, with multiple functions that help maintain health [8]. As previously mentioned, seaweed polysaccharides differ in their properties and compositions from one type of algae to another [46], so their effects on the human GM will also differ. The GM, especially in its most distal parts, harbors many bacteria, archaea, protozoa and viruses, which along with their genetic material, is collectively referred to as the gut microbiome (GMB) [77]. This GM is composed of up to 12 different bacterial phyla of which more than 90% belong to the Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes [78], while the remaining phyla are much less constant and numerous [79]. The most frequent bacterial species in the colon, which is where the highest bacterial concentration exists [78], belong mainly to the families Bacteroidaceae, Prevotellaceae, Rikenellaceae, Lachnospiraceae and Ruminococcaceae [77]. The GM presents a diverse set of functions important to human health, such as the extraction of energy from a broad spectrum of nutrients, the production of vitamins, the promotion of immune homeostasis and the prevention of colonization of the intestine by pathogens [80]. One of the most important functions of the GM is in the prevention of chronic low-grade inflammation [81]. Host genetics define the chemistry and physics of the GM, including the availability of nutrients and the threshold of activity required to induce an immune response. Consequently, intestinal microbial communities are composed of species that have evolved to occupy specific ecological niches in the gut, including the ability to metabolize specific molecules available from the host or to evade host defenses [77]. Nutrients can interact directly with the GM to promote or inhibit its growth. In this sense, the ability of the GM to extract energy from specific components of the diet offers a direct competitive advantage to specific members of the GM, allowing them to proliferate at the expense of other members [81]. Thus, diet affects not only the composition and absolute abundance of intestinal bacteria but also their growth kinetics [82]. In this context, the most influential nutrients are indigestible carbohydrates, which can be of both terrestrial and marine algae origin [81]. The human genome encodes a limited number of hydrolases capable of hydrolyzing the glycosidic bonds of polysaccharides in dietary fibre (collectively referred to as CAZymes). Consequently, many polysaccharides, such as resistant starch, inulin, lignin, pectin, cellulose and FOS, reach the large intestine undigested. In contrast, the GMB codes tens of thousands of CAZymes. In the 120 Molecules 2020, 25, 1004 presence of bacteria harboring key enzymes involved in carbohydrate metabolism, these complex polysaccharides can thus be degraded and metabolized in vivo [83]. The bacteria able to degrade these complex polysaccharides are called primary degraders and include members of the genera Bacteroides, Bifidobacterium and Ruminococcus, Roseburia, Facealibacterium, Anaerostides or Coprococcus. A relative abundance of these genera in our GM infers that during a food shortage, these bacteria can alternate between energy sources by using sensors and regulatory mechanisms that control gene expression [21, 81]. Hydrolases act on polysaccharides to generate oligosaccharides and monosaccharides. Secondary fermentation of these compounds by the GM produces SCFA, specifically acetic, propionic, butyric, lactic and succinic acids, which initiate a complex metabolic network [81]. The GM of hunter–gatherer, rural and agricultural populations are usually more bacterially diverse than in modernized urban societies [84] and so require a greater functional repertoire to maximize their energy intake from dietary fibres. Conversely, the consumption of a diet composed mainly of products of animal origin causes an enrichment in the GM of genera of bile-tolerant bacteria, such as Alistipes, Bilophila and Bacteroides, and the almost total exhaustion of bacteria that metabolize polysaccharides, such as Roseburia, Eubacterium rectale subgroup and Ruminococcus bromii [81]. Clinical studies investigating prebiotic effects have some disadvantages with respect to ethical constraints, as well as limited sampling possibilities from the colon and limited measurements of in situ SCFA production. These concerns are commonly avoided by applying an in vivo approach [41], that are the most common in the investigation of seaweed effects on human GM, as is described below. Contrariwise, in vitro studies show important limitations because only represent the first step of a long process, and the results observed in vitro can be magnified, diminished, or totally different in a more complex and integrated system [48]. An additional limitation is that, due to the short fermentation time in in vitro studies, they fails to capture the complete picture of cross-feeding interactions between gut microbes, and which may not fully correlate with the long-term effects of seaweed compounds on GM [41]. 4.1. Polysaccharides from Brown Seaweeds Although the prebiotic and immuno-modulation properties of brown algae have been studied both in animal models and in vitro, humans intervention studies are also needed to assess whether there is a direct association between these uses of algae and the human GM, but are currently restricted due to ethical concerns [78]. The most relevant results obtained from examining the impacts of brown seaweeds on the GM can be found in Table 1. In this table it were included results about the prebiotic effect of brown seaweed species from genus Ecklonia [7,85], Sargassum [86–88], Laminaria [82,89–92], Ascophyllum [93–95], Fucus [23,63], Undaria [90], Saccorhiza [96] or Porphyra [97]. As can be seem in Table 1, in most cases, the administration of whole brown seaweed or brown seaweed-extracted polysaccharides resulted in an increase of SCFA production, stimulating of beneficial bacteria grown such as Lactobacillus [7,82,85,86,95], Bifidobacterium [7,82,85,87,92] or Faecalibacterium [7,58,87]. In some cases, the brown seaweed or brown seaweed-extracted polysaccharides also inhibited the growth of potentially pathogen bacteria [73,86]. In some cases, it were reported other beneficial effects not strictly related with action on GM, such as reducing serum inflammatory markers [23], reducing serum levels of lipopolysaccharide-binding protein [44], increasing CAZymes [44], reducing activity of fecal bile salt hydrolase activity [96], or reduced the expression or diabetes-related genes [15]. Laminar storage polysaccharides, typical of brown seaweeds, are low-molecular-weight, linear polysaccharides composed of glucose units with a low degree of branching [79]. Besides affecting mucin composition and SCFA concentration, laminins can affect the adherence, translocation and proliferation of bacteria in the gut [98,99]. At the same time, laminins stimulate the proportion of Bifidobacterium, which generates a prebiotic potential. In other research, laminarin has been shown to promote an immune response [98], and could be useful for inhibiting the production of putrefactive substances from undigested proteins [100]. In vitro batch fermentation of laminarin for 24 h promoted an increase in Bifidobacterium and Bacteroides, and propionate and butyrate production [42]. Contradicting results by other researchers indicated that laminarin was not selectively fermented by Lactobacillus and 121 Molecules 2020, 25, 1004 Bifidobacterium, but could modify the composition, secretion and metabolism of the jejunal, ileal, caecal and colonic mucosa to protect against bacterial translocation [32]. In addition, laminarin increased the presence of Clostridium spp. and Parabacteroides distasonis in rats [101]. An in vitro study conducted with the species Sargassum thunbergii revealed a dramatic increase in the population of beneficial bacteria (from 17% to 28%), while a group of harmful Firmicutes decreased from 75% to 64% after 48 h of fermentation [87]. No noticeable changes were found in Proteobacteria or Actinobacteria. At the genus level, an increase in Lactobacillus, Bifidobacterium, Roseburia, Parasutterella and Fusicatenibacter appeared after incubation for 24 h, followed by an increase in Faecalibacterium and Coprococcus at 48 h of incubation [87]. Bifidobacterium, Coprococcus and Parasutterella have been negatively correlated with non-alcoholic steatohepatitis, hepatocellular carcinoma and diabetes [102], while Ruminococcus, Roseburia and Faecalibacterium are producers of butyric acid and are facilitate the degradation of polysaccharides and fibres [103]. Fusicatenibacter was positively associated with increased serum leptin in obese rats [104], which reduces their appetite. All these findings highlight the prebiotic potential of S. thunbergii by its modulation of the composition and abundance of beneficial GM. An in vitro study using S. wightii in MRS broth evaluated their antioxidant activity and prebiotic score comparing L. plantarum and Salmonella Typhimurium relative growths. The study showed that the prebiotic activity score was positive, promoting selectively the growth of L. plantarum with respect to the pathogen S. Typhimurium. Specifically, a prebiotic effect by 1.42-fold more growth stimulation of L. plantarum than S. Typhymurium [86]. In other work Chen et al. [58] showed an increase in fucoidan from A. nodosum in an in vitro assay simulating the human digestive tract was due to an increase in Bacteroidetes, Firmicutes and SCFA. At the genus level, the genera Bacteroides, Phascolarctobacterium, Oscillospira and Faecalibacterium increased, while the levels of Fusobacterium, Megamonas, Parabacteroides, Clostridium and Dorea decreased relative to the samples to which the algae A. nodosum had not been added [46]. demonstrated the in vitro prebiotic activity of a mixture of fucoidans and alginates obtained from A. nodosum, leading to an increase in the growth rate of L. delbrueckii and L. casei to levels similar to those observed after administration of inulin, a standard commercial prebiotic [46]. Other authors [93] conducted a study in rats, which were administered polysaccharides extracted from A. nodosum, and they were seen an increase in both acetate, propionate and butyrate SCFAs. According to Zaporozhets et al. [76], fucoidans obtained from F. evanescens stimulate the colonic growth of beneficial Bifidobacterium species, such as B. longum B379M and B. bifidum 791B. Lean et al. [23] administered F. vesiculosus-derived fucoidan extracts to mice and, interestingly, found a reduction in markers associated with inflammatory bowel diseases. When Wister rats were fed with feed enriched with alginates or laminarins, An et al. [101] found a notable decrease in the number of metabolites resulting from putrefaction, such as indole, H2 S and phenol. This result was subsequently confirmed in both in vitro and rat models by Nakata et al. [100], who also found a decrease in ammonium levels with alginate. At the phyla level, alginate increased the levels of Actinobacteria, while laminarins increased the levels of Proteobacteria. At the genus level, Bacteroides was markedly more abundant in the group fed with alginate, and B. capillosus was the most frequent species. In rats fed with laminarin-enriched feed, Parabacteroides, Lachnospiraceae and Parasutterella bacterium were detected in greater abundance than in control rats. Nguyen et al. [44] studied laminarin supplementation in a mice high-fat diet. They could see a decrease in Firmicutes and an increase in the Bacteroidetes phylum, especially the genus Bacteroides. Ramnani et al. [94] performed in vitro fermentation with A. nodosum-derived alginates, which increased Bifidobacterium and SCFAs. An increase in the proportion of Bacteroidetes to Firmicutes was observed as well in fermentations added with sulphated polysaccharides extracted from A. nodosum versus controls. Increased levels of Bacteroidetes and decreased levels of Firmicutes have been associated with a reduced risk of obesity in humans [79]. 122 Crude polysaccharide contained carbohydrates 22.7%, sulphate content of 17.1% and protein content 1.34%. Hydrolysates showed 25.1–26.7% carbohydrates, 25.3–25% sulphate and 1.7–1.4% protein contents Average molecular weight 31.0 and 56.0 kDa 21% sulfate content; 1330 KDa molecular weight; 7.3% Man, 24.1% GlcA, 1.5% Glc, 7.2% Gal, 1.3% Xyl, 58.6% Fuc 48.7% total fibre, 16.1% non-digestible non-starch polysaccharides, 1.3% total starch, 43% total sugar, 3.8% protein and 4.5% total phlotorannin Polysaccharides extracted from 20 g of Ascophyllum nodosum in a single dose, compared to blank and FOS-added samples 0.2 g of polysaccharides extracted from A. nodosum per rat for 4 days, comparing crude polysaccharide with crude polysaccharide hydrolysates, alginate and hydrolyzed alginate 1% w/v low molecular weight polysaccharide derivatives extracted from A. nodosum for 24 h. Inulin was used as positive control and cellulose as negative control 100 mg/kg/day of fucoidans obtained from A. nodosum for 6 weeks. Control group received saline solution 1.5% w/v of enzyme-assisted extracted polysaccharides from Ecklonia radiata for 24 h. Inulin and resistant starch were used as positive controls and glucose and cellulose were used as negative controls In vitro fermentation system using fresh fecal samples from four healthy donors In vivo trials using 3 Wistar male rats per sample, comparing effect of A. nodosum crude polysaccharide with hydrolyzed A. nodosum polysaccharides. SCFA were generated by fermentation with Lactobacillus plantarum BCC 5493 and Enterococcus faecalis BCC 39,179 In vitro fermentation system using fresh fecal samples from three healthy donors In vivo trial using 18 male C57BL/6 mice. 6 mice received fucoidans extracted fro A. nodosum, and 6 acted as blank group In vitro fermentation system using fresh fecal samples from 3 healthy donors 123 Increase of total bacteria, Bifidobacterium, Lactobacillus Fucoidans administration resulted in a much more diverse cecal microbiota, increase on Lactobacillus and Talassospira, whereas No significant changes in GM Not provided Increase in SCFA, acetate and propionate in A. nodosum added polysaccharides with respect to blank samples and FOS-added samples Increase in Bacteroidetes and Firmicutes. At genus level, increase of Bacteroides, Phascolarctobacterium, Oscillospira, Faecalibacterium, while decreased Fusobacterium, Megamonas, Parabacteroides, Clostridium, Dorea Total carbohydrate 42.3%; uronic acid 11%; protein 1.4% and sulfate content 23.9%; Monosaccharides content were composed of Man, GlcA, Glc, Gal, Xyl, and Fuc at a molar ratio of 16.65, 20.34, 1.60, 9.69, 3.44, and 48.29 Increase in total SCFA, acetic and propionic acids Fucoidans decreased the serum levels of lipopolysaccharide-binding protein Increase in total SCFA, acetic and propionic acids Increase in both acetic, propionic and butyric acids, in this order. SCFA were higher in the case of polysaccharides with lower molecular weight Significant Changes in Related Metabolites Significant Changes in Gut Microbiota Polysaccharides Characterization Seaweed, Dosage and Time of Exposure Type of Study Table 1. Prebiotic effect of different species of brown seaweed. [85] [95] [94] [93] [58] Reference Molecules 2020, 25, 1004 124 Fucoidans extracted from Fucus vesiculosus intraperitoneally (10 mg/kg/day) or orally (10 mg/kg/day for high purity fucoidan or 400 mg/kg/day for focus-polyphenol) for 7 days 0.8 g of fucoidans obtained from Laminaria japonica for 48 h. Blank samples contained no polysaccharide In vitro fermentation system using fresh fecal samples from three healthy donors In vitro fermentation system using fresh fecal samples from three3 healthy donors In vivo trail using 10 C57BL6 mice per group, with previously induced colitis by supplementing 3% w/v of dextran sulphate sodium in the drinking water for 8 days Crude extract fraction: 14.4%, fibre, 5.6% non-digestible non-starch polysaccharides, 20.6% sugar, 0.2% ManA, 0.5% Man, 17.2% Glc, 0.5% Gal, 0.3% Xyl, 1.8% Fuc, 4.6% phlorotannin. Phlorotannin-enriched fraction: 3.4% fibre, 3.4% sugar, 3.4% Glc, 13.4% phlorotannin. Low molecular weight polysaccharide-enriched fraction: 0.5% fibre, 0.4% starch, 22.7% sugar, 22.7% Glc, 2.5% phlorotannin. Polysaccharides extracts obtained by microwave-intensified enzymatic process from 4.5 g of crude E. radiata for 24 h. Four different seaweed fractions were employed (crude extract fraction, phlorotannin-enriched fraction, low molecular weight polysaccharide-enriched fraction and high molecular weight polysaccharide-enriched fraction. Inulin was used as positive control and cellulose as negative control Not provided Fucus-polyphenol: 40.2% neutral carbohydrates; 21.8% sulfates; 26.2% polyphenols; 3.6% uronic acids and 203.1 kDa peak molecular weight.High purity fucoidan: 59.5% neutral carbohydrates; 26.6% sulphates; <0.5% polyphenols; 1.4% uronic acids; 61.8 kDa molecular weight. High molecular weight polysaccharide-enriched fraction: 62.4% fibre, 22.8% non-digestible non-starch polysaccharides, 0.3% starch, 42.1% sugar, 1.9% GulA, 7.2% ManA, 2.1% Man, 1.1% GlcA, 17.1% Glc, 1.7% Gal, 1.5% Xyl, 9.4% Fuc, 1.7% phlorotannin. Polysaccharides Characterization Seaweed, Dosage and Time of Exposure Type of Study Table 1. Cont. Decrease in Enterobacter spp. while increase in beneficial bacteria as Lactobacillus and Bifidobacterium Not provided Increase of Bifidobacterium, Lactobacillus, Clostridium coccoides in all tested fractions with respect to negative controls. Low molecular weight polysaccharide-enriched fraction showed the better fermentative results, obtaining better counts that positive controls for Lactobacillus, Faecalibacterium prausnitzii, C. coccoides and Firmicutes Significant Changes in Gut Microbiota Decrease in pH and increase in lactic acid and SCFA, including acetic and butyic acids Both oral fucoidan reduced cytokines associated with inflammatory bowel disease such as interleukin-1α, interleukin-1β, interleukin-10, macrophrage inflammatory protein-1α, macrophrage inflammatory protein-1β, granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor Total SCFA were higher in crude fraction than all other fractions after 24 h fermentation. All fractions except phlorotannin-enriched fraction significantly increased SCFA production with respect to negative controls Significant Changes in Related Metabolites [82] [23] [7] Reference Molecules 2020, 25, 1004 Not provided Not provided Not provided Not provided 2% w/w of laminarins for 2 weeks. Black samples received control diet 1 g laminarins from Laminaria digitata for 24 h. Glucose was used as negative control and FOS as positive control Supplementation with 10% of dried L. japonica for 4 weeks. Control rats were fed with basal diet Mice received normal diet, high-fat diet or high-fat diet added with laminarins at 1% w/w in a high-fat diet ad libitum for 4 weeks. After finishing, highly-fat diet was provided for an additional 2 weeks Polysaccharides isolated from Laminaria digitata crude or depolymerized (1% w/v for 48 h). Cellulose was used as negative control and FOS as positive control In vivo trial using six male Wistar rats per group In vitro fermentation system using fresh fecal samples from three healthy donors and an in vivo trial using 20 Wistar rats In vivo trial using 28 female Sprague-Dawley rats In vivo trial using six female BALB/C mice per group In vitro fermentation system using fresh fecal samples from three healthy donors Not provided Fucoidans from L. japonica 18.4% sulfate content; 310 KDa molecular weight; 11.2% Man, 7.3% GlcA, 5.2% Glc, 19.3% Gal, 2.9% Xyl, 54.1% Fuc 100 mg/kg/day of fucoidans obtained L. japonica for 6 weeks. Control group received saline solution In vivo trial using 18 male C57BL/6 mice. Six mice received fucoidans extracted Laminaria japonica, and six acted as blank group Polysaccharides Characterization Seaweed, Dosage and Time of Exposure Type of Study Table 1. Cont. 125 Increase in total SCFA, and butyric acid. Lower production of acetic acid propionic acids Reduction in Firmicutes to Bacteroidetes ratio and decrease of pathogenic bacteria such as Clostridium, Escherichia and Enterobacter Increase Parabacteroides, Fibrobacter and Lachnospiracease and decrease in Streptococcus, Ruminococcus and Peptostreptococcaceae in laminarin-added samples Increase in SCFA with respect to cellulose-added samples, but similar SCFA content or even lower with respect to FOS-added samples Mice fed with laminarin supplementation showed significantly higher CAZyme families in feces [89] Increase in total SCFA in laminarin-added culture medium than in glucose-added. Laminarins supplementation increased the colon luminal content of mucin, while decreased luminal mucin in jejunum, ileum and caecum in rats No significant differences were obtained in the in vitro trial for GM composition. Decrease in Firmicutes and increase in Bacteroidetes phylum, especially the genus Bacteroides in laminarin-added fed mice with respect to controls [101] Increase organic acids, specially propionate, whereas decreased cecal putrefactive compounds (indole, phenol and H2 S) Increase of Bacteroides capillosus, Clostridium ramosum y Parabacteroides distasonis [91] [44] [90] [95] Reference Decreased in the serum levels of lipopolysaccharide-binding protein Significant Changes in Related Metabolites Increase in the abundance of Ruminococcaceae Significant Changes in Gut Microbiota Molecules 2020, 25, 1004 Reduced activity of fecal bile salt hydrolase activity and secondary bile acids S. confussum administration significantly reduced the gene expression of JNK1 and JNK2 in hepatic cells and increased expression of IRS1 and PI3K Not provided Increased gut bacterial diversity in treated hamsters. Significant increase in Barnesiella, Tannerella, Eubacterium and Clostridium XIVa, with significant decrease in Allobaculum, Bacteroides, and Clostridium IV in the S. confussumadded group. Not provided Not provided Sulfated oligosaccharide containing galactose, sulfated galactose, sulfated anhydrogalactose and methyl sulfated galactoside Not provided Polysaccharides extracted from Porphyra haitanensis (250 mg/kg) for 2 weeks. Control mice received 0.9% normal saline at a dose of 20 mL/kg/day. Positive controls received the same plus combined Bifidobacterium, Lactobacillus and Streptococcus thermophilus tables, 500 mg/kg. 5% w/w polysaccharides extracted from Saccorhiza polyschides with high-fat diet for 8 months 150 mg/kg body weight of Sargassum confussum solution once daily for 60 days by intragastric administration Extracts from Sargassum multicum (1% w/v for 24 h). FOS was used as positive control and no carbon source was added to the negative control 1% w/v of enzymatic-extracted polysaccharides from Sargassum wightii in MRS broth for 48 h In vivo trial using 24 male C57BL/6J mice In vivo trial using 16 male C57BL/6 mice fed with a high-fat diet In vivo trial using Syrian golden hamsters In vitro fermentation system using fresh fecal samples from three healthy donors 126 In vitro test comparing the growth of L. plantarum NCIM 2083 with respect to Salmonella Typhimurium MTCC 3224 53.5% fiber, 13.2% protein, 2.3% fat, 28.9% ash. Content of Cel, Fru and Gluc (not specific proportions) Prebiotic effect by 1.42-fold more growth stimulation of L. plantarum than Salmonella Typhimurium Increase Bacteroides and Prevotella, and decrease in Clostridium coccoides and Eubacterium rectale Increase of Prevotellaceae Rikenellaceae and Lactobacillus, while decreased Lachnoclostridium or Lachnospiraceae Inhibition of Salmonella Typhimurium, Listeria monocytogenes or Vibrio parahaemolyticus adhesion and invasion Not provided Increase in SCFA and lactic acid production with respect to negative controls Not provided Not provided Increase of SCFA, acetic propionic and butyric acids and decrease of pH in a similar way of the other beta-glucans assayed Glu residues with degree of polymerization between 22 and 25 and 5 kDa as average molecular weight Increase of all Bifidobacteria with respect to initial counts in a similar way of the other beta-glucans assayed Supplementation with 0.5% w/v and 0.1% w/v of sodium alginate and laminarins extracted from Eisenia bicyclis for 18 h. β-Glucan > 95%, protein 3%; monosaccharides: 98% Gluc; 2% Man. 6 kDa as average molecular weight In vitro fermentation system in cellular lines using human-enterocyte-like29-Luc cells Significant Changes in Related Metabolites Beta-glucans from L. digitata (0.5% w/v for 24 h) compared to barley β-glucan, Curdlan from Alcaligenes faecalis, mushroom sclerotia from Pleurothus tuber-regium and inulin Significant Changes in Gut Microbiota In vitro fermentation system with individual bifidobacteria including B. infantis JCM 1222; B. longum JCM 1217 and B. adolescentis JCM 1275 Polysaccharides Characterization Seaweed, Dosage and Time of Exposure Type of Study Table 1. Cont. [86] [88] [15] [96] [97] [73] [92] Reference Molecules 2020, 25, 1004 Increase in total SCFA, and butyric acid. Lower production of acetic acid propionic acids Reduction in Firmicutes to Bacteroidetes ratio and decrease of pathogenic bacteria such as Clostridium, Escherichia and Enterobacter 68.3% carbohydrate; 0.3% protein; 3.5% sulfate: Monosaccharide molar ratio: 3.9% arabinose; 6.2% Gal, 3.2% Glc, 15.6% Xyl, 14.8% Man 15.6% GulA, 40.6% GlcA. Average molecular weight 4.8 kDa Not provided 200 mg polysaccharides extracted from Sargassum thunberguii for 48 h Supplementation with 10% of dried Undaria pinnatifida and for 4 weeks. Control rats were fed with basal diet [90] [87] Reference BCC: Biotec Culture Collection; BCC: British Culture Collection; Cel: Cellobiose; Gal: galactose; GlcA: galacturonic acid; Glc: glucose; GulA: guluronic acid; FOS: fructooligosaccharides; Fru: fructose; Fuc: fucose; JCM: Japan Collection of Microorganism; kDa: kilodaltons; ManA: Mannuronic acid; Man: mannose; MRS: Man, Rogosa and Sharpe; MTCC: Microbial Type Culture Collection and Gene Bank; NCIM: National Centre of Integrative Medicine; SCFA: Short chain fatty acids; Xyl: Xylose. In vivo trial using 28 female Sprague-Dawley rats Decrease of pH and increase in total SCFA and acetic, propionic, butiric and n-valeric acids Decrease of Firmicutes, while increase of Bacteroidetes and beneficial bacteria such as Bifidobacterium, Roseburia, Parasutterella and Fusicatenibacter after 24-h fermentation, and increase of Faecalibacterium and Coprococcus after 48-h fermentation In vitro fermentation system using fresh fecal samples from three healthy donors Significant Changes in Related Metabolites Significant Changes in Gut Microbiota Seaweed, Dosage and Time of Exposure Type of Study Polysaccharides Characterization Table 1. Cont. Molecules 2020, 25, 1004 127 Molecules 2020, 25, 1004 Evidence that probiotic bacteria in the gastrointestinal tract utilize dietary alginate was reviewed by Shang et al. [38]. Among the studies, Kuda et al. [73] found that supplementation with sodium alginate and laminarin of brown algae inhibited the adhesion and invasion of pathogens, such as S. Typhimurium, Listeria monocytogenes or Vibrio parahaemolyticus. Other authors reported an increase in Lactobacillus and Ruminococcus in the intestine of mice fed fucoidans from A. nodosum, besides a reduction in the opportunistic Peptococcus bacteria [95]. In vitro fermentation experiments conducted by Charoensiddhi et al. [85] demonstrated the growth-promoting effect of E. radiata extracts on beneficial bacteria, such as Bifidobacterium, Lactobacillus and Clostridium coccoides, and SCFAs production was stimulated as well. Later, the same authors [7] found increased levels of beneficial bacteria, such as Bifidobacterium, Lactobacillus and C. coccoides associated with the phlorotannin-enriched fermentation of E. radiata. Higher numbers of Lactobacillus, Faecalibacterium prausnitzii, C. coccoides, Firmicutes and E. coli were observed for phlorotannin-supplemented fermentation compared with inulin fermentation [7]. In contrast, the number of Enterococcus in both fermentations decreased approximately ten-fold relative to the initial counts. Other authors tested the effects of supplementation of two brown algae (U. pinnatifida and L. japonica) on the GM and body status of laboratory rats [90]. In both instances, the animals’ body weight was reduced, which was thought to be mediated by the influence of the seaweed on the composition of the intestinal microbial communities associated with obesity, reducing the proportion of Firmicutes with respect to Bacteroidetes, and the populations of pathogenic bacteria, such as Clostridium, Escherichia and Enterobacter [90]. Similarly, L. japonica increased beneficial bacteria and SCFA, and decreased the pH level [82], while β-glucans extracted from L. digitata increased Bifidobacterium and propionic and butyric acids in vitro, in addition to lowering pH [92]. β-Glucans obtained from another Laminaria species (L. digitata) were able in an in vitro test [92] to increase Bifidobacterium and propionic and butyric acids, in addition to lowering pH. A study by Strain et al. [91] in vitro investigated the effect of a polysaccharide-rich raw extract obtained from L. digitata. A significant alteration of the relative abundance of several families, including Lachnospiraceae and genera such as Streptococcus, Ruminococcus and Parabacteroides of human faecal bacterial populations was seen. Concentrations of acetic acid, propionic acid, butyric acid and total SCFA were significantly higher. Finally, Huebbe et al. [96] conducted a study on mice that were administered polysaccharides from S. polyschides with a high-fat diet. A metabolic improvement was seen including normalization of blood glucose, reduction of plasma leptin, reduction of fecal bile salt hydrolase activity and secondary bile acids in these mice. 4.2. Polysaccharides from Red Seaweeds The most relevant results obtained from the investigation of red seaweeds effect on GM can be found in Table 2. In this table, results about the prebiotic effect of red seaweed species from genus Acanthopora [86], Gracilaria [105,106], Kappaphycus [107], Euchema and Grateloupia [10], Chondrus [57], Gelidium [94], or Osmundea [88] were included. As can be seem in Table 2, as was described previously for brown seaweeds, administration of red seaweeds or seaweed-extracted polysaccharides resulted in an increase of SCFA production, stimulating of beneficial bacteria grown such as Lactobacillus [86] or Bifidobacterium [57,94,107], whereas inhibited the growth of potentially pathogen bacteria [57,86]. It was also reported red seaweeds activity on the prevention of naproxen-induced gastrointestinal damage [106]. Agarose stands out among the polysaccharides isolated from red algae that cannot be digested by human intestinal enzymes. When seaweed is consumed, whether as an edible food or food additive, agarose reaches the most distal portions of the gastrointestinal tract, where it is fermented and metabolized by the GM [108,109]. As described by Ramnani et al. [94], low-molecular-weight agarose exerted a prebiotic effect in vitro by promoting the growth of Bifidobacterium and increasing SCFA concentrations in the medium. 128 Molar mass distribution was found to be within 2.6 × 106 and 3.8 × 105 g/mol, while the soluble carbohydrate, protein, and sulfate contents were 85.5%, 2.5%, and 8.4%, respectively Average molecular weight 923.3 kDa, sugar content 0.11% 100 mg of sulphated polysaccharides obtained from Gracilaria rubra for 24 h. Basal nutrient medium was used for control negative group and FOS was used for control positive group In vivo trial using male Wistar rats (six per group) In vitro fermentation system using fresh fecal samples from four healthy donors 62.1% sugar; 21.4% sulfate; Monosaccharides at molar ratio: 0.01 Man, 0.01 GluA, 1% Gal, 0.09% Xyl, 0.01% Fuc, 0.03% Glu Rats were pretreated with 0.5% carboxymethylcelloluse (controls) or 0.5% w/v of of sulphated polysaccharides from Gracilaria birdiae, twice daily for 2 days. After 1 h, naproxen (80 mg/kg) was administered twice a day for 2 days 0.1–0.5% w/v Eucheuma spinosum for 24 h In vitro test in MRS broth for Lactobacillus and Bifidofacterium compared to MHB broth for Staphylococcus aureus and Escherichia coli Not provided 41.9% sugar; 20.6% sulfate; Monosaccharides at molar ratio: 0.01 Man, 0.02 GluA, 1% Gal, 0.1% Xyl, 0.05% Fuc, 0.07% Glu Fed added with 0.5–2.5% (w/w) whole Chondrus crispus for 21 days In vivo trial using male Sprague-Dawley rats (six per group) 45.9% fiber, 10.9% protein, 1.6% fat, 39.4% ash. Only Glu was found as monosaccharide 0.1–0.4% w/v Grateloupia filicina added in culture media for 24 h 1% w/v of enzymatic-extracted polysaccharides from Acanthopora spicifera in MRS broth for 48 h In vitro test comparing the growth of Lactobacillus plantarum NCIM 2083 with respect to Salmonella Typhimurium MTCC 3224 Polysaccharides Characterization In vitro test in MRS broth for Lactobacillus and Bifidofacterium compared to MHB broth for S. aureus and E. coli Seaweed and Dosage Type of Study 129 Prevention of naproxen-induced gastrointestinal damage determined by macro- and microscopic findings Increase in total SCFA and acetic, propionic and isobutyric acids Increase of Bacteroidetes, Bacteroidaceae, Prevotellaceae, Ruminococcaceae and propionic acid, while decrease Fusobacteriaceae and Lachnospiraceae. At genus level, increase of Bacteroides, Prevotella and Phascolarctobacterium Not provided Increase in beneficial bacteria at all concentrations, without significant differences between 0.4% and 0.5%. No inhibition was detected against pathogens No relevant variation was observed in GM populations Not provided [105] [106] [10] [10] [57] Increase in total SCFA and acetic, propionic and butiric acids in rats fed with C. crispus at 0.5% and 2.5%. Higher concentrations of all SCFA were found in the case of rats fed added 2.5% of C. crispus with respect to rats fed added with 0.5% of C. crispus Increase of Bifidobacterium brevis and decrease of pathogens such as Clostridium septicum and Streptococcus pneumoniae Increase in beneficial bacteria with better results at 0.1% concentration. No inhibition was detected against pathogens [86] Reference Not provided Significant Changes in Metabolites Prebiotic effect by 0.84-fold more growth stimulation of L. plantarum than S. Typhimurium Significant Changes in Gut Microbiota Table 2. Prebiotic effect of different species of red seaweed. Molecules 2020, 25, 1004 Average molecular weight 143.8 kDa Average molecular weight 20.1 kDa and 6.5 kDa, respectively Not provided Not provided 1% w/v low molecular weight polysaccharide derivatives extracted from Gracilaria spp. for 24 h. Inulin was used as positive control and cellulose as negative control 1% w/v low molecular weight polysaccharide derivatives extracted from Gelidium sesquipidale for 24 h. Inulin was used as positive control and cellulose as negative control 1% w/v Kappaphycus alvarezii for 24 h Extracts from Osmundea pinnatifida (1% w/v for 24 h). FOS was used as positive control and no carbon source was added to the negative control In vitro fermentation system using fresh fecal samples from three healthy donors In vitro fermentation system using fresh fecal samples from three healthy donors In vitro fermentation system In vitro fermentation system using fresh fecal samples from threehealthy donors Increase in SCFA, acetic and propionic acids Increase in SCFA Increase in Bifidobacterium, decrease in Clostridium coccoides and Eubacterium rectale Increase in Bifidobaterium counts Both G. sesquipidale extracts (20.1 kDa and 6.5 kDa molecular weight) significantly increased total SCFA, acetic and propionic acids Increase in total SCFA, acetic and propionic acids Significant Changes in Metabolites Only G. sesquipidale of 6.5 kDa significantly increased Bifidobaterium counts No significant changes in GM Significant Changes in Gut Microbiota [88] [107] [94] [94] Reference Gal: galactose; Glc: glucose; GulA: guluronic acid; FOS: fructooligosaccharides; Fuc: fucose; GM. Gut microbiota; kDa: kilodaltons; Man: mannose; MRS: Man, Rogosa and Sharpe; MTCC: Microbial Type Culture Collection and Gene Bank; NCIM: National Centre of Integrative Medicine; SCFA: Short chain fatty acids; Xyl: Xylose, Polysaccharides Characterization Seaweed and Dosage Type of Study Table 2. Cont. Molecules 2020, 25, 1004 130 Molecules 2020, 25, 1004 Bajury et al. [107] conducted an in vitro colon model in which they evaluated the prebiotic capacity of K. alvarezii. This study showed an increase in SCFA (particularly acetate and propionate) and Bifidobacterium. In the other hand, decrease in C. coccoides and E. rectale. These results suggested that K. alvarezii might have the potential as a prebiotic ingredient. A study published by Zhang et al. [110] focused on the beneficial effect of low-melting-point agarose (in the form of neoagaro-oligosaccharides) on the GM during the relief of intense exercise-induced fatigue in mice. Results showed the abundance of Bacteroidetes and Proteobacteria increased and decreased, respectively, during the attenuation of fatigue and its associated gastrointestinal problems. Ladirat et al. [111] found that mice fed 2.5% (w/v) neoagaro-oligosaccharides for seven consecutive days achieved a much more pronounced increase in the population of Lactobacillus spp. and Bifidobacterium spp. in their GM relative to those fed 5% (w/v) FOS for 14 consecutive days. Likewise, it was demonstrated that agaro-oligosaccharides could be used as a prebiotic to encourage the growth of beneficial strains of bacteria, such as B. adolescentis ATCC 15703 and B. infantis ATCC 15697. Low-molecular-weight agar has demonstrated a bifidogenic effect, along with an increase in SCFA acetate and propionate concentrations, after 24 h of in vitro fermentation with human faeces inoculant [94]. Another type of polysaccharide with prebiotic function found in red algae are the group of carrageenans, which are derived from D-galactose, and approved as food additives [79]. In rats fed 2.5% C. crispus, of which carrageenan is a major polysaccharide, B. brevis, as well as SCFA, increased considerably, while pathogens Clostridium septicum and Streptococcus pneumonia noticeably decreased compared with the basal diet [57]. Elevation of plasma immunoglobulin levels was also found in rats fed with C. crispus, resulting in improved host immunity. Consistent with the prebiotic activity of carrageenan, carrageenans isolated from red algae G. filicina and E- spinosum promoted the growth of Bifidobacterium [10]. Research led by Di et al. [105] found that the polysaccharides of Gracilaria rubra increased the relative abundances of Bacteroides, Prevotella and Phascolarctobacterium in vitro compared with the control group. Bacteroides spp. assists the host with degrading polysaccharides and contains codifying genes of glucosidase enzymes [39]. Prevotella is another beneficial genus with the potential to participate in the metabolism and utilization of plant polysaccharides. The genus Phascolarctobacterium is associated with the production of SCFA [110]. Many other bacterial genera, such as Legionella, Sutterella, Blautia, Holdemania, Shewanella and Agarivorans, were decreased as a consequence of intake of C. crispus supplements in rats [57]. Decreases in the presence of Streptococcus were also observed. In conclusion, carrageenans from C. crispus could act as a fermentable substrate for probiotic bacteria present in the gastrointestinal tract, thereby promoting the growth of probiotic groups, while inhibiting certain groups of pathogenic bacteria [57]. Another study in chickens described an overall impact of administering whole red algae (Sarcodiotheca gaudichaudii and C. crispus) on the intestinal mucosa, increasing the height and surface of the villi in these animals [112]. Moreover, the abundance of beneficial bacteria, such as B. longum and Streptococcus salivarius increased, while some harmful bacteria species, such as C. perfringens, decreased [112] Rodrigues et al. [88] used extracts from the red algae O. pinnatifida and S. muticum in an in vitro fermentation system, which increased the production of acetate and propionate, and the population of Bifidobacterium. In work published by Silva et al. [106], in which extracts of sulphated polysaccharides from G. birdiae were administered to laboratory rats, gastrointestinal damage induced by naproxen was prevented, although it did not produce notable variations in the GM of these rats. An in vitro study using A. spicifera in MRS broth evaluated their antioxidant activity and prebiotic score with L. plantarum and S. Typhimurium. The study showed that the prebiotic activity score was positive, promoting the growth of L. plantarum and suppressing the growth of the pathogen S. Typhimurium. Specifically, a prebiotic effect by 0.84-fold more growth stimulation of L. plantarum than S. Typhymurium [86]. 131 Molecules 2020, 25, 1004 4.3. Polysaccharides from Green Seaweeds Unlike brown and red algae, the current evidence for the fermentation capacity of green algae and their polysaccharides is scarce, partly because their fermentation requires a specific activity of α-l-rhamnosidase in the gastrointestinal tract, which is infrequent [113]. The most relevant results obtained from the investigation of green seaweeds effect on GM can be found in Table 3. In this table, results about the prebiotic effect of green seaweed species from genus Enteromorpha [38,82,86,113–115] and Ulva [115] were included. Administration of green seaweeds or seaweed-extracted polysaccharides also resulted in an increase of SCFA production, stimulating of beneficial bacteria grown such as Lactobacillus [38,86,115], Bifidobacterium [38], or Akkermansia [38] whereas inhibited the growth of potentially pathogen bacteria [81,114]. Other beneficial actions were reported such as decrease lipopolysaccharide-binding protein in female mice [38], diminished histopathological lesions of inflammatory infiltrations in distal colon [114], or modulating diabetes-related genes expression in diabetic mice [22]. Ulvans are one of the most frequent polysaccharides in green algae. This polysaccharide is a water-soluble sulphated heteropolysaccharide [79]. Ulvans contains sulphate and uronic acids, and so produce undigestible ionic colloids, has ion-exchange capacity and can bind to bile acids, consequently increasing the excretion of bile acids with cholesterol-lowering or antihyperlipidemic effects [2,79]. Antioxidant and immunomodulatory properties are other beneficial actions elicited by ulvans [116,117]. Ulvans has also been studied for its possible prebiotic potential. Kong et al. [82] performed an in vitro assay using Enteromorpha with a high content of ulvans, but there were no noticeable variations in the populations of Enterococcus, Lactobacillus and Bifidobacterium compared with controls. In contrast, in a recent in vitro faecal fermentation analysis, ulvans stimulated the growth of Bifidobacterium and Lactobacillus populations and promoted the production of SCFA, such as lactic and acetic acids [42]. In a previous study by Ren et al. [114], both whole Enteromorpha and polysaccharides extracted from Enteromorpha improved inflammation associated with loperamide-induced constipation in mice. In those mice, the GM showed an increase in Firmicutes and Actinobacteria compared with the control mice, whereas the relative amounts of Bacteroidetes and Proteobacteria decreased. In work by Shang et al. [38], an extract of E. clathrata was administered to mice, resulting in marked decreased concentrations of genera, such as Enterobacter, Staphylococcus and Streptococcus. Surprisingly, such supplementation also dramatically reduced the population of A. muciniphila in the intestine. These observations indicate a possible unfavorable effect of these polysaccharides on the GM. Contrarily, these polysaccharides were reported to increase the abundance of A. muciniphila, Bacteroides, Alloprevotella, Ruminococcaceae and Blautia in the intestinal tract of mice, and decrease the abundance of Peptococcus, Rikenellaceae and Alistipes [96,109]. An in vitro faecal fermentation of xylans derived from Palmaria palmata reported that xylose was fermented after 6 h, and the SCFA content increased simultaneously [32]. This study did not determine the bacterial composition. Nonetheless, Xylans and xylo-oligosaccharides extracted from terrestrial plants, such as wheat husks and corn, are considered potential prebiotics due to evidence of bifidogenesis, improved plasma lipid profile and positive modulation of immune function markers in healthy adults [118]. 132 133 Not provided Not provided Not provided Not provided Polysaccharides extracted from Ulva prolifera (250 mg/kg) for 2 weeks. Control mice received 0.9% normal saline at a dose of 20 mL/kg/day. Positive controls received the same plus combined Bifidobacterium, Lactobacillus and Streptococcus thermophilus tables, 500 mg/kg. 0.8 g of fucoidans obtained from Enteromorpha prolifera for 48 h. Blank samples contained no polysaccharide Loperamide at a dosage of 9.6 mg/kg/twice a day via oral gavage for 2 weeks was provided to mice to induce slow-transit constipation in mice. Afterwards, E. prolifera and polysaccharides extracted from E. prolifera added in fed at a 1:5 w/v ratio was administered for 7 days Treated mice were fed with high sucrose/high fat diet for 5 weeks. Next type-2 diabetes was induced by intraperitoneal administration of streptozotocin at 45 mg/kg for 3 days. Diabetic mice were administered with 150 mg/kg E. prolifera extracts or its flavonoid-rich fractions less than 3 kDa, respectively, for 4 weeks In vivo trial using 24 male C57BL/6J mice In vitro fermentation system using fresh fecal samples from three healthy donors In vivo trial using 24 Kunming female mice In vivo trial using 24 Kunming male mice Flavonoids from E. prolifera reduced blood glucose in mice, reduced mRNA expressions of JNK1/2 gene and increased the expression of PI3K, IRS1 and AKT genes in diabetic mice Both E. prolifera and E. prolifera extracts diminished histopathological lesions of inflammatory infiltrations in distal colon. Both E. prolifera and E. prolifera extracts reduced serum levels of nitric oxide (inhibitory neurotransmitter) and showed laxative effects E. prolifera increased bacterial diversity, Bacteroidales, Firmicutes, Actinobacteria, and decreased Bacteroidetes and Proteobacteria. Extracts from E. prolifera increased Prevotellaceae, Firmicutes, Actinobacteria, and decreased Bacteroidetes and Proteobacteria E. prolifera extracts increased the proportion of Alistipes, Lachnospiraceae and Odoribacter, while both extracts reduced the proportion of Ruminiclostridium and Akkermansia in GM of diabetic mice Not significant changes Not provided Not provided Decrease in Enterobacter spp. in E. prolifera fucoidans-added samples Polysaccharides supplementation decreased Tenericutes, and Cyanobacteria. At genus level, decreased Lachnospiraceae, Lactobacillus, Mollicutes and Mucispirillum, while increased Prevotellaceae and Rickenellaceae Prebiotic effect by 1.44-fold more growth stimulation of Lactobacillus plantarum than Salmonella Typhimurium [22] [114] [82] [115] [86] [38] Reference Gal: galactose; GlcA: glucuronic acid; Glc: glucose; GM: gut microbiota; kDa: kilodaltons; Man: mannose; MRS: man Rogosa and Sharpe; MTCC: Microbial Type Culture Collection and Gene Bank; NCIM: National Centre of Integrative Medicine; Rha: rhamnose; SCFA: Short chain fatty acids; Xyl: Xylose. 60.6% fiber, 16.9% protein, 1.2% fat, 25.4% ash. Monosaccharide content included cellobiose, fructose, glucose and maltose 1% w/v of enzymatic-extracted polysaccharides from Enteromorpha compressa in MRS broth for 48 h In vitro test comparing the growth of Lactobacillus plantarum NCIM 2083 with respect to Salmonella Typhimurium MTCC 3224 E. clathrata supplementation decreased lipopolysaccharide-binding protein in female mice but not in male mice Molecular weight 11.67 kDa; 14.7% sulfate content. Monosaccharide composition: 1.0% Man, 49.7% Rha, 10.8% GlcA; 29.9% Glc; 1.3% Gal; 7.2% Xyl Enteromorpha clathrata, 100 mg/kg/day or 50 mg mg/kg/day for 4 weeks Increase of Akkermansia muciniphila, Bifidobacterium spp., and Lactobacillus spp. E. clathrata supplementation induced much less alteration in the composition of female GM than in male GM In vivo trial using 36 C57BL/6J mice, 18 males and 18 females in different trials Significant Changes in Metabolites Significant Changes in Gut Microbiota Seaweed, Dosage and Time of Exposure Type of Study Polysaccharides Characterization Table 3. Prebiotic effect of different species of green seaweed. Molecules 2020, 25, 1004 Molecules 2020, 25, 1004 E. clathrata is an edible green seaweed possessing polysaccharides with numerous bioactivities, including anticoagulant, immunomodulatory, antioxidant, anticancer and anti-obesity effects [38]. It was reported that the polysaccharides of E. clathrata exerted diverse prebiotic effects on A. muciniphila, Bifidobacterium and Lactobacillus in male and female mice [38]. The results were most evident in the male mice because of a sex-specific effect on the GM, as sex hormones play a key role in determining the composition of intestinal microorganisms [109]. In other work from the same authors, male mice were supplemented with polysaccharides of E. clathrata in the diet, which increased the abundance of Bacteroides, Prevotella, Alloprevotella, Eubacterium and Peptococcus, and decreased the proportion of the cancer-related Helicobacter [109]. In the female counterparts, the abundance of Odoribacter, Clostridium IV, Oscillibacter and Alistipes spp. increased, and the proportions of beta-proteobacteria decreased [38]. An in vitro study using E. compressa in MRS broth evaluated their antioxidant activity and prebiotic score with L. plantarum and S. Typhimurium. The study showed that the prebiotic activity score was positive, promoting the growth of L. plantarum and suppressing the growth of the pathogen S. Typhimurium. This seaweed exhibited the highest score of prebiotic activity (1.44-fold), stimulating the growth of L. plantarum than S. typhimurium [86]. The natural products of marine macroalgae have shown notable antidiabetic potential by interfering with carbohydrate metabolism. For example, E. prolifera contains many bioactive compounds, such as sulphated polysaccharides, which could improve glucose metabolism, in addition to displaying anti-inflammatory, antiviral and anticoagulant functions [22]. 5. Conclusions Although substantial evidence of the prebiotic effect of seaweed and seaweed extracts has been published in recent years, these studies have been performed using in vitro digestion systems simulating the human colon, or in animal models. Animals, such as mice or rats, differ widely from humans in the GM composition, immune function, diets, metabolism and other key aspects, so extrapolating the results obtained from animal models to humans may not be valid. In vitro systems replicate more similarly the human intestinal microbiota, but are less-dynamic systems than the real human colonic environment. Additionally, other factors should be considered, such as the possible effect of other secondary compounds contained in seaweeds on the GM composition, or the potential to transfer genes from marine bacteria to human GM bacteria coding for enzymes that could degrade seaweed polysaccharides. Thus, not all people will respond equally after seaweed ingestion. The decrease in terrestrial agriculture and disposable water is likely to increase the consumption of algae by humans in the near future. 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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/). 140 molecules Review Avocado Oil: Characteristics, Properties, and Applications Marcos Flores 1, *, Carolina Saravia 2, *, Claudia E. Vergara 1 , Felipe Avila 3 , Hugo Valdés 4 and Jaime Ortiz-Viedma 5, * 1 2 3 4 5 * Departamento de Ciencias Básicas, Facultad de Ciencias, Universidad Santo Tomás, Avenida Carlos Schorr 255, Talca 3473620, Chile; claudiavergarava@santotomas.cl Escuela de Nutrición y Dietética, Facultad de Salud, Universidad Santo Tomás, Avenida Carlos Schorr 255, Talca 3473620, Chile Escuela de Nutrición y Dietética, Facultad de Ciencias de la Salud, Universidad de Talca, Talca 3460000, Chile; favilac@utalca.cl Centro de Innovación en Ingeniería Aplicada, Departamento de Computación e Industrias, Universidad Catolica del Maule, Avenida San Miguel 3605, Talca 3480112, Chile; hvaldes@ucm.cl Department of Food Science and Chemical Technology, Faculty of Chemical and Pharmaceutical Sciences, University of Chile, Santos Dumont 964, Santiago 8380494, Chile Correspondence: marcosflores@santotomas.cl (M.F.); csaraviam@santotomas.cl (C.S.); jaortiz@uchile.cl (J.O.-V.); Tel.: +56-7123-424-18 (M.F.) Academic Editor: Jose M. Miranda Received: 9 May 2019; Accepted: 8 June 2019; Published: 10 June 2019 Abstract: Avocado oil has generated growing interest among consumers due to its nutritional and technological characteristics, which is evidenced by an increase in the number of scientific articles that have been published on it. The purpose of the present research was to discuss the extraction methods, chemical composition, and various applications of avocado oil in the food and medicine industries. Our research was carried out through a systematic search in scientific databases. Even though there are no international regulations concerning the quality of avocado oil, some authors refer to the parameters used for olive oil, as stated by the Codex Alimentarius or the International Olive Oil Council. They indicate that the quality of avocado oil will depend on the quality and maturity of the fruit and the extraction technique in relation to temperature, solvents, and conservation. While the avocado fruit has been widely studied, there is a lack of knowledge about avocado oil and the potential health effects of consuming it. On the basis of the available data, avocado oil has established itself as an oil that has a very good nutritional value at low and high temperatures, with multiple technological applications that can be exploited for the benefit of its producers. Keywords: avocado oil; oil extraction; antioxidants compounds; fatty acid profile 1. Introduction Avocado (Persea americana Mill.) is a fruit native to Central America, grown in warm temperate and subtropical climates throughout the world. The pulp of this fruit contains about 60% oil, 7% skin, and approximately 2% seed [1,2]. The main producers of avocado oil in the world are New Zealand, Mexico, the United States, South Africa, and Chile [3]. Avocado oil has sparked a growing interest in human nutrition, food industry, and cosmetics. The lipid content, mainly of monounsaturated fatty acids, is associated with cardiovascular system benefits and anti-inflammatory effects [4,5]. There are no internationally defined parameters for avocado oil. The values that are commonly used are those recommended for olive oil. The quality standard for olive oil is available in the Codex Alimentarius and the International Olive Oil Council (IOC) [6]. Molecules 2019, 24, 2172; doi:10.3390/molecules24112172 141 www.mdpi.com/journal/molecules Molecules 2019, 24, 2172 Woolf et al. [7] proposed a classification for avocado oil based on its extraction method and fruit quality. Avocado oil of a higher quality, “extra virgin”, corresponds to that produced from high-quality fruit, extracted only with mechanical methods, using a temperature below 50 ◦ C and without the use of chemical solvents. “Virgin” avocado oil is produced with fruit of a lower quality (with small areas of rot and physical alterations), extracted by mechanical methods, using a temperature below 50 ◦ C and without the use of chemical solvents. “Pure” avocado oil is a type of oil for the production of which the quality of the fruit is not important; it is a bleached and deodorized oil, infused with the natural flavor of herbs or fruits. Finally, “mixed” avocado oil is combined with olive, macadamia, and other oils. Therefore, it presents sensory and chemical characteristics that are variable. The Mexican norm [8] states that the “crude oil of avocado” is a slightly amber-colored fatty liquid, obtained by physical extraction of the pulp and the seed of the fruit (Persea americana). “Pure” edible avocado oil is a product with at least 98.5% refined avocado oil. In this work, a systematic review of the literature was carried out to collect, select, evaluate, and summarize all available evidence regarding the processes and properties of avocado oil. The research question was: What are the most published topics on avocado oil? The answer to this question allowed us to include topics, such as: extraction methods (i.e., cold pressed method, ultrasound-assisted aqueous extraction method, supercritical CO2 method, CO2 subcritical method, enzymatic extraction, and solvent extraction), procedures of conservation, contamination/adulteration, technological applications, composition (characteristics according to the variety and origin of the fruit, physicochemical characterization, avocado seed oil, and comparison with other oils), and biological effects (human health effects and experimental studies in animals). Figure 1 shows the exponential increase of scientific interest in avocado oil. On the topic, “avocado oil” (from 1980 to date), 180 and 224 articles have been published in the Web of Science (WoS) and Scopus, respectively. Figure 1. Graphic of articles (topic: “avocado oil”) from 1980 to 2019 in the Web of Science (WoS) and Scopus. The purpose of this research is to produce a complete profile on avocado oil, including extraction methods, physicochemical characteristics, nutritional properties, as well as various applications in the food and medicine industries. Avocado oil has proven to be a vegetable oil with a composition of major and minor components that are highly appreciated by the population, either at low or high temperatures, with multiple technological applications. 2. Extraction Methods for Avocado Oil The information presented in this section focuses on the process efficiency, improvement of production performance, and product quality as well as applications in the food industry. 142 Molecules 2019, 24, 2172 Considering the high humidity percentage of avocado (around 70 to 80%), the influence of the pulp drying method prior to oil extraction has been studied [9]. The quality parameters (peroxide value, iodine value, amount of oleic acid, refractive index, electrical conductivity, content of carotenoids, chlorophyll, phenolic compounds, and antioxidant activity) have shown better results when the pulp is dried at 60 ◦ C under vacuum, and the extraction is performed by the Soxhlet method. Meanwhile, the bioactive compounds were best preserved when the avocado pulp was dried at 60 ◦ C with air ventilation and mechanical pressing [10]. On the other hand, avocado pulp oil, pressed and dried in a microwave, presented a better quality—determined by the acidity index, peroxide index, and oxidative stability—when compared with oil obtained by extraction with ethanol. The composition of fatty acids did not differ significantly when analyzing oil obtained by drying under microwaves or in a drying oven with forced air circulation [11]. According to Chimsook and Assawarachan [12], the studied drying method of the avocado pulp, prior to the extraction of the oil, does not significantly influence the composition of fatty acids. However, changes were determined in the antioxidant activity and vitamin E content of cold-pressed avocado oil (from Thailand). Higher antioxidant activities and a higher vitamin E content were observed in oil, the pulp of which was dried with hot air, when compared to oils obtained by an air-dried and vacuum process. This study is consistent with the fact that oils from the fortune avocado variety, obtained by the pulp drying lyophilization method, resulted in lower concentrations of α-tocopherol, squalene and β-sitosterol, as well as higher relative concentrations of campesterol and cycloartenol acetate, compared to oils obtained through hot air-drying processes [13]. 2.1. Cold Pressed Method According to the CODEX STAN 19-1981 [14], the method of extraction of edible vegetable oils is characterized by mechanical procedures, for example, extrusion or pressing, without the application of heat. In addition, the oil can only be purified by washing, sedimentation, filtration, and centrifugation. In the cold pressing method, oil recovery is only obtained from the parenchyma cells of the pulp; its rupture begins in the first stages of grinding and it can be seen that the idioblastic cells (oil carriers) remain intact during the process of extraction. The extraction yield increases when the pulp is beaten at 45.5 ◦ C for 2 h [15]. In this method, a lower extraction yield is obtained, although with higher concentrations of α-tocopherol and squalene, as well as lower contents of campesterol and cycloartenol acetate, compared to the Soxhlet method [13]. Drying by lyophilization and subsequent extraction by the Soxhlet method allows for a better extraction performance. However, when drying by lyophilization and extracting by cold pressing, oils with a greater concentration of antioxidants, and other bioactive compounds were obtained [13]. 2.2. Ultrasound-Assisted Aqueous Extraction Method (UAAE) This method uses the cavitation forces produced by acoustic waves to break down the cell walls of the oil-containing cells. This process allows for the generation of an emulsion, which facilitates oil extraction. This method can be carried out using an ultrasonic bath or an ultrasonic horn transducer [16]. The high frequency ultrasound conditioning (0.4, 0.6, and 2 MHz, 5 min, 90 kJ/kg) of the avocado puree can improve the oil separation and potentially reduce the beating time in industrial processes, without affecting the quality of the oil. If this treatment is applied after shaking, the extractability of the oil increases by between 2% and 5%. The oils obtained from sonicated purees showed free fatty acids (FFA) and peroxide values below the levels of industrial specification (peroxide less than 20 meqO2 /kg) and an increase in total phenolic compounds after a 2 MHz treatment [17]. The ultrasound-assisted aqueous extraction (UAAE) of low virgin avocado oil in FFA, considered as virgin avocado oil, is that obtained by mechanical or natural means at low temperatures (<50 ◦ C) and without chemical refining [7]. The optimal UAAE parameters to produce the highest extraction of virgin avocado oil was 6 mL/g water-dried pulp powder, 30 min of sonication time at 35 ◦ C. The sonicated virgin avocado oil was lighter and had a higher level of unsaturated fatty acids, compared to the avocado oil extracted by the Soxhlet method [16]. 143 Molecules 2019, 24, 2172 2.3. Supercritical CO2 Method This method of extraction is based on the use of supercritical fluids, substances that are, in certain circumstances, in a state in which they have intermediate properties between liquid and gas. Supercritical CO2 (scCO2 ) is a totally innocuous gas, which becomes a powerful solvent under conditions of pressure and at a temperature above its critical point [18]. Extraction with scCO2 presents a higher performance at a pressure of 400 bar. The use of ethanol as a co-solvent favors the extraction of residual oil, benefiting the extraction of a fraction enriched in tocopherols [19]. Some authors [20,21] proposed the combined extraction of avocado oil and active compounds present in peppers (capsanthin) and tomatoes (lycopene) using scCO2 in order to enrich the avocado oil. For this, a fixed bed extractor was used, where the lipids and the desired active ingredient were subjected to the extraction process, simultaneously with scCO2 . First, both the scCO2 and the oil extracted from the avocado passed through the avocado bed and then through the second bed, where the plant component to be co-extracted was found. The lipids obtained in the first chamber served as a co-solvent with scCO2 for extraction in the second chamber. In the case of the simultaneous extraction of edible avocado oil and the capsanthin (carotenoid) of red pepper, the higher concentration of oil improved the extraction yield of capsanthin. However, a less concentrated extract was obtained, since the carotenoid was diluted in the product. In the case of the extraction of avocado oil rich in lycopene, the extraction yield of lycopene increased as the proportion of avocado in the first extraction chamber increased, being the best condition for the extraction of lycopene present in tomato pomace at 400 bar and 50 ◦ C. Restrepo et al. [22] evaluated the quality of avocado oil extracted by Soxhlet, cold pressed, and scCO2 methods, determining the quality of the oil in terms of free fatty acid titration, peroxide index, iodine index, saponification, and specific gravity, according to the American Oil Chemists’ Society (AOCS) standards. Extraction with supercritical fluids was the technique by which the highest yields and quality were obtained. Oils extracted by scCO2 were characterized as possessing a lower acidity index (0.48%), low oxidation of unsaturated fatty acids (16.87 meqO2 /kg) and higher iodine index (80.18 cgI2/g), when compared with the other methods. In addition, extraction by cold pressing showed better results in terms of vitamin E content. Regarding extraction by pressurized fluids, the extraction with the liquefied gas of compressed oil (LPG), constituted by a mixture of propane, n-butane, isobutane, ethane, and other hydrocarbons, showed a higher oil extraction performance in less time and with a lower solvent consumption than the scCO2 method. On the other hand, the oil obtained by compressed LPG presented higher concentrations of Stigmasterol, licopersene, palmitic acid, oleic acid, and linoleic acid. However, scCO2 provided a higher yield in terms of antioxidant activity, which was determined by means of the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical assay [23]. 2.4. CO2 Subcritical Method Extraction with sCO2 operates under the same principle as the scCO2 extraction, but with a temperature below 31.1 ◦ C and CO2 pressure of 72.9 bar [24]. In this part of the review, a comparison of the physicochemical properties of avocado oil, extracted through sCO2 , UAAE, and conventional solvent (AOAC 920.39 [25]) will be analyzed. Extraction with sCO2 was performed at 27 ◦ C and 68 bar CO2 , UAAE was performed with 60 mL of distilled water, an ultrasonic power of 240 W and a frequency of 40 kHz for 30 min at 35 ◦ C, followed by a final pressing. Compared to solvent extraction, oils extracted using sCO2 and UAAE had higher iodine index values but lower melting points, determined by slip, free fatty acid content, and saponification index values. The oils extracted by sCO2 and UAAE have a clear color and higher levels of unsaturated fatty acids than the oil extracted with hexane. Regardless of the extraction method, the main fatty acids in avocado oils were oleic and palmitic acids, while the main triacylglycerols in avocado oils were palmitoyl-dioleoyl-glycerol (POO; 22.48–23.01%) and palmitoyl- Oleoyl-linoleoyl-glycerol (POL; 17.64–18.23%) [24]. 144 Molecules 2019, 24, 2172 2.5. Enzymatic Extraction In order to improve the performance of extraction by centrifugation, the incorporation of enzymes, such as pectinases, α-amylase, proteases, and cellulase, to avocado paste have been considered. The yield varies depending on the concentration and type of enzyme used and the reaction time and percentage of water used. It is emphasized that this method improves oil by up to 25 times, in comparison with the performance of a non-enzymatic centrifugation [26]. 2.6. Solvent Extraction Reddy et al. [27] compared four extraction methods to produce avocado oil (Hass and Fuerte variety). They analyzed: (1) the extraction with traditional solvent using Soxhlet (5.0 g of dried avocado sample with 250 mL of hexane for 24 h); (2) Ultrasonic Soxhlet extraction (5.0 g of dry avocado sample, sonicated in a water bath at 60 ◦ C, with hexane as the solvent, for 1 h); (3) Soxhlet extraction, combined with a microwave treatment (avocado paste 5 mm thick, extended in the rotating plate of a domestic microwave oven, heated to the maximum power for 11 min, with 5 g of the resulting mass subsequently extracted by means of the Soxhlet method with hexane); and (4) extraction with supercritical fluid (Argon and scCO2 used as extraction fluids, extractions performed for 2 h, with a fluid flow rate of 2.8–3.5 mL/min). The traditional Soxhlet extraction method yields the most reproducible results, whereas the microwave extraction showed a higher extraction yield and higher fatty acid content (69.94%). Meyer and Terry [28] performed a sequential extraction and quantification of fatty acids and avocado sugars. The average oil yield using Soxhlet extraction, with ethanol as the solvent, was significantly higher than the oil obtained by homogenization with hexane, and the fatty acid profiles for the two methods were similar. As the maturity of the fruit increases, the extraction of oil is improved. After lipid removal, methanolic extraction was superior in terms of the sucrose and perseitol obtained, compared to extraction with 80% ethanol (v/v). The extraction of mannoheptulose was not affected by any of the solvents used. The yield of avocado oil extraction has been assessed, comparing four extraction methods using solvents of different polarities. The extraction was performed using the Soxhlet method, with (1) petroleum ether, (2) homogenization with petroleum ether, (3) homogenization with a mixture of chloroform/methanol (2:1 v/v), and finally (4) extraction with chlorine-naphthalene and ball milling. It was determined that methods that only use petroleum ether as an extraction medium presented lower yields (6–9% less) than the last two methods. Saponifiable residues were lower when the method using the chloroform/methanol mixture was employed. However, this method did not completely eliminate the residual oil from the fruit [29]. Ortiz-Moreno et al. [30] analyzed the effect of four extraction methods on the chemical-physical quality of avocado oil, namely, (1) the method of microwave extraction and manual pressing, (2) extraction with hexane using the Soxhlet methodology, (3) microwave extraction, combined with the Soxhlet methodology, using hexane as the solvent, and (4) extraction with acetone. The method with the highest oil extraction performance was the third method. The amount of trans fatty acids produced by the first method was the lowest and the latter method is also the one that generates the least physicochemical alterations. When analyzing the effect of the drying method and avocado oil extraction process, ripe fruit, independent of the drying method, presents a higher extraction performance than immature fruit. This is influenced by the enzymatic degradation of the cell wall of the parenchyma during maturation. Freeze drying improves the amount of oil extracted for the scCO2 extracts and, to a lesser extent, for the hexane extracts. Extraction with hexane has been shown to have a higher oil extraction yield than scCO2 due to the lower degree of selectivity of this solvent, which completely penetrates the plant material [31]. Regarding the performance of the avocado oil extraction process, methodologies have been proposed for developing countries. One is carried out with boiling petroleum ether (30–60 ◦ C) and another extraction with distilled water (avocado paste, diluted at a ratio of 3:1 and 5:1 (w/w), heated in a water bath of 75 to 98 ◦ C, with subsequent centrifugation. In the extraction methods, calcium chloride, sodium chloride, 145 Molecules 2019, 24, 2172 calcium carbonate and calcium sulfate were used as extraction aids. The presence of inorganic salts at a low concentration improves the extraction performance, provided that it does not exceed 5%; otherwise, it has an adverse effect. The most efficient extractions were obtained with a water/avocado ratio of 5:1, pH of 5.5 and centrifugal force of 12,300× g, with the addition of 5% calcium carbonate or calcium sulfate. At higher heating temperatures (75–98 ◦ C), the oil release time decrease. In addition, the gravity sedimentation for four days at 37 ◦ C, followed by centrifugation, improves the oil extraction performance [32]. Considering the use of organic solvents in avocado oil extraction processes could alter the quality of fatty acids by inducing the formation of trans isomers. Ariza-Ortega et al. [33] proposed the application of infrared spectroscopy by Fourier transform (FTIR) to study trans fatty acids in the avocado oils of the Hass, Fuerte, and Criollo varieties. For this, oil extraction was performed by centrifugation at 40 ◦ C and extraction with hexane at 70 ◦ C for 4 h. The method using centrifugation did not increase the deterioration of fatty acids. A strong band at 723 cm−1 was documented, which is attributable to the cis functional groups, where the green color was maintained. On the other hand, the infrared spectroscopy with Fourier transform (FTIR) analysis identified an absorption band, located at 968 cm−1 , which is associated with fatty acids, with trans isomerism for the Fuerte variety extracted with hexane. 3. Procedures for the Conservation of Avocado Oil The conservation of oils is a necessary issue to address, since it allows for increasing the useful life of the products. One of the efforts made to improve the conservation of avocado oil has been the use of physical techniques, such as the electric field. The electric field (voltage 9 kV cm−1 , frequency 720 Hz, time of 5 and 25 min) allows the polyphenol oxidase enzyme present in the avocado pulp to be inactivated, preserving the components present in the avocado oil. The modifications in the quality of the refined oil (established according to the acidity index, peroxides, and iodine) are minimal, considering the electric field method as an alternative for the addition of synthetic antioxidants [34]. The oxidative stability (determined by finding the antioxidant activity reducing ferric ion, FRAP), during the storage of cold-extracted avocado oil in the presence of the oleoresins of Capsicum annuum L. (vegetable material rich in carotenoids), was assessed. It was determined that the optimal extraction of carotenoids was at a concentration of 1:3 (w/v: Capsicum annuum L/avocado oil) for 48 h in darkness at room temperature. The behavior of the oil under stronger conditions (45 ◦ C, 30 days) showed the following characteristics: (1) the extracts were stable to lipid oxidation, with a Totox index total value of 27.34, (2) 85.6% of carotenoids were conserved, (3) 80.66% of the antioxidant activity was retained, and (4) there was a color change (ΔE) of 1.783. The oleoresins obtained by extraction with avocado oil can be considered as an economic and sustainable alternative for the extraction of carotenoids, with a good oxidative stability, compared with organic solvents [35]. 4. Use of Analytical Techniques in the Quantification, Adulteration, and Contamination of Avocado Oil There is growing interest among consumers in accessing quality and authentic products. Vegetable oils can suffer from contamination and/or adulteration, which causes the product to have components not specific to the oil. It is here that the development, implementation, and application of analytical technologies are very useful. The components present in avocado oil, such as fatty acids and phytosterols, have been quantified, mainly by gas chromatography, coupled with a flame ionization detector (GC-FID). In addition, techniques, such as ultra-high-performance liquid chromatography (UHPLC), coupled with mass spectrometry (UHPLC-MS) or a photodiode array detector (UHPLC-PDA), as well as Inductively Coupled Plasma Mass Spectrometry (ICP-MS), have been used for the identification and/or quantification of analytes, such as polyphenols, squalene and minerals, respectively [36,37]. Other analytical techniques have been used for the qualitative determination of the components present in avocado oil, including 13 C nuclear magnetic resonance spectroscopy (NMR), which has been used for the identification of its major components, including fatty acids [38]. At the same time, 1 H Nuclear 146 Molecules 2019, 24, 2172 magnetic resonance spectroscopy (1 H-NMR) has been used for the detection of the minor components present in other vegetable oils [39]. Therefore, the development of new analytical methodologies for quantifying the analytes present in avocado oil represents a major challenge. Rohman et al. [40] studied the purity of avocado oil, adulterated with palm oil and canola oil, through FTIR, combined with chemometric techniques. FTIR combined with multivariate calibrations can be used to detect and quantify the adulteration of avocado oil in binary mixtures with palm oil and canola oil. The adulteration of avocado oil with soybean oil or grape seed oil can be determined using mid-infrared spectroscopy, combined with the statistical method of partial least squares discriminant analysis. This methodology allows for a simple and fast discrimination of avocado oil in binary mixtures and Tertiary oils. The frequency selected for the authentication of avocado oil was 1500–750 cm−1 , with a precision of 100% for the analysis of the mixture of two oils and 93.3% for the mixture of three oils [41]. Organophosphorus pesticides in samples of commercial avocado oil were determined using atmospheric pressure microwave-assisted liquid–liquid extraction (APMAE), with solid-phase extraction or low-temperature precipitation, as the clean-up step. The analysis was carried out by gas chromatography-flame photometric detection and gas chromatography-tandem mass spectrometry. Chlorpyrifos residues were detected in one of four samples of commercially packaged avocado oil, produced in Chile [42]. While spectroscopic techniques have focused on determining the adulteration of avocado oil with the presence of other types of vegetable oil, according to the literature reviewed here, there is a research deficiency related to the modification of the composition of avocado oil, including the study of its major components, such as triacylglycerides and/or fatty acids, in addition to its minority components, such as phytosterols, alkanes, aliphatic alcohols, polyphenols, and others. This could provide information for detecting the contamination of avocado oil with other oils of a different quality. 5. Technological Applications of Avocado Oil At the industrial level, there is a constant demand for the production of healthy foods that can maintain their nutritional properties over time, as well as environmentally friendly technological solutions. Avocado oil is mainly sold for direct consumption due to its interesting contribution of fatty acids, vitamins, antioxidants, among other compounds. Efforts have been made to develop products based on avocado oil. Arancibia et al. [43] propose the development of O/W nanoemulsions using the natural emulsifiers, lecithin and synthetic tween 80, systems that improve the characteristics with respect to traditional emulsions, such as (i) increased dispersibility of water in the encapsulated oils, which generates slightly turbid emulsions and an easy production, and (ii) a good physical and chemical stability, as well as a high bioavailability of its lipid components. Another interesting technological application for avocado oil has been the production of structured lipids. Caballero et al. [44] propose the elaboration of triacylglycerides of the MLM type, using regio-specific immobilized commercial lipases sn-1.3, where M corresponds to saturated medium-chain fatty acids (6–12 carbon atoms) at positions sn1 and sn3 of glycerol. L corresponds to saturated or unsaturated long-chain fatty acids (14–24 carbon atoms) in the sn2 position. The increased interest in this type of lipids is due to the low caloric intake (average caloric density for this family of lipids 5 kcal/g). According to the literature, there are no negative effects associated with the ingestion of MLM lipids for both animals and humans. Finally, avocado oil has also been used in the production of biodegradable polymers. Polyhydroxyalkanoates (PHAs) are linear polyesters, produced by a large number of bacteria under stress conditions, with different thermal and mechanical properties, which depend on their molecular structure. Flores-Sanchez et al. [45] prepared PHAs through a fermentative process using the bacterium C. necátor H-16 with avocado oil and fructose, as a carbon source. The highest yield in obtaining polymers was obtained when the addition of avocado oil was 20% v/v, which demonstrates the feasibility of using this oil as a renewable carbon source for the PHA production process. 147 Molecules 2019, 24, 2172 6. Composition of Avocado Oil There is a growing interest in avocado oil, including the determination of the composition of major and minor components. Therefore, for a total understanding of the nutritional and functional properties that this oil presents, it is important to consider the different varieties and parts of the fruit. 6.1. Characteristics According to the Variety and Origin of the Fruit Avocado is a fruit grown mainly in warm temperate and subtropical climates throughout the world, so it is interesting to study how the climate and country of origin can affect the fruit quality and therefore, the oil. Thus, the oil from the fruit of the Hass variety, originating from crops from Mexico, Australia, the United States, and New Zealand, was characterized by a high content of 62% lipids, of which oleic (42–51%) and palmitic (20–25%) lipids were present in a greater proportion. Among the predominant triacylglycerols were OOO (21–34%) and OOP (19–24%), where O and P denote oleic and palmitic acids, respectively. On the other hand, Hass avocado oil from New Zealand contained a significant amount of natural pigments and unsaturated compounds, compared to oils from Mexico, Australia, and the United States [1]. Studies carried out in South America on the analysis and characterization of avocado oil showed a high content of monounsaturated fatty acids (69.4%) and a lower amount of polyunsaturated and saturated fatty acids, which were 16.6% and 14%, respectively. These studies indicate that avocado oil has a thermal stability close to 176 ◦ C and has a lower concentration of total phenolic compounds than olive oil. Despite this, the antioxidant activity of avocado oil is similar to that of olive oil. Olive oil has a high concentration of polyphenols, such as tyrosol and hydroxytyrosol [46]. Galvão et al. [47] analyzed the fatty acid composition of the pulp, seed, and skin oil of the Fortuna, Collinson, and Barker varieties, indicating that there was a small variation in the composition of monounsaturated fatty acids in the skin oil among the cultivars. However, the seed oil of the Collinson variety was the best due to the lower SFA content. The SFA content for pulp oil corresponded to 22.3, 29.4 and 41.3% in the Fortuna, Collinson, and Barker varieties, respectively. In this sense, it was possible to affirm that the pulp oil of the Fortuna and Collinson varieties presented a better quality, in terms of fatty acid profile, than the Barker variety. In Mexico, avocado oil from six local creole varieties (BTancitaro, Irapuato, Orgánico, Puerto, San José, and STancitaro) were analyzed and compared with oil from the Hass variety. It was observed that the Mexican creole genotypes had a greater thermal stability, properties resistant to oxidation, and a greater phenolic content, in comparison with the commercial oil from the Hass variety. In addition, these varieties showed intense fluorescent peaks at 675 and 720 nm, as well as broad absorption bands centered at 465 and 510 nm, which can be used as an identification parameter for these oils [48]. Yanty et al. [49] indicated that the avocado oil originating from three Malaysian varieties was found in a significantly lower proportion than in the Australian Hass variety. In addition to being in a semi-solid form, all these oils had a higher proportion of oleic acid, although they also had different proportions of palmitic and linoleic acids. Regarding the composition of TAG for local varieties, the highest was POO, followed by POL, OOO, and PPO, while in the Hass variety, the distribution was OOO, followed by PPO, OOL, and POL. As a result of these different compositions in TAG, differences were found in the iodine index, melting point by slip and melting and solidification characteristics. 6.2. Physicochemical Characterization Table 1 shows the composition of the common fatty acids of the oils from different varieties and origin of avocados, discussed in this work. However, only some varieties have the C6:0, C7:0, C8:0, C9:0, C10:0, C11:0, C12:0, C13:0, C14: 0, C14:1, C15:0, C15:1, C16:1, C17:0, C17:1, C19:0, C20:0, C20:1, C20:3, C20:4, C22:0, C22:1, C22:2, C23:0, and C24:0 fatty acids in a low proportion, ranging from traces (<0.06%)–3.58%. Dreher and Davenport [50] refer to the fact that the oil coming from the Hass variety can contain up to 71% of monounsaturated fatty acids (MUFA), 13% of polyunsaturated fatty acids (PUFA), and 16% of saturated fatty acids (SFA). 148 149 0,49 0.6 0.63 ± 0.0002 0.37 ± 0.00 0.45 ± 0.16 0.24 ± 0.02 0.30 ± 0.01 0.93 ± 0.08 0.48 0.5 0.38 ± 0.01 0.43 ± 0.01 18,62 13.4 17.37 ± 0.0015 18.17 ± 0.02 13.7 ± 1.5 25.63 ± 0.11 22.59 ± 0.23 20.61 ± 0.16 22.24 ± 0.05 10. 75 20.5 12.16 ± 0.04 16.93 ± 0.03 23.66 21.312 ± 0.550 12.37 ± 0.01 19.9–21.3 28.12–34.48 30.37 ± 0.06 18.18 18.87 18.74 ± 0.06 12.87 0.762 ± 0.021 0.51 ± 0.01 0.23–1.07 1.30 ± 0.01 0.40 0.59 0.51 ± 0.00 1.45 Estearic 18:0 Palmitic 16:0 8,47 3.9 7.52 ± 0.0002 4.03 ± 0.01 3.4 ± 0.4 7.29 ± 0.05 11.63 ± 0.13 10.31 ± 0.03 13.14 ± 0.01 3.14 6.8 6.57 ± 0.01 7.33 ± 0.05 3.58 2.391 ± 0.188 7.58 ± 0.00 2.7–7.0 6.64–8.5 5.22±0.02 6.56 4.16 7.88 ± 0.01 3.86 Palmitoleic 16:1 Ω7 60,17 65.3 62.89 ± 0.0019 51.76 ± 0.04 67.4 ± 3.0 42.59 ± 0.16 49.19 ± 0.57 50.97 ± 0.30 47.69 ± 0.03 74.32 60.6 61.72 ± 0.30 57.39 ± 0.18 47.20 64.436 ± 0.666 64.62 ± 0.20 57.1–64.5 40.73–42.72 43.65 ± 0.04 60.25 63.07 54.40 ± 0.10 57.44 Oleic 18:1 Ω9 10,97 15.2 10.64 ± 0.0004 11.12 ± 0.01 14.4 ± 1.8 20.87 ± 0.10 14.72 ± 0.06 16.10 ± 0.11 14.47 ± 0.01 10.03 13.2 8.30 ± 0.02 8.25 ± 0.02 13.46 9.147 ± 0.030 8.46 ± 0.02 10.6–11.0 15.52–18.88 17.45 ± 0.04 13.03 11.83 10.87 ± 0.01 18.70 Linoleic 18:2 Ω6 0,98 1.3 0.72 ± 0.0001 0.59 ± 0.00 1.1 ± 0.01 3.19 ± 0.06 1.63 ± 0.16 1.72 ± 0.02 1.54 ± 0.00 0.85 0.44 ± 0.00 0.56 ± 0.00 1.60 0.467 ± 0.016 0.47 ± 0.00 0.4–0.6 1.51–2.14 2.03 ± 0.01 1.40 1.32 0.61 ± 0.00 0.92 α linolenic 18:3 Ω3 [51] [47] [52] [52] [54] [55] [52] [10] 1 [24] 2 [49] [51] [51] [3] [53] [1] [51] [46] [37] [52] [53] 3 Ref. composition studied under different extraction methods (cold pressed and solvents); 2 composition studied under different extraction methods (solvents, SCO2 , and UAAE); 3 in conditions similar to natural ripening. 1 BREDA CRIOLLA MEXICANA DE MALASIA REED ANTILLANA VARIETY NO INDICATED FUERTE BACON PINKERTON MARGARIDA FORTUNE Australia México New Zealand United States HASS Varieties and/or Country of Origin Table 1. Composition (%) of the common fatty acids of avocado oil. Molecules 2019, 24, 2172 Molecules 2019, 24, 2172 In order to study the fatty acid profile of the avocado oil from the fruits harvested and artificially ripened, the avocado oil was extracted from preserved fruit at 5 ◦ C in a controlled atmosphere and was analyzed (4% of O2 and 6% of CO2 ). Postharvest ripening was stimulated in the presence of exogenous ethylene (0 or 100 ppm) at a temperature of 18 ◦ C for 24 h and then preserved at 15 and 20 ◦ C. It was concluded that post-harvest conservation and ripening by means of a controlled atmosphere did not have a detrimental effect on the fatty acid profile or the amount of oil obtained, when compared with the one commonly applied in the ready-to-eat market [53]. This information is very important for the fresh product industry, where avocados move long distances at low temperatures. When carrying out the physicochemical evaluation of two Hass avocado oils sold in Chile and labeled as extra virgin, it was demonstrated that both oils presented significant differences in the content of tocopherols, total phenols, oil stability, measured as the induction time, UV absorption coefficients, peroxide index, free acidity, total chlorophyll, total carotenoids, and polar compounds. In addition, the presence of 3,5-stigmastadiene in one of the samples, a compound that has been associated with a high degree of refining or exposure to high temperatures of oils, indicated a disparity in the quality parameters and a lack of regulation of avocado oil in the local market [37]. On the other hand, monovarietal oils from the Bacon, Fuerte, Hass, and Pinkerton varieties, obtained in Spain, were compared with commercial oils originating in Brazil, Chile, Ecuador and New Zealand. The content of triacylglycerols, fatty acids, aliphatic, and terpene alcohols, desmethylmethyl, methyl and dimethyl sterols, squalene and tocopherols were determined. The main triacylglycerols were those with ECN48 (48 equivalents of carbon atoms). The oleic, palmitic, and linoleic fatty acids were the most abundant fatty acids, and the desmethyl sterols were the main quantified minor compounds. Small amounts of aliphatic and terpene alcohols were observed. The concentrations of squalene were higher in the oils of the Bacon, Fuerte, and Pinkerton varieties than in the other varieties. The most abundant tocopherol was α-tocopherol [52]. While the fruit reaches a minimum of 8% fat at the time of its extraction from the tree, during vegetative ripening, values of 20% or more are reached, depending on the variety. In this sense, it is recommended that the avocado oil industry ripen the fruit on the tree, since climacteric ripening, in comparison with commercial ripening, influences not only the increase in oil content, but also the profile of the fatty acids, increasing the amount of unsaturated fatty acids, such as oleic acid, and decreasing the amount of saturated fatty acids, such as palmitic and palmitoleic acids [56]. This situation seems controversial with respect to the previously discussed studies. However, it is well known that the composition of fruits depends on environmental and growth conditions. In addition, the quantification of the different analytes depends on the conditions of extraction, processing, and detection limits of the analytical equipment. Martinez-Nieto and Moreno-Romero [57] have analyzed the sterol composition of the unsaponifiable matter of avocado oil in 4 varieties (Reed, Bacon, Fuerte, and Hass) by means of gas chromatography. The results showed that the Fuerte and Reed varieties contained 2% more of β-sitosterol than the Bacon and Hass varieties. In relation to the cholesterol content, the smallest amount was present in the Fuerte variety and the largest in the Hass variety. In this sense, the unsaponifiable sterol composition can be a guiding tool to determine the authenticity of oil. Martínez-Nieto et al. [51] analyzed the composition of different fractions in an industrial extraction process using a continuous method, under conditions similar to those used in the extraction of olive oil, for ripe avocados of the Fuerte, Reed, Hass, and Antillana varieties. They concluded that it is possible to obtain a good net oil yield with the industrial equipment used prior to specific modifications in the grinding stage and in the decanter, as well as good quality parameters, such as the acidity index, peroxide index, and absorbance coefficients, for both virgin oil and refined avocado oil. When evaluating the physicochemical characteristics of the avocado oil obtained from the Bantul, Purwokerto, and Garut varieties, originating in Indonesia, by extraction with solvents, the Garut variety presented a better quality in terms of the iodine index, which is associated with a greater amount of unsaturated fatty acids. The conjugated dienes and trienes were significantly different 150 Molecules 2019, 24, 2172 between the samples. The p-anisidine index did not have significant differences between the samples. The saponification index was higher in the Purwokerto variety, and the peroxide index was higher in the Bantul variety. In addition, the analysis using differential scanning calorimetry (DSC) showed that the three samples had different melting and crystallization profiles [58]. 6.3. Avocado Seed Oil In relation to avocado seed oil, Barrera-López and Arrubia-Vélez [59] pointed out that the Lorena variety contained about 8.47% oil, and the unsaponifiable matter was 76.9%. The phytosterols quantified in a greater proportion were ergosterol, 5α-cholestane and stigmasterol. Avocado seed presents, in its composition, a large number of extractable polyphenols, which have attracted attention due to their high antioxidant capacity. It was determined that, with a higher power of the ultrasound (0–104 W) and increase of the temperature (20–60 ◦ C), the polyphenol content and antioxidant capacity was increased [60]. When performing a physicochemical analysis of the seed oil of the Hass variety, cultivated in Peru and obtained by the Soxhlet method, it was found that it had a high fatty acid profile in linoleic acid (48.77%) and linolenic acid (12.17%). While the antioxidant activity, determined by the DPPH method, was low, it was higher in the saponifiable fraction than in the unsaponifiable fraction, which was attributable to the presence of polyphenols and steroids. In addition, it was determined that the quality parameters, such as acidity, peroxide, saponification, iodine, and specific gravity indexes, were similar to those for extra virgin olive oil [61]. When comparing the composition of oil from the pulp and seed of the Fuerte variety, cultivated in the region of Northeastern Brazil, a great difference in the lipid content between the pulp and the seed can be seen (15.39% v.s. 1.87%, dry base). It was determined that the parameters of oil quality, refractive index, gravity, and peroxide index were similar for both oils, but the iodine, acid index, and saponification index were higher in seed oil than in pulp oil. Gas chromatography showed that seed oil had a greater variety of fatty acids than pulp oil. Additionally, the fatty acid profile of the pulp was much more concentrated in monounsaturated fatty acids than that of seed, and conversely, the seed oil is much more concentrated in polyunsaturated fatty acids than pulp oil [55]. 6.4. Comparison with Other Oils Dubois et al. (2007) [62] compared 80 varieties of vegetable oils, including avocado oil, indicating that it was composed of more than 60% of monounsaturated fatty acids, a characteristic shared with olive oil, hazelnut, and macadamia nut profiles. In comparison with olive oil, avocado oil possessed a higher proportion of saturated fatty acids (16.4%), with a predominance of palmitic acid (15.7%), a lower proportion of monounsaturated fatty acids (67.8%), with a predominance of oleic acid 60.3% and a higher proportion of polyunsaturated fatty acids (15.2%), the most important of which was linoleic acid at 13.7%. Additionally, similar fatty acid profiles have been published by Berasategi et al. [3], who also showed that avocado oil had a higher PUFA/SFA and higher omega-6/omega-3 ratios than olive oil. Berasategi et al. [3] showed that the phytosterol content was higher in avocado oil (3.3 g to 4.5 mg/g of oil) than in olive oil, of which the most abundant was β-sitosterol, followed by sitostanol, cycloartenol, cycloeucalenol and D7-avenasterol. The number of sterols in the avocado oil was higher, 4-demethyl-sterols being the most abundant, reaching 80% of the total fraction of sterols. This greater proportion was even maintained under drastic conditions of deterioration (180 ◦ C). Moreover, avocado oil has a lower proportion of vitamin E, compared to olive oil. This study indicates that the thermal stability of avocado oil is similar to that of olive oil. From Table 2, it is possible to appreciate a summary of the different antioxidant components present in the avocado oils of the different varieties shown in this work. 151 PINKERTON VARIETY NO INDICATED ANTILLANA REED BACON FUERTE HASS Varieties α-Tocopherol 86.75 ± 0.62 103.11 ± 6.87 51.90 ± 0.04 45.62 ± 0.19 - β-Sitosterol 91.917 ± 0.027 95.2 82.95 ± 0.06 94.767 ± 0.012 92.9 80.56 ± 0.08 92.189 ± 0.012 82.6 ± 0.03 94.605 ± 0.027 91.2 89.3 84.08 ± 0.08 9.02 ± 0.09 20.35 ± 1.22 71.61 ± 0.57 13.71 ± 0.56 - γ-Tocopherol 6.63 ± 0.07 8.81 ± 0.03 9.16 ± 0.03 5.86 ± 0.01 9.42 ± 1.69 Δ5-avenasterol 6.091 ± 0.026 4.7 5.88 ± 0.01 5.043 ± 0.012 6.4 4.62 ± 0.02 6.096 ± 0.010 3.71 ± 0.01 5.123 ± 0.021 8.6 10.6 6.00 ± 0.01 18.36 ± 1.44 Campesterol 0.001 ± 0.001 0.13 ± 0.00 0.001 ± 0.001 0.15 ± 0.00 0.011 ± 0.001 0.40 ± 0.01 0.001 ± 0.001 0.11 ± 0.00 1.11 ± 0.12 Estigmasterol Table 2. Antioxidant compounds present in avocado oil. Concentration [=] mg × kg−1. 0.46 ± 0.01 0.57 ± 0.04 0.58 ± 0.04 0.41 ± 0.03 2.19 ± 0.22 Sitoestanol 0.04 ± 0.00 0.04 ± 0.00 0.05 ± 0.00 0.04 ± 0.00 0.43 ± 0.03 Campestanol [57] [51] [52] [57] [51] [52] [57] [52] [57] [51] [51] [52] [3] Ref. Molecules 2019, 24, 2172 152 Molecules 2019, 24, 2172 7. Biological Effects The presence of compounds with nutritional interest, such as unsaturated fatty acids (MUFA and PUFA), as well as compounds with biological activity, such as tocopherols, tocotrienols, phytosterols, carotenoids, and polyphenols, have made avocado oil of growing interest for research on the possible biological effects of avocado oil, with the aim of preventing and treating diseases through the diet of the population. 7.1. Human Health Effects A study in 13 healthy adults with a habitual hypercaloric and hyperlipidic diet, where butter was replaced by avocado oil extracted at 35 ◦ C from the pulp alone, was conducted. The incorporation of avocado oil for a period of six days reflected an improvement in the postprandial profile of insulin, glycemia, total cholesterol, low-density lipoproteins, triglycerides, and inflammatory parameters, such as C-reactive protein (CRP) and interleukin-6 [63]. Avocado pulp oil (Mexican creole genotypes) has shown anti-inflammatory activity by inhibiting the enzymes COX 1 and COX 2 in a similar way to the drug, ibuprofen, and extra virgin olive oil [48]. Additionally, when avocado oil was added to vitamin B12 skin cream preparation, it was well tolerated and had the potential for long-term topical therapy of psoriasis [64]. 7.2. Experimental Studies in Animals When administering avocado oil to Wistar diabetic rats for 90 days (1 gr/250 g weight), it was observed that it promoted an improvement in the functionality of the electron transport chain and decreased the generation of free radicals in the liver, attenuating the harmful effects of oxidative stress [65]. In the brain, an improvement in mitochondrial function has been observed, as well as a decrease in free radical levels, lipid peroxidation, and an improvement in the reduced/oxidized glutathione ratio. These results demonstrate that supplementation with avocado oil prevents mitochondrial dysfunction in the brain and liver of diabetic rats [66]. Ortiz-Avila et al. [67] demonstrated that supplementation with avocado oil in Wistar rats led to a reduction of the alterations in the electron transport chain at the renal level, attenuating the oxidative damage, although a protective effect on lipid peroxidation was not evidenced. Some authors [68–70] studied the relationship between avocado oil and collagen metabolism in both the skin and liver, finding that oil obtained from intact fruit (pulp and seed), refined with hexane, was associated with fibrosis in the liver, an increase in liver enzymes and consequently hepatotoxicity. In addition to an increase in the solubility of collagen, behavior that is attributed to a decrease in the activity of lysyl oxidase was observed [66]. A similar conclusion was proposed by Lamaud et al. [71], stating that the mixture of soybean and avocado oils decreased the degree of collagen cross-linking, a process that has been associated with a delay in wound healing. However, Oliveira et al. [53] indicated that the healing activity of a semi-solid formulation of avocado oil (50/50 Vaseline) and avocado pulp oil promoted the increase of collagen synthesis and decreased the number of inflammatory cells during the process of wound healing. 153 Molecules 2019, 24, 2172 Regarding the effects on cardiovascular health, it is possible to point out that the atherogenic power of avocado oil could be similar to that of olive oil and lower than that of corn and coconut oil [72]. On the other hand, an avocado oil-rich diet modifies the fatty acid content in cardiac and renal membranes in a tissue-specific manner in Wistar rats. The rise in renal arachidonic acid suggests that diet content can be a key factor in vascular responses [73]. Márquez-Ramírez et al. [74] studied the antihypertensive effect of avocado oil in rats with induced hypertension. They were subsequently treated with the administration of 1 mL of oil per 250 g of rat weight or 40 mg of losartan potassium per kg of rat weight. Avocado oil significantly decreased both systolic and diastolic pressure in hypertensive rats, but not in controls. Avocado oil mimicked the effects of the drug, losartan, on blood pressure, vascular performance, and oxidative stress. In rats fed with sucrose, it was observed that the addition of avocado oil in the diet reduced the levels of triglycerides, VLDL and LDL, without affecting the levels of HDL. It also reduced the level of ultrasensitive CPR, indicating that the inflammatory processes associated with metabolic syndrome were partially re-established [4]. Additionally, a diet high in sucrose, which causes liver alteration in Sprague-Dawley Weaned rats, was partially reversible by the administration of avocado oil, obtained by centrifugation and extracted by solvents. This effect was similar to that of olive oil, but would not be extended to the pancreatic level [5]. In relation to insulin resistance induced by a diet high in sucrose in Wistar rats, it was possible to reduce it through the dietary addition of 5–20% avocado oil [75]. In female Wistar rats exposed to prolonged androgenic stimulation, avocado oil improved triglyceride, VLDL and HDL levels, generating a direct regulatory effect on the lipid profile [76]. The influence of avocado oil on cell damage, induced by methyl methanesulfonate (MMS) and doxorubicin, can be carried out using in vitro (V79 cells) and in vivo models (Swiss mice). Avocado pulp oil had no genotoxic effects. The oil was effective in reducing the chromosomal damage induced by MMS and doxorubicin. However, an increase in liver tissue damage was observed when evaluating a high dose of avocado oil (measured as an increase in the hepatic enzyme aspartate aminotransferase), a phenomenon attributable to the high concentration of palmitic fatty acid [77]. Table 3 shows a summary of the biological effects presented in this study. 154 Daily administration of avocado oil was 1 mL/250 g of weight for a period of 3, 6 and 12 months to Winstar and Goto-Kakizaki rats. Swiss mice were given avocado oil in different concentrations of 250, 500, 1000 and 2000 mg/kg in an in vivo model. In vitro model was applied to different concentrations of avocado oil: 100, 200 and 400 μg/mL. Administration of a diet with different concentrations of avocado oil to insulin resistant rats. Administration of avocado oil (1 g/250 g weight) to diabetic rats for 90 days. Diet supplemented with avocado oil to rats exposed to prolonged androgenic stimulation. Administration of a diet with olive oil and avocado oil to rats exposed to a diet rich in sucrose. Administration of a diet with olive oil and avocado oil to rats exposed to a diet rich in sucrose. Diabetic male rats Goto-Kakizaki; the control rats were Wistar males. In vitro (Chinese hamster lung fibroblasts/V79 cells) and in vivo models (Swiss mice). Male Wistar rats Male Wistar rats 155 Wistar rats Wistar rats Wistar rats Protocol Used Daily administration of 1.0 mL/250 g of avocado oil by gavage or losartan at 40 mg/kg for 45 days. Animal Model Male Wistar rats Conclusions In the liver: (a) A diet high in sucrose causes hepatic impairment, partially reversible by the administration of avocado oil, which does not occur at the pancreatic level. (b) Avocado oil (independent of its extraction method) exhibits effects similar to those of olive oil in the fatty acid profile. At the cardiovascular level: In rats fed with sucrose, it was observed that avocado oil reduces the levels of triglycerides, very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL), without affecting the levels of high-density lipoprotein (HDL). It also reduces the level of ultrasensitive CRP, indicating that the inflammatory processes associated with metabolic syndrome are partially reestablished. (a) Avocado oil mimics the effects of losartan. (b) Effects of avocado oil could be mediated by decreased actions of Angiotensin-II in mitochondria. (c) The intake of avocado oil could mitigate the harmful effects of hypertension in kidneys. In the brain: (a) Improvement of mitochondrial function. (b) Decreased levels of free radicals and lipid peroxidation. (c) Improvement of the reduced/oxidized glutathione ratio. (d) Prevention of mitochondrial dysfunction. (a) Avocado pulp oil has no genotoxic effects. (b) The oil was effective in reducing the chromosomal damage induced by methyl methanesulfonate and doxorubicin. However, an increase in hepatic enzyme aspartate aminotransferase was found to be a marker of liver damage. (a) The dietary addition of 5–20% avocado oil can reduce glucose tolerance and insulin resistance, induced by the high sucrose diet, in Wistar rats. (b) The addition of 5–30% avocado oil reduced the body weight gain induced by the high sucrose diet in Wistar rats. In the liver: (a) Promoted an improvement in the functionality of the electron transport chain. (b) Decreased the generation of free radicals. (c) Diminished the harmful effects of oxidative stress in the liver. Avocado oil exerted a direct regulatory effect on the lipid profile. Table 3. Experimental studies on the biological effects of avocado. [4] [5] [76] [65] [75] [77] [66] [74] Ref. Molecules 2019, 24, 2172 The control group received a laboratory pellet, while the treated group received a diet rich in 10% avocado oil (weight/weight) for a period of two weeks. Rabbits were fed a semi-purified diet containing 0.2% cholesterol and 14% oil for 90 days (corn, coconut, olive and avocado). Rats and chicks were fed 10% avocado oil (w/w). Male Wistar rats Growing rats were fed for eight weeks with 10% (w/w) refined or unrefined avocado oil or soybean oil, using different extraction media. The rats were fed diets containing 10% avocado oil (w/w) for four weeks. Application of oil mixtures in the skin of the dorsal area of rats for 15 days, treatment with a mixture of 2/3 of soybean oil and 1/3 avocado in a 5% solution of sweet almond oil. Charles river female rats. Charles river female rats. Rats Female rats and chicks. Male rabbits of the New Zealand White strain. Protocol Used Avocado oil administration: 1 mL of avocado oil/250 g of weight daily for a period of 90 days. Animal Model Male Wistar rats Table 3. Cont. Conclusions Rats and chickens fed unrefined avocado oil showed a significant decrease in total collagen solubility in the liver. Rats fed with unrefined avocado oil extracted with hexane from intact fruit (unsaponifiables) or avocado seed oil showed significant increases in the content of soluble collagen in the skin, although the total collagen content was not affected. Rats fed refined or unrefined soybean oils showed no effects. The consumption of avocado oil extracted from intact fruits can cause changes in metabolism in the liver. Serum alkaline phosphatase activity increased in rats fed seed oil, oil extracted with unrefined solvent of intact fruit or unsaponifiables, and aspartate aminotransferase activity increased significantly in the group fed avocado seed oil. The mixture of soybean and avocado oils decreases the degree of collagen cross-linking, delaying wound healing. At the renal level: (a) Protection against induced oxidative stress. (b) ROS generation decrease. (c) Improvement of the activities of complexes II and III. (d) Lower peroxidizability index in diabetic mitocondria. The administration of a diet rich in avocado oil to rats for two weeks modifies the content of fatty acids in cardiac and renal membranes. The atherogenic power of avocado oil is equivalent to that of olive oil and lower than that of corn and coconut oil. [71] [69] [70] [68] [72] [73] [67] Ref. Molecules 2019, 24, 2172 156 Molecules 2019, 24, 2172 8. Conclusions While the composition and quality of the avocado oil depends on the origin, weather conditions, variety, and extraction methods, it is characterized as a mainly monounsaturated oil, with an adequate proportion of polyunsaturated fatty acids, similar to olive oil. In addition, it contains other bioactive compounds, present in the unsaponifiable fraction, such as tocopherols, polyphenols, and a remarkable proportion of phytosterols. This oil has also been shown to perform well at high temperatures. All these characteristics indicate that avocado oil has nutritional properties that are greatly appreciated by the population, even for technological applications. This makes it a product of increasing exploitation interest for producers. This growing interest also requires a greater development of studies on the adulteration and contamination of avocado oil, as well as the further assessment of the promising biological effects of the different components present in the oil, either in animals or humans. Furthermore, considering the presented information, it is necessary to inquire into the international regulations concerning the different qualities of this oil. Author Contributions: Conceptualization, M.F. and J.O.-V.; methodology, C.E.V., C.S. and J.O.-V.; software, H.V.; validation, C.E.V., F.A., and H.V.; formal analysis, M.F. and F.A.; investigation, M.F., C.S. and J.O.-V.; resources, M.F.; data curation, C.S. and C.E.V.; writing—original draft preparation, C.S.; writing—review and editing, M.F., F.A., H.V. and J.O.-V. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. Abbreviations UAAE sCO2 scCO2 LPG FFA MUFA PUFA SFA TAG O P L COX PCR FTIR IOC DPPH APMAE PHAs ECN48 VLDL LDL HDL Ultrasound-assisted aqueous extraction Subcritical CO2 Supercritical CO2 Liquefied gas of compressed oil Free fatty acid Monounsaturated fatty acid Polyunsaturated fatty acid Saturated fatty acid Triacylglycerol Oleic acid Palmitic acid Linoleic acid Cyclooxygenase enzyme C reactive protein Infrared spectroscopy with Fourier transform International olive council 2,2-Diphenyl-1-picrylhydrazyl Atmospheric pressure microwave-assisted liquid–liquid extraction Polyhydroxyalkanoates 48 equivalents of carbon atoms Very low-density lipoprotein Low-density lipoprotein High-density lipoprotein References 1. Tan, C.; Tan, S.; Tan, S. Influence of Geographical Origins on the Physicochemical Properties of Hass Avocado Oil. J. Am. Oil Chem. Soc. 2017, 94, 1431–1437. [CrossRef] 157 Molecules 2019, 24, 2172 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 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[CrossRef] Salazar, M.J.; El Hafidi, M.; Pastelin, G.; Ramírez-Ortega, M.C.; Sánchez-Mendoza, M.A. Effect of an avocado oil-rich diet over an angiotensin II-induced blood pressure response. J. Ethnopharmacol. 2005, 98, 335–338. [CrossRef] Márquez-Ramírez, C.; Hernández de la Paz, J.; Ortiz-Avila, O.; Raya-Farias, A.; González-Hernández, J.; Rodríguez-Orozco, A.; Salgado-Garciglia, R.; Saavedra-Molina, A.; Godínez-Hernández, D.; Cortés-Rojo, C. Comparative effects of avocado oil and losartan on blood pressure, renal vascular function, and mitochondrial oxidative stress in hypertensive rats. Nutrition 2018, 54, 60–67. Del Toro-Equihua, M.; Velasco-Rodríguez, R.; López-Ascencio, R.; Vásquez, C. Effect of an avocado oil-enhanced diet (Persea americana) on sucrose-induced insulin resistance in Wistar rats. J. Food Drug Anal. 2016, 24, 350–357. [CrossRef] [PubMed] De Souza-Abboud, R.; Alves-Pereira, V.; Soares da Costa, C.A.; Teles-Boaventura, G.; Alves-Chagas, M. The action of avocado oil on the lipidogram of wistar rats submitted to prolonged androgenic stimulum. Nutr. Hosp. 2015, 32, 696–701. Nicolella, H.D.; Neto, F.R.; Corrêa, M.B.; Lopes, D.H.; Rondon, E.N.; Dos Santos, L.F.; De Oliveira, P.F.; Damasceno, J.L.; Acésio, N.O.; Turatti, I.C.; et al. Toxicogenetic study of Persea americana fruit pulp oil and its effect on genomic instability. Food Chem. Toxicol. 2017, 101, 114–120. [CrossRef] [PubMed] © 2019 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/). 161 MDPI St. Alban-Anlage 66 4052 Basel Switzerland Tel. +41 61 683 77 34 Fax +41 61 302 89 18 www.mdpi.com Molecules Editorial Office E-mail: molecules@mdpi.com www.mdpi.com/journal/molecules MDPI St. Alban-Anlage 66 4052 Basel Switzerland Tel: +41 61 683 77 34 Fax: +41 61 302 89 18 www.mdpi.com ISBN 978-3-03928-765-9 

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Indonesian Journal of International Law Volume 5 Number 3 Treaty and National Law Article 6 4-30-2008 Kedudukan Hukum Internasional dalam Sistem Hukum Nasional Melda Kamil Ariadno Follow this and additional works at: https://scholarhub.ui.ac.id/ijil Part of the International Law Commons Recommended Citation Ariadno, Melda Kamil (2008) "Kedudukan Hukum Internasional dalam Sistem Hukum Nasional," Indonesian Journal of International Law: Vol. 5: No. 3, Article 6. DOI: 10.17304/ijil.vol5.3.179 Available at: https://scholarhub.ui.ac.id/ijil/vol5/iss3/6 This Article is brought to you for free and open access by the Faculty of Law at UI Scholars Hub. It has been accepted for inclusion in Indonesian Journal of International Law by an authorized editor of UI Scholars Hub. 

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Global Journal of Health Science; Vol. 8, No. 5; 2016 ISSN 1916-9736 E-ISSN 1916-9744 Published by Canadian Center of Science and Education Psychometric Testing of the Greek Version of the Clinical Learning Environment-Teacher (CLES+T) Evridiki Papastavrou1, Maria Dimitriadou1 & Haritini Tsangari2 1 School of Health Sciences, Department of Nursing, Cyprus University of Technology, Cyprus 2 School of Business, Department of Economics and Finance, University of Nicosia, Cyprus Correspondence: Maria Dimitriadou, School of Health Sciences, Department of Nursing, Cyprus University of Technology, Cyprus. Tel: 357-9967-1898; Fax: 357-25-002-864. E-mail: maria.dimitriadou@cut.ac.cy Received: June 1, 2015 doi:10.5539/gjhs.v8n5p59 Accepted: June 24, 2015 Online Published: August 31, 2015 URL: http://dx.doi.org/10.5539/gjhs.v8n5p59 Abstract Clinical practice is an important part of nursing education, and robust instruments are required to evaluate the effectiveness of the hospital setting as a learning environment. The study aim is the psychometric test of the Clinical Learning Environment+Teacher (CLES+T) scale-Greek version. 463 students practicing in acute care hospitals participated in the study. The reliability of the instrument was estimated with Cronbach’s alpha coefficients. The construct validity was evaluated using exploratory factor analysis (EFA) with Varimax rotation. Convergent validity was examined by measuring the bivariate correlations between the scale/subscales. Content, validity and semantic equivalence were examined through reviews by a panel of experts. The total scale showed high internal consistency (α=0.95). EFA was identical to the original scale, had eigen values larger than one and explained a total of 67.4% of the variance. The factor with the highest eigen value and the largest percentage of variance explained was “supervisory relationship”, with an original eigenvalue of 13.1 (6.8 after Varimax rotation) and an explanation of around 38% of the variance (or 20% after rotation). Convergent validity was examined by measuring the bivariate correlations between the scale and a question that measured the general satisfaction. The Greek version of the CLES+T is a valid and reliable instrument that can be used to examine students’ perceptions of the clinical learning environment. Keywords: clinical learning environment, nurse education, psychometric testing, validation 1. Introduction Nurse education is an integral part of any healthcare delivery system as it has a major role to play in the educational development of graduates who can deliver safe, good quality nursing care. Nursing professionals’ knowledge, attitudes and skills are acquired both through formal education in institutions and through experience in the clinical area (Gaberson & Oermann, 2010). The clinical area represents an environment where the student nurse interact emotionally, physically and cognitively with their surroundings and fulfill their learning outcomes. Under these circumstances the clinical area is referred to as the ‘clinical learning environment’. Clinical learning environment (CLE) is viewed as an invaluable resource in familiarizing students with the reality of their professional role (Henderson, Cooke, Creedy, & Walker, 2012). Students being exposed to a range of clinical experiences (Edwards, Smith, Courtney, Finlayson, & Chapman, 2004) helps to broaden and deepen both cognitive and psychomotor skills (Souza, Venkatesaperumal, & Radhakrishnan, 2013), develop caring relationships and aid nurses in their professional evolution, as embodied within the attitudes of the nursing workforce (Haugan, Sørensen, & Hanssen, 2012). Interest in the concept of clinical education as a determinant of quality nursing has gained increasing attention since 1980, leading to various nursing education reforms (Pollard, Ellis, Stringer, & Cockayne, 2007). Historically, a number of researchers investigated the effectiveness of the clinical learning environment (CLE) from students’ perspectives, employed quantitative (Saarikoski, Marrow, Abreu, Riklikiene, & Özbicakçi, 2007; Melender, Jonsén, & Hilli, 2013; Papathanasiou, Tsaras, & Sarafis, 2014; Dimitriadou, Papastavrou, Efstathiou & Theodorou, 2015), qualitative (Papp, Markkanen, & Von Bonsdorff, 2003; Mattila, Pitkäjärvi, & Eriksson, 2010) and mix methodology (Bisholt, Ohlsson, Engström, Johansson, & Gustafsson, 2014; Ip & Chan, 2005). The pedagogical atmosphere characterized by respect, acceptance and opportunities for learning with the mentor and clinical teacher alike have a stake in making clinical learning successful and reliable (Papp et al., 2003; 59 www.ccsenet.org/gjhs Global Journal of Health Science Vol. 8, No. 5; 2016 Saarikoski et al., 2007; Dimitriadou et al., 2015). Also, student satisfaction was consistently identified as the most reliable index of a “good” clinical learning environment (Chan, 2001; Papastavrou, Lambrinou, Tsangari, Saarikoski, & Leino-Kilpi, 2010; Dimitriadou et al., 2015). For that reason, a variety of instruments for evaluating CLE from students’ perspective have been developed such as the CLEI-Clinical Learning Environment Inventory (Chan, 2001); the CLESDI-Clinical Learning Environment Diagnostic Inventory scale (Hosoda, 2006); the BES-CPE (Levett-Jones, 2009); the SECEE - Student Evaluation of the Clinical Education Environment (Sand-Jecklin, 2009); and the CLES - Clinical Learning Environment and Supervision Scale. The CLES is a 27-item scale (Saarikoski & Leino-Kilpi, 2002) developed to measure both the clinical learning environment and clinical supervision. Later, this scale was reviewed by the developers and a new sub-dimension added culminating in a 34-item CLES + T scale (Saarikoski, Isoaho, Warne, & Leino-Kilpi, 2008) and assesses 5 factors. They are as follows- pedagogical atmosphere, leadership style of the ward manager, supervisory relationship, the premises in the ward, and the role of the nurse teacher. The CLES+T instrument was used mainly and extensively in Europe in an effort to develop a powerful, multilingual tool for evaluating the quality of clinical learning. To date, the CLES+T scale has been translated into nine different languages, i.e., English, Finnish, Italian, Greek, Swedish, Dutch, Norwegian, German and Spanish. It has been demonstrated to be a valid and reliable tool among different international samples (Saarikoski et al., 2008; Johansson et al., 2010; De Witte, Labeau, & De Keyser, 2011; Henriksen, Normann, & Skaalvik, 2012; Tomietto et al., 2012; Bergjan & Hertel 2013; Saarikoski et al., 2013; Watson et al., 2014; Vizcaya-Moreno, Pérez-Cañaveras, De Juan, & Saarikoski, 2015). Since the quality of clinical learning depends on how well the practice curriculum is structured, the use of a reliable tool is helpful in obtaining information regarding the effective organization of clinical practice and the quality of it’s supervision. With this in mind, CLES+T is selected to test the psychometric properties with the aim to enhance cross-cultural collaboration. 2. Methods 2.1 Aim The current study aim was to test the psychometric properties of the Greek version of the Clinical Learning Environment Scale + Teacher (CLES+T-GR) of Greek Cypriot students. 2.2 Participants and Settings The target population of the study included all nursing students enrolled at the three Universities offering a Bachelor’s degree in Nursing within Cyprus. Only students who were practicing in hospitals were recruited, and those who were practicing in primary health care centers and other community care settings were excluded. 664 students fulfilled the inclusion criteria and were given the questionnaire. 463 questionnaires were returned, giving a response rate of 70.33%. 2.3 Ethical Considerations and Data Collection The research proposal was submitted to the National Bioethics Committee, which is responsible for all research projects in Cyprus according to the Law (3558/2001). The permission for access to the field research was obtained by the Chairs of Nursing from each university. The aims of and the rationale for the study, and assurances that the data would be processed anonymously were included in the information letter supplied with the questionnaire. The questionnaires were given to the students individually immediately after they had completed their clinical placement, during a nursing laboratory lesson. After completion, each questionnaire was returned in a closed envelope. The completion of the questionnaire was considered as an informed consent for participation in the study. 2.4 Research Instrument As mentioned the CLES+T scale consists of 34 items classified into 5 subscales: (1) pedagogical atmosphere on the ward; (2) supervisory relationship; (3) leadership style of the ward manager; (4) premises of ward nursing; and (5) role of the nurse teacher in clinical practice (Saarikoski et al., 2008). Respondents are asked to score their perception to each item on a 5-point Likert-type scale ranging from “very dissatisfied” to “very satisfied”. Also demographic data, hospital and ward type, length of clinical placement, number of meeting with the nurse teacher, motivational level on clinical placement, and level of satisfaction were collected. 2.5 Data Analysis For demographic data and scale items, descriptive statistics (frequencies, percentages, means and standard deviations), skewness and kurtosis were used. The internal consistency of the Greek version of the instrument and each dimension was estimated with Cronbach’s alpha coefficients. Also, item analysis was conducted on the data, providing item-to-total correlations and Cronbach’s alpha if the item was deleted from the scale. Construct 60 www.ccsenet.org/gjhs Global Journal of Health Science Vol. 8, No. 5; 2016 validity was evaluated using exploratory factor analysis (EFA) with Varimax rotation. Principal components analysis (PCA) was implemented as the extraction method in EFA. Convergent validity was examined by measuring the bivariate correlations between the scale/subscales and the question about the general satisfaction of nurses. Content, validity and semantic equivalence were examined through review by a panel of experts of the content of each item, its wording and the meaning of the items, after translation, in the context of the Cypriot culture (Squires et al., 2013). 3. Results 3.1 Sample Characteristics The final sample included 463 participants. Among those, 38.7% were males and 61.3% females, with ages ranging from 18 to 34 years, a mean of 21.08 years and standard deviation 2.23 years. 149 participants studied in private universities, and 314 in the single public university. 3.2 The Individual Scale Items Descriptive statistics (mean and standard deviation) for individual items were calculated. In order to examine the variability of the answers and test for significant deviations from normality, item skewness and kurtosis were also reported. Acceptable values of skewness and kurtosis, based on George and Mallery (2001), are those between -1.5 and +1.5, whereas values between -1 and +1 are considered excellent. The highest mean was of an item on the supervisory relationship subscale (item 18 in the scale - My mentor showed a positive attitude…), with a mean of 4.3, and a (low) standard deviation of 1.00. This item had marginal values of skewness and kurtosis as well. This verified that most of the answers were “agree” or “fully agree”, as opposed to negative attitudes, but the values were within the acceptable range. Although, in most of the items, there was a weak trend towards the positive attitudes (agree and strongly agree), there were no critical values (high positive or negative) of kurtosis or skewness for any item with all values being in the acceptable range. Therefore, transformations were not deemed necessary and all items were included in the analysis. These results are presented in Table 1. 3.3 Internal Consistency and Reliability-Item Analysis Cronbach’s alpha was used for testing the reliability of the 34-item scale, as well as for the five subscales, where values close to one are considered satisfactory. Also, item analysis was conducted to provide information about how well each individual item correlated to other items in the sub-scale where corrected item-to-total correlations below 0.30 are usually considered unacceptably low (Polit & Beck, 2011). Also, all of the inter-item correlations were examined with the reliability of each item being considered by finding if the it’s Cronbach’s alpha was deleted. The results showed high internal consistency for the total scale (α=0.95). Similarly, the reliability of each sub-category was found to be high, ranging from 0.81 (“nursing care”) to 0.96 (“supervisory relationship”). The latter results are presented in table 1. Item analysis showed that when any item was deleted from the scale, the alpha was slightly lower or approximately the same as compared to when all the items were included, suggesting that deleting any item does not change the overall reliability significantly, and as such, that all the items contribute to the high reliability of the scale. A slight increase was seen in items “I felt comfortable going to the ward at the start of my shift” and “the staff learned how to know the students by their names”, however small (from 0.949 to 0.950). The items can thus be considered reliable. Corrected item-to-scale correlations varied from 0.38 to 0.71, showing that all correlations were satisfactory, that is, above 0.3. Finally, out of more than 250 inter-item correlations between the 34 items (not reported), only two correlations exceeded 0.80 (in the supervisory relationship factor). Thus, in general, the results suggest that within the scale no items duplicated each other. The corrected item-to-scale correlations and Cronbach’s alpha if the item was deleted appear in Table 1. 61 www.ccsenet.org/gjhs Global Journal of Health Science Vol. 8, No. 5; 2016 Table 1. Descriptives, Internal consistency and Reliability (sequence as presented in the questionnaire) Item Mean Std. Deviation Skewness Kurtosis Corrected Item-Total Correlation Cronbach Alpha if item Deleted Pedagogical Atmosphere, 9 items, Cronbach’s alpha 0.875 The staff were easy to approach 3.7646 1.06635 -.843 .183 .522 .948 I felt comfortable going to the ward at the start of my shift 3.0606 1.22013 -.159 -.893 .385 .950 During staff meetings(e.g. before shifts) I felt comfortable taking part in the discussions 4.0475 1.03296 -1.160 1.093 .493 .948 There was a positive atmosphere on the ward 3.8013 1.04210 -.840 .426 .620 .948 The staffs were generally interested in student supervision 3.2505 1.19427 -.248 -.733 .605 .948 The staff learned to know the students by their personal names 2.4514 1.31973 .500 -.870 .403 .950 There were sufficient meaningful learning situations on the ward 3.4190 1.08981 -.356 -.525 .620 .948 The learning situations were multi-dimensional in terms of content 3.3826 1.08331 -.289 -.449 .613 .948 The ward can be regarded as a good learning environment 3.6609 1.09773 -.508 -.476 .613 .948 Leadership style in ward management, 4 items, Cronbach’s alpha 0.849 The WM regarded the staff on his/her ward as a key resource person 3.9391 1.03774 -.970 .620 .546 .948 The WM was a team member 3.5502 1.18464 -.628 -.351 .490 .949 Feedback from the WM could easy be consider a learning situation 3.3275 1.13437 -.323 -.525 .584 .948 The effort on individual employee was appreciated 3.3348 1.09000 -.286 -.425 .531 .948 Nursing care, 4 items, Cronbach’s alpha 0.812 The ward nursing philosophy was clearly defined 3.6811 .98259 -.569 .090 .576 .948 Patients received individual nursing care 3.8304 1.02769 -.696 -.099 .512 .948 There were no problem in the information flow related to patients’ care 3.7609 1.01158 -.636 -.015 .471 .949 Documentation of nursing ( e.g. nursing plans, daily recording of nursing procedures etc.) was clear 3.8824 .99743 -.730 .129 .589 .948 Supervisory Relationship, 8 items, Cronbach’s alpha 0.849 My supervisor showed a positive attitude towards supervision 4.2673 1.05593 -1.482 1.504 .658 .947 I felt that I received individual supervision 3.7733 1.18752 -.734 -.326 .647 .947 62 www.ccsenet.org/gjhs Global Journal of Health Science Vol. 8, No. 5; 2016 I continuously received feedback from supervisor 3.9399 1.16111 -.857 -.219 .683 .947 Overall I am satisfied with the supervision I received 3.9690 1.22435 -1.057 .110 .705 .947 The supervision was based on a relationship of equality and promoted my learning 3.9498 1.15256 -.921 -.048 .703 .947 There was a mutual interaction in the supervisory relationship 3.9569 1.14764 -.987 .251 .714 .947 Mutual respect and approval prevailed in the supervisory relationship 4.0597 1.11053 -1.098 .456 .681 .947 The supervisory relationship was characterized by a sense of trust 4.0072 1.14320 -1.001 .173 .703 .947 Role of nurse teacher, 9 items, Cronbach’s alpha 0.937 In my opinion, the NT was capable of integrating theoretical knowledge and everyday practice of nursing 3.8366 1.18799 -.834 -.179 .607 .948 The NT was capable of operational sing the learning goals of this placement 3.8293 1.18870 -.855 -.107 .601 .948 The NT helped me to reduce the theory-practice gap 3.6800 1.22331 -.704 -.389 .543 .948 The NT was like a member of the nursing team 3.4181 1.27918 -.417 -.804 .501 .949 The NT was able to give his or her expertise to the clinical team 3.5730 1.19551 -.561 -.455 .550 .948 The NT and the clinical team worked in supporting my learning 3.4900 1.22629 -.442 -.718 .573 .948 The common meetings between myself mentor and NT were comfortable experience 3.8491 1.15296 -.794 -.174 .594 .948 In our common meetings I felt that we are colleagues 3.8604 1.14160 -.776 -.232 .632 .947 Focus on meetings was in my learning needs 3.5643 1.25568 -.553 -.699 .552 .948 3.4 Content Validity and Semantic Equivalence The Greek Version of the CLES-GR was translated and back-translated following a specific step procedure (Papastavrou et al., 2010) after obtaining consent from the authors. Although the content validity of the Greek version of the CLES-GR has been established, the questions were reviewed by five experts as the questionnaire was modified by the designers and re-named to include the nurse teacher. The expert panel agreed that the CLES+T-GR reflected the situation in the clinical practice environment, i.e., that the items were suitable and relevant to be tested on Cypriot students and it was of acceptable face validity. 3.5 Construct Validity Construct validity was evaluated using exploratory factor analysis (EFA) with Varimax rotation. Principal components analysis (PCA) was implemented as the extraction method in EFA. The procedure used is similar to the psychometric studies published for the CLES+T (Saarikoski et al., 2008; Johansson et al., 2010; Henriksen et al., 2012; Bergjan & Hertel, 2013; Vizcaya-Moreno et al., 2015). First, the assumptions regarding the suitability 63 www.ccsenet.org/gjhs Global Journal of Health Science Vol. 8, No. 5; 2016 of the data for factor analysis were examined, including the sample size, Kaiser–Mayer–Olkin (KMO) measure of sampling adequacy and Bartlett’s test of sphericity. The sample size was satisfactory, considering the rule-of-thumb for determining a priori sample size to be a “participant- to- item” ratio of 10:1 (Costello & Osborne, 2005). The current study, included 34 items and 463 participants, therefore this ratio was satisfied. The data were found to be appropriate for factor analysis, since the KMO measure was equal to 0.931, larger than 0.5, indicating high sampling adequacy, whereas Bartlett’s test of sphericity was significant (p<0.001), thus rejecting the null hypothesis of an identity correlation matrix (Field, 2009). The criteria for factor selection included eigenvalues being higher than 1, as well as the percentage of variance explained by the factors, where each factor was expected to explain at least 5% of the variance to be included (Polit & Beck, 2011; Field, 2009). In the current study, the five-factor structure of the model that was obtained from EFA was identical to the original scale, where all the items loaded on each component agreed completely with the five sub-dimensions of the scale in Saariskoski et al. (2008). The five components had eigenvalues larger than one and explained a total of 67.4% of the variance. The most important factor for the learning environment of Cypriot nurses, which had the highest eigenvalue and the largest percentage of variance explained, was “supervisory relationship”, with an original eigenvalue of 13.1 (6.8 after Varimax rotation) and an explanation of around 38% of the variance (or 20% after rotation). The factor loadings in this component ranged from 0.78 up to 0.88. The loadings in all the other components were similarly very high and thus satisfactory, in that all were higher than 0.5. All the results (factor loadings of each item, eigenvalues and % of variance explained) appear in the Table 2 below: Table 2. Factor loadings Items on factor Supervisory relationship (factor 1) My supervisor showed a positive attitude towards supervision .844 I felt that I received individual supervision .783 I continuously received feedback from supervisor .826 Overall I am satisfied with the supervision I received .864 The supervision was based on a relationship of equality and promoted my learning .866 There was a mutual interaction in the supervisory relationship .864 Mutual respect and approval prevailed in the supervisory relationship .880 The supervisory relationship was characterized by a sense of trust .833 Role of nurse teacher (factor 2) The NT was capable of integrating theoretical knowledge and everyday practice of nursing .812 The NT was capable of operational sing the learning goals of this placement .834 64 Pedagogical atmosphere (factor 3) Premises of nursing in the ward (factor 4) Leadership style of the Ward manager (factor 5) www.ccsenet.org/gjhs Global Journal of Health Science The NT helped me to reduce the theory-practice cap .805 The NT was like a member of the nursing team .757 The NT was able to give his or her expertise to the clinical team .824 The NT and the clinical team worked in supporting my learning .820 The common meetings between myself mentor and NT were comfortable experience .695 In our common meetings I felt that we are colleagues .698 Focus on meetings was in my learning needs .690 The staff was easy to approach .727 I felt comfortable going to the ward at the start of my shift .659 During staff meetings(e.g. before shifts) I felt comfortable taking part in the discussions .725 There was a positive atmosphere on the ward .755 The staffs were generally interested in student supervision .693 The staff learned to know the students by their personal names .609 There were sufficient meaningful learning situations on the ward .524 The learning situations were multi-dimensional in terms of content .516 The ward can be regarded as a good learning environment .508 Vol. 8, No. 5; 2016 The ward nursing philosophy was clearly defined .640 Patients received individual nursing care .741 There were no problem in the information flow related to patients’ care .691 Documentation of nursing ( e.g. nursing plans, daily recording of nursing procedures etc.) was clear .684 .685 The WM regarded the staff on his/her ward as a key resource person 65 www.ccsenet.org/gjhs Global Journal of Health Science Vol. 8, No. 5; 2016 The WM was a team member .843 Feedback from the WM could easy be consider a learning situation .794 The effort on individual employee was appreciated .616 Eigenvalues 13.065 3.750 3.179 1.654 1.270 % of variance explained (total=67.405%) 38.427 11.029 9.349 4.864 3.736 Eigenvalues (after rotation) 6.817 5.990 4.435 2.989 2.687 % of variance explained (total=67.405%) (after rotation) 20.049 17.618 13.045 8.791 7.902 It should be noted that although item analysis did not show any significant deviations from normality, the principal axis factoring was implemented in EFA since it is a method without any distributional assumptions (e.g., Saarikoski et al., 2008; Johansson et al., 2010). However, the results from the two methods (principal components analysis and principal axis factoring) were identical in terms of the factor structure and grouping of items, although with slightly altered factor loadings, and therefore the results from the principal axis factoring are not reported. In addition, it should be mentioned that, similar to Saarikoski et al. (2008), different models also were compared, namely with four and six factors. The 4-factor results were similar to the results reported by Saarikoski et al. (2008) where three dimensions were identical to the 5-factor solution and the fourth combined ward management and nursing care together, but in addition to the concern that one factor included too many items, the 4-factor model also explained a lower percentage of variance (63.7%). Finally, the 6-factor model, which was originally obtained, included an extra factor which included items 6-9 from the list of pedagogical atmosphere items, but the first two items (6 and 7), loaded similarly on the Pedagogical Atmosphere factor, with high loadings on both factors, therefore only the last three items (7-9), loaded more significantly on the sixth factor. Including this extra factor, with only these three items did not make sense intuitively. Therefore, the five-factor solution was the preferred and most appropriate model structure overall. 3.5 Convergent Validity Convergent validity was examined by measuring the bivariate correlations between the scale/subscales and the question that measured the general satisfaction of nurses (replies to which optionally ranged from “fully dissatisfied” to “fully satisfied”). Spearman’s rho correlation coefficient was significant with p<0.001 between the overall satisfaction and all the scales, showing that the scale and subscales have convergent validity and indicating that all the subscales are important and need to be included in the scale. These results are presented in Table 3. Table 3. Bivariate Correlations between the item on “total satisfaction” with all the scales/subscales. Total satisfaction Scale Pedagogical Atmosphere Ward Management Nursing care Supervisory relationship Role of the Nurse Teacher Spearman’s Correlation coefficient 0.610** 0.521** 0.388** 0.385** 0.550** 0.432** p-value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 ** Correlation is significant at α=0.01. 3.6 Correlations Between the Subscales Pearson correlation coefficient was used to examine the relation between the subscales. All the scales were highly significantly positively related, with p-values<0.001. These correlations are presented in Table 4. 66 www.ccsenet.org/gjhs Global Journal of Health Science Vol. 8, No. 5; 2016 Table 4. Correlations between the CLES-T subscales Pedagogical Ward Scale Nursing Atmosphere Management care Supervisory Role of the relationship Nurse Teacher PearsonCorrelation 0.794** 0.685** 0.708** 0.791** 0.748** p-value p<0.001 p<0.001 p<0.001 p<0.001 p<0.001 PedagogicalAtmosph PearsonCorrelation ere p-value WardManagement Nursing care Supervisory relationship 0.554 ** p<0.001 0.577 ** p<0.001 ** PearsonCorrelation 0.537 p-value p<0.001 0.472 ** p<0.001 0.431 ** p<0.001 ** PearsonCorrelation 0.459 p-value p<0.001 0.402** p<0.001 0.381** p<0.001 0.378** p<0.001 PearsonCorrelation 0.460** p-value p<0.001 ** Correlation is significant at the 0.001 level. 4. Discussion Examination of the psychometric properties of the CLES+T-GR demonstrated the internal consistency of the total scale, and its subscales as well, which was similar to results in other languages (Bergjan & Hertel, 2013; Henriksen et al., 2012; Johansson et al., 2010; Saarikoski et al., 2008; Vizcaya-Moreno et al., 2015). In the present study, three validation processes were applied: content, construct and convergent validity, indicating the degree to which scores measure what they claim to measure. The results have shown that CLES+T-GR is a multidimensional instrument which consists of five factors, and is very similar to the first scale CLES-GR used without the “teacher-T” section (Papastavrou et al., 2010). In that study, the construct validity of the CLES-GR was examined using a sample of 350 students with an exploratory factor analysis. The total percentage of variance that the factor model explained was high (67%) and the questions loaded on the same factors as the factors in the original questionnaire. The reliability of the CLES-GR was satisfactory (Cronbach’s alpha =0.95) and the alpha values of the sub-dimensions ranged from 0.79 to 0.95. The CLES+T is an extension of the CLES-GR that gives more possibilities to evaluate the new role dimensions of the nurse teacher in the clinical area. As nursing education has moved to the higher education systems so the CLES+T is thus more suited to the Cyprus Nursing educational system currently relied upon. The factor model with a total explanation percentage of 67.4% of the variance found in our study was comparable with factor models of other studies in Europe, namely Germany 73% (Bergjan & Hertel, 2013) Spain 66.4% (Vizcaya-Moreno et al., 2015). Italy 67% (Tomietto et al., 2012) and Finland 64% (Saarikoski et al., 2008). The strongest factor was found in the subcategory “supervisory relationship” with high loadings ranging from 0.833 to 0.844, followed by the “role of the nurse teacher” factors with loadings ranging from 0.690 to 0.834. It is interesting that “supervisory relationship” has been identified as the strongest factor in most of the validated versions of the CLES+T (Saarikoski et al., 2008; Johansson et al., 2010; Henriksen et al., 2012; Bergjan & Hertel, 2013; Vizcaya-Moreno et al., 2015). Only in the Italian version (Tomietto et al., 2012) the strongest factor was the “pedagogical atmosphere” and “supervisory relationship” was the forth in sequence. Another interesting finding of our study is that “role of the nurse teacher” is the second strongest factor and as such is similar to the Norwegian sample (Henriksen et al., 2012). However, loadings were weaker than the Cypriot sample, ranging from 0.498 to 0.816, and also the question related to the nurse teacher cooperation loaded in another factor, that of the supervisory relationship. The least important factor of the clinical learning environment in our study was the “leadership style of the ward manager”, which in combination with the high importance given to the “role of the nurse teacher” in our study may be considered as shifting all of the learning responsibility to the nurse teacher, in turn minimizing the role of the ward manager. Initially the ward manager was recognized as a key person in the establishment of the ward as a good learning environment for student nurses in 1980’s (Orton, 1983), although subsequent studies found that the pedagogical activities of the clinical nursing staff are more important in the supervision of students (Mattila et al., 2010). Empirical evidence also gives more emphasis to models of “one to one” supervision rather than the traditional model of group 67 www.ccsenet.org/gjhs Global Journal of Health Science Vol. 8, No. 5; 2016 supervision, and likewise to the role of the staff nurse in the clinical supervision process (Saarikoski et al., 2007). The strong support given to the role of the nurse teacher in our study also gives the message that students regard their teacher as more important in the process of learning and the person who will fulfill their expectations to reduce the theory-practice gap. As mentioned, the CLES+T is validated in seven European countries and languages and despite the differences in examining the psychometric differences, the results show that in general almost all the items of the scale loaded on the same factor, although the robustness of each factor in each country varies. This is understandable in terms of the differences and complexities of health care and hospital organization systems that formulate the practice environment and influence student learning in each country. Also, although the cultural and social contexts of the learning environments in the countries mentioned is so varied, the advantage of CLES+T lies in the opportunity to use an instrument that measures all aspects of these diverse environments in a consistent way. This means that CLES+T is a strong, valid and reliable instrument that can be safely administered to all nurse students to evaluate their perceptions of various clinical settings as learning environments. Bachelor degrees in nurse education in Europe are characterized by different structures, standards and approaches to the relationship between theoretical and practice-based learning (Salminen et al., 2010). Hence, systematically processed empirical data collected with valid, reliable instruments are needed urgently for national and international comparisons, and to increase the pressures on policy makers. 4.1 Limitations The limitations of the study include the issue of different study settings in that students were practicing in five different hospitals and many dissimilar wards. This means that the results may be different if analyzed at a hospital or unit level rather than nationally. Also, the comparisons made with validation studies from other countries need to be viewed with caution, as those studies did not implement any statistical comparative analyses of the factor loadings and so it is possible that certain results occurred by chance Finally, it would be interesting to employ other statistical methods of validation, as a confirmatory factor analysis. 5. Conclusions The Greek version of the CLES+T has been shown to be reliable and valid. The CLES+T-GR evaluates the learning context of the clinical practice environment taking into consideration the multidimensional nature of the clinical area which involves aspects of the ward such as the pedagogical atmosphere, nursing care and ward management as well as supervision aspects and inter-staff relationships. The role of the teacher is central to the concept of the clinical learning environment, especially in integrating theoretical knowledge and everyday practice of nursing. CLES+T-GR enables researchers to use a methodological tool to assess the process of learning in applied science, such as nursing, that relies heavily on practice. Additionally, it facilitates the design of more effective, targeted interventions for improving the clinical learning environment. The results further suggest that there is still much to be done to promote improvements in the clinical area that will facilitate student learning and further research is needed into this area in the Greek context. Acknowledgments The authors would like to thank Mikko Saarikoski for the permission given for using CLES+T, the nursing students that participated in the study and Mr Gerry Barrett for the language editing. Author Contributions Study Design: EP, MD Data Collection and Analysis: MD, EP, HT Manuscript Writing: EP, MD, HT Funding The research was granted by Technological University of Cyprus. Conflict of Interest The authors declare that there is no conflict of interests regarding the publication of this paper. References Bergjan, M., & Hertel, F. (2013). Evaluating students' perception of their clinical placements - testing the clinical learning environment and supervision and nurse teacher scale (CLES + T scale) in Germany. Nurse Educ. 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SciPost Physics Submission Dual applications of Chebyshev polynomials method: Efficiently finding thousands of central eigenvalues for many-spin systems Haoyu Guan1 , Wenxian Zhang1* , 1 School of Physics and Technology, Wuhan University, Wuhan, Hubei 430072, China * wxzhang@whu.edu.cn June 28, 2021 Abstract Computation of a large group of interior eigenvalues at the middle spectrum is an important problem for quantum many-body systems, where the level statistics provide characteristic signatures of quantum chaos. We propose an exact numerical method, dual applications of Chebyshev polynomials (DACP), to simultaneously find thousands of central eigenvalues, which are exponentially close to each other in terms of the system size. To cope with the neardegenerate problem, we employ twice the Chebyshev polynomials, to construct an exponential semicircle filter as a preconditioning step and to generate a large set of proper basis states in the desired subspace. Numerical experiments on Ising spin chain and spin glass shards confirm the correctness and efficiency of DACP. As numerical results demonstrate, DACP is 30 times faster than the state-of-the-art shift-invert method for the Ising spin chain while 8 times faster for the spin glass shards. In contrast to the shift-invert method, the computation time of DACP is only weakly influenced by the required number of eigenvalues, which renders it a powerful tool for large scale eigenvalues computations. Moreover, the consumed memory also remains a small constant (5.6 GB) for spin-1/2 systems consisting of up to 20 spins, making it desirable for parallel computing. Contents 1 Introduction 2 2 Dual applications of Chebyshev polynomials method 2.1 Exp-semicircle filter 2.2 Chebyshev evolution 2.3 Subspace diagonalization 3 4 6 6 3 Numerical results 7 4 Conclusion 12 A Chebyshev polynomials 13 B Detailed deductions of Eq. (9) 14 1 SciPost Physics Submission C Evaluation of matrix elements and solution of the generalized eigenvalue problem 15 D Comparison with a hybrid method 16 References 17 1 Introduction Energy level statistics provides an essential characterization of quantum chaos [1, 2]. Integrable systems often imply level clustering and a Poisson distribution of energy level spacings [2], while chaotic systems exhibit strong level repulsion and a Wigner-Dyson distribution [3]. Other useful statistical tools like the δ3 statistic [4] and the power spectrum of the δn statistic also depend on the level distribution [5]. Numerical observation of eigenvalues is important to exactly characterize these level statistics, because direct theoretical derivation is not available. In addition, the need to access individual eigenstates at the middle of the spectrum, which correspond to the “infinite temperature” limit, is emphasized in studying the phenomenon of many-body localization (MBL) [6–8]. Numerical simulations remain instrumental in understanding quantitatively many aspects of the MBL problem. As the many-body problems of interest often involve a huge Hilbert space dimension growing exponentially in terms of system size, it is rather challenging to conduct a full diagonalization of the Hamiltonian or to solve the time-independent Schrödinger equation. Seeking to resolve the eigenvalue problem in a small part of the spectrum is thus an unavoidable and desirable substitution. To obtain the interior eigenstates, a hybrid strategy of matrix spectroscopy is often invoked [9–11]. It aims at computing eigenstates in selected regions of the spectrum and couples the ground state solvers with a spectral filter, where the filter is designed to cast the selected interior regions to the edges of the spectrum. Among these filters, the Green function (H − λI)−1 is an excellent one. After applying the Green function filter, the cluster of eigenvalues near the test energy λ is mapped to very large positive and negative values. The level spacings near λ are greatly amplified, which thus improves the convergence. The Lanczos method [12] has been coupled with such a filter [9, 10], and the Chebyshev polynomial expansion of the Green function is implemented [13]. In particular, the shift-invert method [14] essentially utilizes this spectral transformation and has been widely used in quantum many-spin systems [8, 15–18]. Moreover, it was even proposed to be the most efficient one for the MBL problem [8]. However, for large systems this method suffers rapid increases in both computation time and memory consumption, due to the factorization of (H − λI)−1 . Other spectral filters have been also proposed, including the Dirac delta function filter δ(H−λI) [19–22] and the rectangular window function filter [23], both of which are expanded by the Chebyshev polynomial. Note that all the methods mentioned above are iterative ones, where the computation time is usually proportional to the required number of eigenvalues, i.e., more eigenvalues means more filtrations and reorthogonalizations [8, 20, 21]. In this paper, we propose an exact numerical method, DACP, to calculate thousands of eigenvalues at the middle of the energy band, which is already helpful to reveal the level statistics [24,25]. For spin systems, such a region usually locates at the peak of the density of states where the energy levels are so close that they are nearly degenerate. It is extremely 2 SciPost Physics Submission challenging to distinguish these near-degenerate eigenvalues without factorizing H−1 . In the DACP method, we construct an exponential of semicircle (exp-semicircle) filter to quickly damp the unwanted part of the spectrum by employing the Chebyshev polynomial for the first time. The second application of the Chebyshev polynomial is to fast search a set of states to span the specific subspace consisting of all the eigenstates corresponding to the desired eigenvalues. The DACP method transforms the original high dimensional eigenvalue problem to a low dimensional one. For practical problems in many-spin systems, the DACP method is very efficient, due to its full exploration of several excellent properties of the Chebyshev polynomial while other methods usually utilize only one of them. For a large class of many-spin systems, the DACP method exhibits a significant increase in the computation speed, up to a factor of 30, in comparison with the shift-invert method. The memory saving is more drastic, up to a factor of 100. Moreover, the DACP method distinguishes itself from those iterative filtering ones, as its convergence time varies only slightly when the required number of eigenvalues is vastly changed. The paper is organized as follows. The detailed formalism of the DACP method, including the exp-semicircle filtration, Chebyshev evolution, and subspace diagonalization, are described in Section 2. Each of the processes relies on a particular property of the Chebyshev polynomial, distinguishing DACP from other filtering methods . In Section 3 we discuss details of two many-spin systems, the Ising model and the spin glass shards, and present the computational results. We conclude in Section 4. 2 Dual applications of Chebyshev polynomials method To access the central eigenvalues of large spin systems, naturally we are restricted by the matrix-free mode, i.e., the matrix of the Hamiltonian must not be explicitly expressed/stored. Instead, we directly treat the Hamiltonian H as an operator whose input and output are states/vectors. Therefore, we shall only operate with the quantum states: |ψ⟩ , H |ψ⟩ , H2 |ψ⟩ , · · · , Hk |ψ⟩ , (1) where k is a positive integer, in the original Hilbert space of dimension up to 5 × 105 . Note that each “matrix-vector product” H |ψ⟩, being a basic operation, consumes considerable time while only a small amount of memory. Moreover, as illustrated in Subsection 2.3, even the states in Eq. (1) are not necessarily required to be stored. Therefore, the requirement of memory in DACP is pretty tiny. In general, we hope to simultaneously find a large scale of central eigenvalues with a minimum k. In this manuscript, we set our goal to find 5, 000 eigenvalues accurately at the middle of the spectrum for the many-spin systems as an illustration. The idea of DACP is fairly straightforward. We first transform a randomly initialized state into a wave packet in the subspace spanned essentially by at least 5, 000 central eigenstates. This transformation is realized by an exp-semicircle filter implemented by the Chebyshev polynomial. Here the Chebyshev polynomial is used to perform an exponential decay. With this particular state in hand, we then generate a large amount of linearly independent states, as large as possible, to approximately span the subspace consisting of the required eigenstates. The Chebyshev polynomial is used again to oscillate (complex exponential) the states, producing approximately linearly independent states. Once the generating set for the desired eigenstates is known, one may explicitly calculate H, the reduced representation of H in this subspace. The remaining operations are then restricted in the subspace of dimension around 104 . Finally, direct diagonalization of H, which is of size 104 × 104 , gives the desired eigenvalues. The detailed procedures and discussions are 3 SciPost Physics Submission 20 10 15 k T (F(x)) 10 10 10 5 10 0 10 −0.1 −0.05 0 0.05 0.1 x Figure 1: The exp-semicircle filters for a = 0.1 (red lines), 0.05 (blue lines), and 0.025 (black lines), with ka = 24. We have set Emax = 1 and Emin −1. The solid lines  = 2 are for the Chebyshev polynomials Tk (F (x)), where F (x) = 2x − (1 + a2 ) /(1 − a2 ), √ while the dashed lines are for the approximations y = exp(2k a2 − x2 ) ≃ Tk (F (x)). The horizontal pink dotted line denotes the half maximum of the logarithm of filters. Note that |Tk (F (x))| ≤ 1 when x ∈ / [−a, a], thus the filter graphs are restricted in [−a, a]. given in the subsections below. 2.1 Exp-semicircle filter We utilize the exponential growth of the Chebyshev polynomials outside the interval [−1, 1] to efficiently construct an exp-semicircle filter, as shown in Figure 1. The filter drastically amplifies the components of a desired range of eigenstates for any randomly initialized states, resulting a new state that sharply localized at the midlle of the spectrum. We note that the Chebyshev filter explores the same property as well, except that it amplifies the lower end of the spectrum [26, 27]. A similar idea has been also applied in the quantum algorithm for finding ground states [28]. For the Hamiltonian H with energy bounded in [Emin , Emax ], where Emin is the minimum energy (Emin < 0) and Emax the maximum energy (Emax > 0), the exp-semicircle filter is designed to amplify the components of the eigenstates corresponding to eigenvalues in the interval [−a, a] and to simultaneously dampen those in the interval [Emin , −a] and [a, Emax ], where a is a real positive parameter. Focusing on the spin systems, we have assumed Emin < −a and a < Emax . After the filtration, a new state mainly consists of the eigenstates with eigenvalues belonging to the interval [−a, a] is generated. For simplicity, let us denote the subspace spanned by the eigenstates contained in [−a, a] as L. Consequently, the ratio of eigenstates outside L is almost negligible, as can be seen in Figure 1. We now introduce the specific implementation details. Note that we want to amplify the middle of the spectrum, but the exponential growth of the Chebyshev polynomial exists only in both ends. To satisfy this goal, we first square the Hamiltonian, obtaining 2 ] (suppose E H2 with a spectrum ranges [0, Emax min = −Emax for simplicity). The middle spectrum [−a, a] of H is transferred to [0, a2 ] for H2 , which lies exactly at the lower end. 4 SciPost Physics Submission 2 ] into [−1, 1] by shift and normalization of Next is to map the dampening part [a2 , Emax H2 . We thus define an operator H2 − Ec F= , (2) E0 2 2 where Ec = (Emax + a2 )/2 and E0 = (Emax − a2 )/2. One may easily affirm this map’s 2 , and correspondingly one has F (x) = correctness by replacing H2 with either a2 or Emax 2 (x − Ec )/E0 . Note that F is simply a polynomial expression of H, so is Tk (F). We then explore the effect of the filtration using Tk (F). As the eigenvalues inside [0, a2 ] 2 of H2 is mapped into [−1 − 2a2 /(Emax − a2 ), −1] of F, we have Tk (F) = (−1)k cosh(kΘ) for the lower end of the spectrum, where   2(a2 − H2 ) −1 Θ = cosh 1+ 2 . (3) Emax − a2 P P Let |ψ⟩ = i ci |ϕi ⟩ + j dj |χj ⟩ be a random initial state, with ci and dj the random coefficients, |ϕi ⟩ the eigenstates inside L, |χj ⟩ the eigenstates outside L. The filtration by Tk (F) is |ψ (k)⟩ = Tk (F) |ψ⟩ c d X  in X  ikθout out in j i = ekθi + e−kθi |ϕi ⟩ + e j + e−ikθj |χj ⟩ 2 2 i j 1 X kθin ≃ e i ci |ϕi ⟩, 2 (4) i 2 2 where θiin = cosh−1 (1 + 2(a2 − Ei2 )/(Emax − a2 )), θjout = cos−1 (2(Ej2 − a2 )/(Emax − a2 ) − 1), Ei and Ej are eigenvalues corresponding to |ϕi ⟩ and |χj ⟩, respectively. In writing Eq. (4) we have ignored (−1)k , as it does not affect the absolute value of coefficients and is a 2 , one may further deduce global phase at the end line. When a is tiny, i.e., a2 ≪ Emax q θiin ≃ 2 a2 − Ei2 /Emax via Taylor’s expansion of cosh−1 (1 + ε), where ε is a small positive number. We thus obtain the exp-semicircle filter 2k Tk (F) ≃ e Emax √ a2 −H2 (5) that peaked sharply at Ei = 0 with a large k, for eigenstates satisfying −a ≤ Ei ≤ a. In Figure 1 the shape of Eq. (5) is presented in dashed lines, which fits pretty well with the exact form. With the initial conditions T0 (F) = 1 and T1 (F) = F, the kth order Chebyshev polynomial can be efficiently determined using the recurrence relation Eq. (17). In this paper, we set the cut-off order K = 18Emax /a. Such a filter exponentially (the fastest rate among all polynomials) amplifies the components of eigenstates inside L [29]. After the normalization, it equivalently dampens those outside L, generating the target state: X |ψE ⟩ ≃ c′i |ϕi ⟩, (6) i where c′i = βeKθi ci and β is the normalization constant. Obviously, the state |ψE ⟩ localizes (in the energy representation) at the middle of the spectrum. We input |ψE ⟩ as the initial state for Subsection 2.2, Chebyshev evolution. in 5 SciPost Physics 2.2 Submission Chebyshev evolution After obtaining a state confined in the small subspace L, we then make use of the oscillation property of the Chebyshev polynomial to efficiently generate a set of distinct states, as many as possible, serving as a complete basis to span L. To achieve this goal, it is necessary to limit Ei ∈ [−1, 1] (corresponding to x in Tk (x)), within which the Chebyshev polynomial behaves as a cosine-like function. This region contrasts with the requirement of the exp-semicircle filter, thus one needs a different transformation of H. Below we describe the specific details for the second application of the Chebyshev polynomial. The original Hamiltonian H needs to be shifted by Ec′ and be rescaled by E0′ , where Ec′ = 12 (Emin + Emax ) and E0′ = 12 (−Emin + Emax ). In a similar way to Eq. (2), we define an operator G = (H − Ec′ )/E0′ , which is definitely bounded by −1 and 1. Assuming Emin = −Emax again, which does not affect the conclusion, we obtain G= H . Emax (7) At the same time, the parameter a is rescaled to ar = a/Emax as well. Let us explore the Chebyshev evolution, i.e., the evolution governed by the operator Tk (G) as k plays the role of time. We input the state Eq. (6) generated by the filtration as an initial state for the Chebyshev evolution. Since ∥G∥ ≤ 1, from Eq. (16) we have Tk (G) = cos(kΩ), where Ω = arccos(G). In this sense, the Chebyshev evolution gives |ψE (k)⟩ = Tk (G) |ψE ⟩  1 X  ikωj + e−ikωj c′j |ϕj ⟩ , e = 2 (8) j where ωj = arccos(Ej /Emax ). Note that both ωj and k are unitless. Certainly, this is not a physical evolution, and Tk (G) is not even a unitary operator. Actually, this evolution essentially represents a superposition of both forward and backward propagation. Each time the polynomial order k increases by 1, the evolution “time” is added by 1 as well. With the aid of the Chebyshev evolution, we are able to construct a complete basis that spans the subspace L. In detail, we collect a set of states as follows n o ˆ sin(X̂), · · · , sin(nX̂), cos(X̂), · · · , cos(nX̂) |ψE ⟩ , I, (9) where X̂ =πG/ar and n is an integer determined by the relation 2n + 1 ≥ d, with d the dimension of L. In practice, one may need a relation 2n + 1 = 1.5d to ensure the completeness of Eq. (9). Here k ≃ mπ/ar serves as the time, with m = 1, · · · , n. Similar to Subsection 2.1, the kth order Chebyshev polynomial Tk (G) can be calculated. The cut-off order (evolution time) K ′ = ⌊nπ/ar ⌋. More details can be found in Appendix B. The duality of the Chebyshev polynomials, as being approximately the trigonometry functions and being the polynomials, plays a vital role in the DACP method. In a certain sense, the Chebyshev evolution is an efficient (possibly the most one among all the polynomials) simulation of the quantum oscillations [30]. 2.3 Subspace diagonalization Computing the basis {|Ψi ⟩ : i = 1, · · · , 2n + 1} (Eq. (9)) by combination of the exp-semicircle filter and the Chebyshev evolution represents the most challenging aspect as well as the most time-consuming part of the DACP method. Once the appropriate basis has been constructed, the task remained is straightforward, i.e., to compute the eigenpairs of the 6 SciPost Physics Submission projected Hamiltonian H. This is equivalent to solving the generalized eigenvalue problem (10) HB = SBΛ. Here, H and S denote the projected Hamiltonian in L and the overlap matrices, respectively, Hij = ⟨Ψi | H |Ψj ⟩ , Sij = ⟨Ψi |Ψj ⟩ . (11) Λ is a diagonal matrix with the eigenvalues in [−a, a] and the matrix B transforms the found basis Eq. (9) to the eigenstates |ϕj ⟩ of H, |ϕj ⟩ = 2n+1 X Bij |Ψi ⟩ . (12) i=1 All these matrices are of size (2n + 1) × (2n + 1). Because of the small size of it, H can be explicitly expressed and stored in core memory. The necessary procedures are conveniently available in the LAPACK library [31]. Importantly, due to the special property of the Chebyshev polynomial, the computation of matrices H and S can even be achieved without an explicit computation and storage of the states |Ψi ⟩. This feature gives rise to a further improvement for the DACP method, both in computation time and memory saving. Besides, for an overcomplete basis Eq. (9), the overlap matrix S is generally singular. We thus employ the singular value decomposition (SVD) to solve it. After the SVD, the eigenvalues of S are discarded if their absolute values sit below the cutoff condition ε = 10−12 . The number of remained eigenvalues effectively counts the linearly independent states in Eq. (9). We present both the explicit expressions (denoted by Tk (G) and |ψE ⟩ only) of matrices H and S, and the solution of the generalized eigenvalue problem in Appendix C. Eigenvalues obtained by the subspace diagonalization may not own the same accuracy when compared to true eigenvalues of the system, thus it is necessary to conduct an independent check to estimate the error bounds. To this end, ifqthe eigenstates |ϕj ⟩ were known, the residual norm (variance of the energy) ||rj || = ⟨H2 ⟩ − ⟨H⟩2 , where H2 = ⟨ϕj | H2 |ϕj ⟩ and ⟨H⟩ = ⟨ϕj | H |ϕj ⟩, is widely used as the parameter measuring the accuracy of the results. It has been shown that ||rj || gives an upper bound on the true error (absolute error) of the computed eigenvalue [32]. 3 Numerical results We apply the DACP method to the quantum spin-1/2 systems with two-body interactions. Such systems are good models for investigating a large class of important problems in quantum computing, solid state theory, and quantum statistics [33–36]. A large amount of exact eigenvalues helps us to obtain the statistical properties, to distinguish quantum chaos from integrability, and serves as a benchmark to evaluate other approximate methods as well. Generally speaking, the DACP method can deal with the spin systems consisting of couplings between any pair of the N spins. Each of the Pauli matrix σ α or the two coupling Pauli matrices σ α ⊗ σ β , where α, β = x, y, z, is properly represented by a specific function. We specify the spin model for two physical systems. One is the disordered onedimensional transverse field Ising model [37], where the Hamiltonian is N −1 N 1X 1X z z x x H= Ji,i+1 σi σi+1 + Γi σi , 4 2 i=1 i=1 7 (13) SciPost Physics Submission with σi the Pauli matrices for the spin i. This system is exactly solvable by Jordan-Wigner transformation [35], making it an ideal correctness checker for the DACP method. The nearest neighbor exchange interaction constants Ji,i+1 are random numbers that uniformly √ √ distributed in [−J/ N , J/ N ] with J = 10. The local random magnetic fields are represented by Γzi , which are random numbers that uniformly distributed in the interval [0, Γ] with Γ = 1. Another system is the spin glass shards [25], which represents a class of global-range interacting systems that require relatively large bond dimensions to be tackled by the DMRG methods [38]. The Hamiltonian describing the system is X X H= Jij σix σjx + Γzi σiz . (14) i<j i All symbols and parameters are the same as that of the above Ising model, except that the first summation runs over all possible spin pairs. This system is interesting because it presents two crossovers from integrability to quantum chaos and back to integrability again. In the limit J/Γ → 0, the ground state is paramagnetic with all spins in the local field direction and the system is integrable [25]. In the opposite limit J/Γ → ∞, the ground state is spin glass and the system is also integrable since there are N operators (σix ) commuting with the Hamiltonian. A quantum chaos region exists between these two limits. J = 10 Γ is approximately the border from the quantum chaos to the integrable (the spin glass side) when N = 20 [25]. By employing the upper-bound-estimator, which costs little extra computation and bounds up the largest absolute eigenvalue E0 , one may estimate Emax = E0 and Emin = −E0 [39]. For this setting we have utilized the symmetry of the density of states (DOS), a bell-shape profile centered at zero, in the many-spin systems. Since we require 5, 000 central eigenvalues, we may set n = 4, 000, corresponding to a dimension 8, 001 and being adequate to span the whole subspace L. The overlap matrix S is generally singular. The approximate distribution of DOS ρ(E) may be efficiently calculated through the Fourier transformation of a time evolved wave function or through a better estimation method given in [40]. The parameter a is appropriately chosen to ensure that the number of eigenstates contained in [−a, a] is a little less than 8, 000 (as illustrated in Figure 2, the precision of some converged eigenvalues may be lower than required). In practice, sometimes there may exist highly near-degenerate eigenvalues, with level spacings as small as 10−7 Γ while the average spacing is 10−5 Γ. It is still hard (two magnitudes longer time) for the Chebyshev evolution to discriminate such close pair of eigenvalues. To circumvent this challenge, we employ the block filter technique [27], which means a block of states is filtered or evolved “simultaneously”, in programming of the DACP method. The idea is that two or several random states in the degenerate subspace are usually linearly independent. For each numerical test, a block of 5 initial trial states is randomly generated and employed with the parameter n being adjusted to n = 800 accordingly. By these settings, we perform numerical tests on the above two systems to show the high exactness and efficiency of DACP method. For this work, we consider only the eigenvalues computations. All the timing information reported in this manuscript is obtained from calculations on the Intel(R) Xeon(R) CPU E7-8880 v4, using sequential mode. In Figure 2, we present the relative error η in logarithmic scale versus the system energy Eexact , for the Ising model with N = 19 and the spin glass shards with N = 17. We have defined the relative error η of the computed central eigenvalues E as η= E − Eexact . Eexact 8 SciPost Physics Submission 0 −6 −8 log10( η ) log10( η ) −4 −8 −10 −12 −12 −14 −16 −3 (a) −2 −1 0 E (10−2Γ) 1 2 −16 −0.12 3 (b) −0.06 0 E (Γ) 0.06 0.12 Figure 2: The relative error η in logarithmic scale of the calculated eigenvalues, for (a) Ising model with N = 19 and (b) spin glass shards model with N = 17. The horizontal axis is the system energy. The number of eigenvalues satisfying η < 10−6 (black dashed line) is 5, 385 for (a) and 5, 000 for (b). We suppose that such a distribution comes from the shape of filters in Figure 1. Exact eigenvalues of both systems have been obtained by other reliable methods. For the Ising model, we make use of the famous Jordan-Wigner transformation to reduce the original 2N × 2N matrix to a 2N × 2N one, and restore the full spectrum of the original Hamiltonian [35]. For the spin glass shards we simply utilize the function eigs of MATLAB, to find 5, 000 eigenvalues closest to E = 0. As for our numerical tests, the parameter a in Figure 2 is 0.036Γ and 0.16Γ for (a) and (b), respectively. In computing the Ising model, the number of eigenvalues satisfying η < 10−6 is not enough (less than 5, 000) by the settings mentioned above. By adjusting the block size to 10 and the parameter n to 500, we then collect enough eigenvalues. The number of converged eigenvalues, i.e., computed eigenvalues satisfying the condition η < 10−6 is 5, 385 for (a) and 5, 000 (all the exact eigenvalues we have) for (b), while the total number of computed eigenvalues is 6, 232 and 5, 910, respectively. The spike around E = 0 for both figures is due to the smallness of the denominator Eexact . The smallest absolute eigenvalue is about 4.4 × 10−6 Γ for (a) and 2.9 × 10−5 Γ for (b). Besides, there is a flat plateau at the middle of the figures, indicating that for those eigenvalues around E = 0 we encounter the numerical error, i.e., the absolute error reaches the limit of the double precision representation. In Figure 2(a) one may observe other spikes, which does not appear in (b). Each of those spikes corresponds to a cluster of eigenvalues lying extremely close to each other. The integrability of the Ising model implies the level clustering and a Poissonian distribution of energy level spacings. We speculate that the Chebyshev evolution does not function well for closely lied eigenvalues, as they give quite similar phase contributions to the final states. To significantly amplify the phase difference exp(i∆Et), it requires t ≳ 1/∆E, where ∆E is the energy difference. An alternative solution is to increase the block size to match up with the dimension of near-degenerate subspace. Ignoring the central plateau and the spikes, the distribution of η shows an inverted shape of the exp-semicircle filter. In Figure 3, we compare the computation time T (CPU time in seconds) of the DACP method with that of the shift-invert approach. The latter is widely used in computing eigenpairs at the middle of the spectrum for quantum spin systems, to name a few, like in [8,15–17]. In our tests, it is implemented by the eigs function of Matlab R2019b, which employs the implicitly restarted Arnoldi method (ARPACK) [41]. Each of the numerical 9 SciPost Physics Submission 6 log10( T ) 5 4 3 Matlab,glass Matlab,Ising DACP,glass DACP,Ising 2 1 13 14 15 16 17 18 19 N Figure 3: Scaling behavior measured by the computation time T (CPU seconds) in logarithmic scale, versus the system size N , with the DACP method (solid lines with squares) compared to the shift-invert method (dahed lines with circles) for the Ising model (red) and the spin glass shards (blue). Each of the numerical tests, represented either by squares or circles, finds 5, 000 central eigenvalues. All the four lines are obtained via linear fitting. Table 1: Scaling constants α by linear fitting of the four curves in Figure 3. α DACP method Shift-invert method Ising 1.49 1.42 Glass 1.50 1.47 tests finds around 5, 000 eigenvalues for either of the two spin systems. For the DACP method, we have discarded the time consumed during the subspace diagonalization, as it is a constant and does not affect the scaling behavior. Specifically, the subspace diagonalization spends roughly 700 CPU seconds in each test. Such a constant is completely negligible for N ≥ 17 systems. As shown in Figure 3, for spin systems with 13 ≤ N ≤ 17, the DACP method is about 30 times faster than the shift-invert approach for the Ising model and 8 times faster for the spin glass shards. Since the shift-invert method essentially finds the ground energy of H−1 , which is usually not a sparse matrix, its computation time T is not affected by the sparsity of the Hamiltonian and the two dashed lines nearly coincide. On the contrary, the DACP method employs the polynomial combination of H acting on the states, its computation time T is heavily influenced by the number of Pauli operators of the specific Hamiltonian. For example, when N = 18 the computation time T for the spin glass shards is 5.5 times that for the Ising model, while the number of Pauli operators is 6.2 times. Considering this effect, the DACP method is still advantageous over the shift-invert approach in the worst case where the exchange interactions run over all possible spin pairs. Furthermore, the scaling among these two methods is comparable. To compare quantitatively, we extract the scaling constants α of the four lines by fitting the numerical results, where α is defined by T = T0 exp(αN ). The values of α are shown in Table 1. 10 SciPost Physics Submission 1500 6000 Ising, solving time T Ising, evolution time TE CPU time (s) Glass, solving time T Glass, evolution time TE 1000 500 1 4000 2 3 2000 4 log10(Number of eigenvalues) Figure 4: Computation time (CPU seconds) required versus number of eigenvalues satisfying the condition η < 10−6 , for the Ising model (red lines with squares, left axis) and the spin glass shards (blue lines with circles, right axis), using the DACP method. For both systems, N = 15. The solid lines show the solving time T = TF + TE + TD , including computation time for the exp-semicircle filtration TF , Chebyshev evolution TE and subspace diagonalization TD . The dashed lines present TE . Indeed, the four scaling constants are quite close. Suppose that α remains unchanged for a bigger N , then the DACP method’s advantage in time cost may keep on for rather large systems. In addition, as illustrated by the numerical tests of the shift-invert method in [8], the factorization time for finding H−1 dominates other computation steps. Considering the factorization part only, the execution time in [8] exhibits a scaling constant α ≃ 1.66, indicating a worse scaling behavior for systems with large spins. The efficiency of the DACP method is thus confirmed. In Figure 4, we show the computation time versus the number of converged eigenvalues, for the two systems of size N = 15. The horizontal axis represents the number of computed eigenvalues satisfying the condition η(E) < 10−6 in each test. Recall that the DACP method is divided into three parts: the exp-semicircle filtration, Chebyshev evolution, and subspace diagonalization. We denote their time consumption as TF , TE , and TD , respectively, while solving time is the full computation time T = TF + TE + TD . One immediately notice that for the two systems, computation time T decreases on the left side, reaching a plateau at the middle region, while quickly increases on the right side. Yet the dashed lines TE present a quite small increasing rate for the whole region, as the horizontal axis is in logarithmic scale. At the middle of the region, where the DACP method performs the best, the solving time T and the evolution time TE roughly coincide with each other. These features are easily understood if one considers the following four facts. (i) Notice that the DOS ρ(E) is usually a bell-shape profile peaked at zero in spin systems and that a is typically a tiny parameter (a/Emax ∼ 10−2 for N = 19), thus one may safely take ρ(E) as a constant ρ̄ in [−a, a]. Consequently, the number of required eigenvalues is R ≃ 2ρ̄a. (ii) Setting the action of the Hamiltonian on the state, H |ψ⟩, as a basic step, and denoting the computation time of the basic step as τ , we may count the filtration time as τ times twice the cut-off order K = 18Emax /a, i.e., TF ≃ 36Emax τ /a ∝ R−1 . (iii) Similarly, we 11 SciPost Physics Submission find the Chebyshev evolution time is TE ≃ ⌊RπEmax /a⌋τ ∝ R0 since R ∝ a. (iv) It is well known that the full diagonalization time is proportional to the cube of the matrix size, i.e., TD ∝ R3 . The combination of these computation times clearly explains the behavior shown in Figure 4. For the left side, the exp-semicircle filtration dominates, as TF is inversely proportional to the parameter R. Whereas, when it comes to the intermediate region where the number of eigenvalues is several hundreds to thousands, the evolution time TE consists of the majority of the computation time while the other two are negligible. As shown in Figure 4, the performance of DACP method is approximately the same in finding 100 to 3, 000 eigenvalues. As R keeps increasing, the subspace diagonalization time TD eventually consumes the most computation time. We remark that the plateau at the middle region in Figure 4 distinguishes the DACP method from those iterative filtering ones [10, 20, 22, 23, 26, 27], although all of them take use of the Chebyshev polynomials. The iterative methods usually require a larger amount of filtration and reorthogonalization, thus a longer convergence time, in finding more eigenvalues [8,20,21]. In fact, as illustrated in Ref. [8], one finds that the computation time is roughly proportional to the number of eigenvalues required for the shift-invert approach. The DACP method is thus highly desirable for large scale eigenvalues computations. Finally, we note that the memory requirements by DACP method are rather small, as it works in a matrix-vector product mode which avoids an explicit matrix representation of the Hamiltonian. In addition, as shown in Appendix C, the whole set of states in Eq. (9) is not preserved in memory at all. The major memory consumption is the storage of elements Hij and Sij , thus the memory requirements of the DACP method relate only to the dimension d of subspace L. By the settings of this work, it occupies around 5.6 GB of memory for appropriately calculating 5, 000 eigenvalues in N ≤ 20 systems. On the contrary, for the shift-invert method, the matrix size of H−1 grows rather fast as the system size N increases (proportional to 4N ), demanding a large amount of memory. For example, in our tests it consumes 573 GB of memory for N = 17 systems, which is already a factor of 100 more than that of the DACP method. 4 Conclusion We propose the DACP method to efficiently calculate a large scale (at least 5, 000) of central eigenvalues with high precision, by employing twice the Chebyshev polynomials. It explores several excellent properties of Chebyshev polynomial, to efficiently filter out those non-central parts of spectrum and to construct the appropriate basis states for the middle of the spectrum. The proposed method is specified to solve the middle of the spectrum, while its efficiency is improved in return. Two key features distinguish DACP from the polynomial filtering methods: its computation time is almost irrelevant to the number of required eigenvalues and its memory requirements are irrelevant to the system sizes. Moreover, as shown in Appendix D, the DACP method is both more stable and more efficient than the Lanczos method applied with the exp-semicircle filter. The numerical tests for the Ising model and the spin glass shards confirm the exactness and efficiency of the DACP method. Compared to the widely used shift-invert method, the DACP method gives a considerable increase in the speed of computations, for the Ising model up to a factor of 30 while for the spin glass shards the increase in speed is less but still considerable (a factor of 8). Besides, the memory requirements are drastically decreased, up to a factor of 100 for N = 17 spin systems and even more for larger ones. As a powerful tool for central eigenvalues calculations, the DACP method may find potential applications in many physical problems, such as many-body localization in condensed 12 SciPost Physics 1 Submission T3(x) (a) 0 −1 −1 (b) 0 0 1 (d) 20 0 −1 −1 1 T5(x) (c) 0 0 1 −1 −1 0 (e) 80 1 (f) 200 0 40 −200 −20 −2 1 T4(x) 0 2 0 −2 0 x 2 −2 0 2 Figure 5: Chebyshev polynomials Tk (x) for k = 3 (red lines), k = 4 (blue lines), k = 5 (black lines). The first row ((a-c) with solid lines) illustrate the oscillations of Tk (x) inside the interval [−1, 1] while the second row ((d-f) with dashed lines) show the rapid increase outside [−1, 1] of Chebyshev polynomials. matter physics [7, 15, 17, 42], and level statistics in quantum chaos [1, 16, 25, 43]. Acknowledgements We thank X.-H. Deng for discussions. The numerical calculations in this paper have been done on the supercomputing system in the Supercomputing Center of Wuhan University. Funding information This work is supported by the National Natural Science Foundation of China (NSFC) Grant No. 91836101, No. U1930201, and No. 11574239. A Chebyshev polynomials In this paper, we limit ourselves to that only the polynomial combinations of H are the allowed operations. We utilize the Chebyshev polynomials to fulfill the tasks mentioned above. It is the key to bridge the combinations of Hk with an approximately exponential function of H, either exp(kH) or exp(ikH). Due to its several remarkable properties, the Chebyshev polynomials may exhaust the potential of this type of methods. 13 SciPost Physics Submission The kth order Chebyshev polynomial of the first kind is defined by
 −1 |x| ≤ 1  cos k cos (x) ,
−1 cosh k cosh (x) , x>1 , Tk (x) =
 k −1 (−1) cosh k cosh (−x) , x < −1 (15) with initial conditions T0 (x) = 1 and T1 (x) = x [44]. It is a piece-wise function containing two different kinds of expression, while being the polynomial function, it is both continuous and smooth. For simplicity, let us set θ = cos−1 (x) (cos θ = x) when x ∈ [−1, 1] and set θ = cosh−1 (x) when x ∈ [1, ∞), the corresponding range of θ is θ ∈ [0, π] and θ ∈ [0, ∞), respectively. In terms of the variable θ, Eq. (15) becomes  cos (kθ) , |x| ≤ 1 Tk (x) = (16) cosh (kθ) , x > 1. One may easily observe that Tk (x) is a sine or cosine-like oscillation function bounded by −1 and 1 inside the interval [−1, 1], as illustrated in Figure 5 (a-c), while it grows extremely fast outside [−1, 1], as shown in Figure 5 (d-f). Note that cosh(kθ) = (ekθ + e−kθ )/2. It is natural to expect an exponential growth of the Chebyshev polynomial outside the interval [−1, 1]. In fact, it is known that among all polynomials with degree ≤ k, the Chebyshev polynomial Tk (x) grows the fastest outside the interval [−1, 1] under comparable conditions [29]. Associated with those properties is a practically useful one: Tk+1 (x) can be efficiently determined by using the 3-term recurrence Tk+1 (x) = 2xTk (x) − Tk−1 (x). (17) All these properties of the Chebyshev polynomial render it a powerful toolbox and are of great use for the DACP method. B Detailed deductions of Eq. (9) Here we derive explicit expressions showing how to construct the set Eq. (9) via the Chebyshev evolution. We focus on the case that a ≪ Emax , which is fairly reasonable for large (N ≥ 15) spin systems. |ψE (k)⟩ = Tk (G) |ψE ⟩ X = cos(kωj )c′j |ϕj ⟩ j ≃ = X kEj ′ kπ )c |ϕj ⟩ − 2 Emax j j  P Ej n ′   (−1) j cos(k Emax )cj |ϕj ⟩ , k = 2n   (−1)n P sin(k Ej )c′ |ϕ ⟩ , j j Emax j k = 2n + 1. cos( (18) Using the fact that when x is small, arccos(x) = π/2−x+o(x), we have ωj = arccos(Ej /Emax ) ≃ π/2 − Ej /Emax at the third line of Eq. (18). Therefore, we have the expression  (−1)n cos(kG), k = 2n (19) Tk (G) ≃ (−1)n sin(kG), k = 2n + 1. We then conduct a Chebyshev evolution with a cutoff order K ′ = ⌊nπ/ar ⌋, recording both Tk−1 (G) |ψE ⟩ and Tk (G) |ψE ⟩ when k = ⌊mπ/ar ⌋ with m = 1, · · · , n. After the evolution is done, the set of states Eq. (9) is automatically generated. 14 SciPost Physics C Submission Evaluation of matrix elements and solution of the generalized eigenvalue problem As shown in Appendix B, we may rewrite the basis Eq. (9), or {|Ψi ⟩ : i = 1, · · · , 2n + 1}, using the Chebyshev polynomials: {I, Tk1 −1 (G), Tk1 (G) · · · , Tkn −1 (G), Tkn (G)} |ψE ⟩ , (20) where km = ⌊mπ/ar ⌋, m = 1, · · · , n, |Ψ1 ⟩ = |ψE ⟩, |Ψ2 ⟩ = Tk1 −1 (G) |ψE ⟩, |Ψ3 ⟩ = Tk1 (G) |ψE ⟩, etc. For simplicity, one may further assume km is an even number. The elements Sij = ⟨Ψi |Ψj ⟩ = ⟨ψE | Tx (G)Ty (G) |ψE ⟩, where x and y are directly determined by i and j. The correspondence is one to one. By making use of the relation [44] 1 Ti (H)Tj (H) = (Ti+j (H) + T|i−j| (H)), (21) 2 one may even find the matrix elements without recording any states during the Chebyshev evolution. Instead, we simply record the two numbers ⟨ψE | Tk (G) |ψE ⟩ and ⟨ψE | HTk (G) |ψE ⟩ at the appropriate time, i.e., when k = km − 2, k = km − 1, k = km and k = km + 1. Finally, we arrive at the explicit expressions of matrix elements
1 ⟨ψE | Tx+y (G) + T|x−y| (G) |ψE ⟩ , 2
1 Hij = ⟨ψE | H Tx+y (G) + T|x−y| (G) |ψE ⟩ . 2 Sij = (22) (23) Also note that since Tx+y is needed, where both x and y may reach the maximum value kn , the cut-off order K is doubled to K = 2kn in this mode. For the generalized eigenvalue problem Eq. (10), the Hermitian matrix S is first diagonalized as S = V Λs V † , (24) where V is the eigenvector matrix for S, V V † = I, and Λs is the associated eigenvalue matrix. Since S is generally singular, we then contract the (2n + 1) × (2n + 1) matrix V by elimination of the columns associated with eigenvalues with absolute value below a cutoff ε = 10−12 . Denoting the number of retained eigenvalues as m, the contracted eigenvector matrix Ve is of order (2n + 1) × m, and e s Ve † . Se = Ve Λ e Since The next step is to form the contracted Hamiltonian matrix H.  1    −2 † − 21 e e e e e I = Λs V S V Λs , (25) (26) −1 e s 2 , the contracted m×m Hamiltonian matrix denoting the transformation matrix U = Ve Λ is e = U † HU. H (27) e of order m × m with m around 103 to 104 is then diagonalized The Hermitian matrix H directly e = Ye Λ e Ye † , H (28) 15 SciPost Physics Submission e and Λ e is a diagonal matrix consisting of those where Ye is the eigenvector matrix of H desired eigenvalues of the original Hamiltonian H contained in [−a, a]. The eigenstates of the projected Hamiltonian H may be obtained through elementary matrix algebra: 2 e e− B = U Ye = Ve Λ s Y. 1 (29) Denoting the Eq. (20) as a 2N × (2n + 1) matrix A with |χi ⟩ being the i-th column, the eigenstates of the original Hamiltonian H contained in [−a, a] is 2 e e− Φ = AB = AVe Λ s Y. 1 (30) e and Φ. We finally get the answers Λ D Comparison with a hybrid method Since the Lanczos method is widely employed to compute the lowest (or highest) eigenvalues, while the exp-semicircle filter provides a means to transform the central eigenvalues to the highest ones, naturally one may combine them together to efficiently compute the central eigenvalues, a way far simpler than the DACP method. But there are several reasons to prefer complicated techniques used in the DACP method to the standard construction of the Krylov space, especially when large scale computations are required. First, it is known that the Lanczos method is not numerically stable under practical conditions. Specifically, although the Lanczos algorithm shows perfect properties in theory, in practice it loses many of its designed features, e.g., global orthogonality and linear independence among Lanczos recursion states [32, 45, 46]. These defects effectively limits the number of eigenvalues which can be computed. In addition, it seems that the emergence of generalized eigenvalue problem (to deal with non-orthogonal base states) is unavoidable due to the error accumulations [47, 48]. The Chebyshev recursion, on the other hand, possesses many interesting properties common in both the ideal and practical calculations [45]. In particular, it is accurate and stable for x ∈ [−1, 1], allowing the propagation in the Chebyshev order domain for tens of thousands of steps without accumulating significant errors [30]. Second, taking the reorthogonalization step into consideration, the efficiency of the Lanczos algorithm decreases. The reorthogonalization step is a necessary part in both the Lanczos and the Arnoldi method. When there is a large amount (e.g., several thousands) of eigenvalues to be computed, the total cost is actually dominated by the reorthogonalization [26, 27, 49]. Suppose the total required number of eigenvalues is R and the Hilbert space dimension is D, then the Lanczos-type methods scale as DR2 , as every generated Ritz vector needs to be reorthogonalized against the existing ones (see also the discussions in Refs. [27,41]). In comparison, we have shown in Section 3 that TF ∝ DR−1 , TE ∝ DR0 , and TD ∝ D0 R3 , where each one of them is better than DR2 (R ≪ D). Although partial reorthogonalization schemes have been proposed, they result in an increased cost in computations as well as memory traffic [20,50], and they are not guaranteed to succeed when the accuracy requirement becomes strict [50]. Moreover, the DACP method and the Lanczos algorithm are different in terms of space complexity, as the former shows max(DR0 , R2 ), while the latter requires DR once the reorthogonalizations are needed [41]. Therefore, the DACP method has superiority over the Lanczos algorithm in both the time and the space complexity. Finally, ignoring the requirement of reorthogonalizations, we find that the two methods are comparable in both the time and the space complexity. Recall that the Chebyshev evolution time, being the dominate term in the DACP method, becomes TE ≃ πREmax τ /a, 16 SciPost Physics Submission where τ is the time for matrix-vector product. We then discuss the time complexity of a combined version, the Lanczos method with the exp-semicircle filter. As shown in Fig. 1 of the Supplemental Material in Ref. [21], and the discussions in Ref. [26], the number of Lanczos steps m shall be at least twice the number of requested eigenvalues R, i.e., m ≥ 2R. Since 2R iterations are needed, and every iteration requires a filter application with TF ≃ 36Emax τ /a, the computation time reads 2R · 36Emax τ /a = 72REmax τ /a. Therefore, the DACP method and the combination of Lanczos with the exp-semicircle filter share a same time complexity. 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Hu et al. Cell Death and Disease (2018)9:979 DOI 10.1038/s41419-018-1013-z Cell Death & Disease ARTICLE Open Access Linc02527 promoted autophagy in Intrahepatic cholestasis of pregnancy Jianguo Hu1, Li Liu1, Yangyang Gong1, Lei Zhang1, Xiaoling Gan1, Xiaodong Luo1, Tinghe Yu1, Xiaocui Zhong1, Xinru Deng1, Lina Hu1, Zhanyu Zhang1 and Xiaojing Dong1 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Abstract LncRNA plays a crucial role in human disease. However, the expression and function of LncRNA in ICP(Intrahepatic cholestasis of pregnancy) is still not fully elucidated. In this study, we found Linc02527 was increased expression in placenta and serum of ICP patients. Ectopically expression of Linc02527 promoted autophagy and proliferate in HTR8 cells. Silencing Linc02527 suppressed the autophagy and proliferate in HTR8 cells. Mechanically study revealed that Linc02527 regulated the expression of ATG5 and ATG7 by sponging miR-3185. Linc02527 directly binding to YBX1 and activated P21. The growth of C57 mouse was retarded when autophagy was activated. In normal condition, inhibited autophagy using chloroquine did not affect the growth of C57 mouse. However, in the condition of autophagy was activated, inhibited autophagy using chloroquine can improve the growth of C57 mouse. Overall, the results of this study identified Linc02527 as a candidate biomarker in ICP and a potential target for ICP therapy. Chloroquine was a potential drug for ICP therapy. Introduction Intrahepatic cholestasis of pregnancy (ICP) is an obstetrical complication. It may lead to intrauterine fetal death. The hazard behind ICP is the excessive accumulation of bile acids in hepatocytes and the inhibition of bile excretion1. Overproduction of bile acids or their impaired removal from hepatocytes may be the reason of this pathology, which leads to increased bile acid serum concentration1–4. Autophagy is an evolutionarily conserved process by which cytosolic components are sequestered into doublemembrane vesicles and consequently fused with lysosomes5,6. Autophagy plays an important role in cellular pathways, including in growth, development and differentiation, aging and cell death7–9. Previous study reported that autophagy was involved in the progress of preeclampsia10. The marker of autophagy was abnormally expression in placenta with hypertensive disorders in pregnancy11. However, little is known about the function of autophagy in ICP. LncRNA has been demonstrated that played an important role in human disease. LncRNA HOTAIR12, lncRNA MACC1-AS113, LncRNA-MALAT114 and LncRNA NEAT115 were involved in cancer migration and invasion. In human placenta, some LncRNAs was vrefiied. Misregulation of LOC391533, LOC284100 and CEACAMP8 may contribute to the mechanism underlying preeclampsia16. lncRHOXF1 is the first lncRNA that regulates the host response to viral infections in human placental progenitor cells17. However, little is known about the expression and function of LncRNA in ICP. In the present study, we investigated LncRNA expression and its potential clinical significance in ICP, trying to clarify the possible function of LncRNA in the pathogenesis. Materials and methods Tissue specimens Correspondence: Xiaojing Dong (xffdoctor@163.com) 1 Department of Obstetrics and Gynecology, Second Affiliated Hospital, Chongqing Medical University, Chongqing, China These authors contributed equally: Jianguo Hu, Li Liu, Yangyang Gong. Edited by G.M. Fimia The placenta tissue and serum sample was were obtained from the second affiliated Department of © The Author(s) 2018 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/. Official journal of the Cell Death Differentiation Association Hu et al. Cell Death and Disease (2018)9:979 Obstetrics and Gynecology, Second Affiliated Hospital, Chongqing Medical University. The Ethics Committee of the Chongqing Medical University approved the study documents and the use of archived placenta tissues and serum sample. Animal models Thirty Sprague-Dawley pregnant rats were randomly divided into two groups: Group NP and Group ICP. The rats provided by animal experiment center of Chongqing medical university. The rats were maintained in collective cages in an appropriate room with controlled temperature and with a 12-h light cycle and fed with standard rat chow and water. They were aged at ~150–180 days and weighed ~250–270 g. From day 13 of gestation, rats in group ICP were injected intraperitoneally with estradiol benzoate (5 mg/kg, Abcam) for 7 days consecutively. Group NP (control group, named Group NP) was injected intraperitoneally with normal saline. On day 13 of gestation, the levels of TBA, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bile acid(TBA) was detected in serum which was drawn from tail vein of pregnant rats. On day 21 of gestation, cesarean section was performed with anesthesia by intraperitoneal injection of 10% hydral with dosage of 3 mL/kg. The placenta was obtained for detecting the expression of LC3-II. Forty C57 pregnant mice were randomly divided into four groups: group NC, group RAPA, group CQ and group CQ + RAPA. Mice were maintained in collective cages in an appropriate room with controlled temperature with a 12-h light cycle, and mice were allowed access to food and water ad libitum. From day 12 of gestation of gestation, mice in group NC were injected intraperitoneally with normal saline for 6 days consecutively. Group RAPA were injected intraperitoneally with rapamycin(5 mg/kg, CST). Group CQ were injected intraperitoneally with chloroquine (50 mg/kg, CST). Group RAPA + CQ were injected intraperitoneally with rapamycin(5 mg/kg, CST) and chloroquine (50 mg/kg, CST). On day 21 of gestation, the pregnant mice were sacrificed. Then, the fetal mice was calculated for body weight, fetus number, bodylenth and placenta weight. Microarray assay Human LncRNA Microarray V4.0 (Agilent, USA) is designed for global profiling of human lncRNAs and protein-coding transcripts. Human miRNA Microarray Release 21.0 (Agilent technologies, USA) is designed for detecting the miRNA expression. The experiments and data analysis were performed by Beijing Boao Crystal Code Biotechnology Co. Ltd. Cell culture, transfection procedure, and reagents Human HTR8 cells were cultured in RPMI 1640 medium (Sigma-Aldrich, R8758) containing 10% fetal Official journal of the Cell Death Differentiation Association Page 2 of 14 bovineserum. The cells were incubated under 5% carbon dioxide at 37 °C. siRNA corresponding to the target sequences were synthesized by Genepharma (Shanghai, China). The following sequences were targeted for human Linc02527 small interfering RNA (siRNA): Linc02527-1: 5’-GGAGAGGCUAUAAACUUCUTT-3′; Linc02527-2: 5′-CCGUGCAUAUUGUCUCCAATT-3′; and NC (negative control) siRNA: 5′-UUCUUCGAAGGUGUCACGU TT-3′. Lentiviral vectors expressing shRNA targeting Linc02527 (named LV3-lnc02527-1 and LV3-lnc02527-2) and the Linc02527-lentiviral expression vector (named LV5-lnc02527) were provided by Genepharma. miR-3185 mimics (sense: 5′-AGAAGAAGGCGGUCGGUCUGCG G-3′) were synthesized at Ruibo Biotechnology(Guangzhou, China). AST detecting using AST Activity Assay Kit (MAK055, Sigma), ALT detecting using ALT Activity Assay (MAK052, Sigma); TBA detecting using Total Bile Acids (TBA) Assay Kit (Colorimetric) (BioVision.Inc, America); CG detecting using CG RIA Kit(YB-10016, Shanghai Zibo Biotechnology Co., Ltd. China). Flow cytometric analysis of cell apoptosis Cells for apoptotic analysis were double stained with Annexin V–FITC and propidium iodide 48 h after transfection and analyzed using a flow cytometer (FACScan; BD Biosciences, Shanghai, China) equipped with CellQuest software (BD Biosciences). Each experiment was performed in triplicate. RNA extraction and Quantitative real-time polymerase chain reaction(RT-qPCR) Total RNA was isolated using a pure high-purity Total RNA Rapid Extraction Kit (Bioteke Corporation, RP1201) according to the manufacturer’s instruction. cDNA was synthesized using the iSCRIPT cDNA synthesis kit (Bio-Rad Laboratories, 4106228). All of the primers used to amplify lncRNAs were synthesized by GenePharma. The real-time PCR kit was purchased from GeneCopoeia. PCR conditions were 95 °C for 10 s, 60 °C for 20 s and 72 °C for 10 s. Each sample was analyzed in triplicate. Relative quantification of mRNA was performed using the comparative threshold cycle (CT) method. This value was used to plot the gene expression using the formula 2−Δ ΔCT. Dual-luciferase reporter gene assay Luciferase reporter gene assay was performed using the Dual-Luciferase Reporter Assay System (Promega Corporation, E1910) according to the manufacturer’s instruction. Luciferase reporter assays for ATG5, ATG7 and Linc02527, wild-type or mutant reporter constructs (termed WT or Mut; obtained from Genepharma) were cotransfected into HTR8 cells in 24-well plates with 100 nM MIR3185 or 100 nM miR-NC and Renilla plasmid Hu et al. Cell Death and Disease (2018)9:979 Page 3 of 14 Table 1 Summary of lncRNA that are differently expressed between placenta from NP and ICP (ICP vs. NP) Gene symbol Chromosomal location Regulation P-value LINC02027 Chr3:810430188-81144798(+) Up 0.02699606 LINC02527 Chr6:112221691-112223054(−) Up 0.01485026 FAM239A ChrX: 3771050-3781615(−) Up 0.025825039 KCNMB2-AS1 Chr3:178447071-178577338(−) Up 0.028023811 YEATS2-AS1 Chr3:183524244-183526729(−) Up 0.0014545523 LINC02050 Chr3: 80814047-80838505(+) Up 0.09413093 TCONS_00005551 Chr3:81143793-81144462(+) Up 0.02998128 TCONS_00011228 Chr6:112221561-112230600(−) Up 0.014197185 LINC01436 Chr21: 36005338-36007838(+) Up 0.010051289 HIT000075265 Chr16:23315293-23360088(+) Down 0.021368366 uc021wnb.1 Chr22: 24245625-24247864(−) Down 0.024611032 by using Endofectin-Plus (Gene-Copoeia, Z01010A). All experiments were performed at least 3 times. Western blotting The expressions of LC3, ATG7, ATG5, YBX1, P21 and actin proteins were analyzed by western blot18,19. The primary antibodies used include polyclonal rabbit antiLC3 (Cell Signaling Technology, 4108); rabbit monoclonal to ATG5 (Abcam, ab109490); rabbit monoclonal to ATG7 (Abcam, ab183188); rabbit monoclonal to YBX1 (Abcam, ab76149); rabbit monoclonal to P21 (Abcam, ab109520); and monoclonal rabbit anti-beta actin (Abcam, ab115777). The band density was analyzed using a gel imaging system and compared with an internal control. Cell proliferation assay Cell proliferation was determined using the EdU assay. It was performed using the Cell-Light TM EdU imaging detecting kit according to the instructions in the kit (Ruibo Biotechnology, Guangzhou, China)20,21. RNA pulldown assay and matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOFMS) To find the proteins bonding to Linc02527 directly, the RNA pull-down assay was performed22,23. The specific operation steps was according to the previous study24. Using SDS-PAGE gel electrophoresis, Silver stain and MALDI-TOF-MS, then, the specific proteins were identified. Statistical analysis All statistical analyses were performed using SPSS software, version 17.0 (Chicago, IL). Each experiment was performed in triplicate. Statistical analysis was performed Official journal of the Cell Death Differentiation Association by Student t test. Pearson correlation analysis was conducted to analyze the association between Linc02527 expression and serum TBA, CG, ALT or AST. Data were presented as mean ± standard deviation. Statistical significance was defined as a p-value less than 0.05. Results Microarray expression profiles of lncRNAs and mRNAs in placenta of ICP patients To explore the potential biological functions of lncRNAs in ICP, we examined the expression patterns of lncRNAs and mRNAs in placenta of ICP patients and controls with full term pregnancy. In total, 358 lncRNAs and 255 mRNAs showed a ≥1.5-fold change (P < 0.05) in 8 samples (Fig. 1a, b). Of these, 100 lncRNAs were upregulated, and 258 were down-regulated; 123 mRNAs were up-regulated, 132 were down-regulated (Table 1 and S1). The differential expression lncRNAs were related to respiratory diseases, hypertensive diseases, cardiovascular diseases and so on (Fig. 1c). These lncRNAs were involved in many cellular process including in sodium channel activity, peptide disulfide oxdoreductase activity and so on (Fig. 1d). They also participated in aldosterone-regulated sodium reabsorption, Stimuli-sensing channels, glutathine metabolism and transmembrane transport of small molecules (Fig. 1e). Linc02527 expression was increased in placenta and serum of ICP We verified the expression of 11 LncRNA in placenta of ICP. We found that Linc02527, KCNMB2-AS1, YEATS2AS1 and TCONS-00011228 were significant increased in placenta of ICP (Fig. 2a, b, Fig. 2S1). We also observed Linc02527, KCNMB2-AS1, YEATS2-AS1 and TCONS00011228 expression was significantly increased in serum Hu et al. Cell Death and Disease (2018)9:979 Page 4 of 14 Fig. 1 Hierarchical clustering of a lncRNA and b mRNA differential expression profiles between the ICP group and control group(NP) in 8 placenta samples. The heat maps are based on expression values of significantly differentially expressed lncRNAs and mRNAs (absolute fold change ≥1.5 and P < 0.05) detected by microarray probes. “Red” and “Green” indicated expression above and below, respectively, relative expression. c Terms enriched disease-related LncRNAs(top 30). d Go and Kegg (e) pathway analysis of differentially expressed LncRNAs(top 30) Official journal of the Cell Death Differentiation Association Hu et al. Cell Death and Disease (2018)9:979 Page 5 of 14 Fig. 2 Linc02527 was detected in placenta and serum of ICP. a The LncRNA was validated by qPCR in placenta of ICP (n = 20) and normal control (n = 20). b Linc02527 was detected in placenta of ICP (n = 20) and normal control (n = 20). c, d The expression of LncRNA was detected in serum of ICP (n = 20) and normal control (n = 20). e–h The serum TBA, CG, ALT and AST were detected in ICP patients (ICP; n = 20) and normal control (NP; n = 20). i–l The expression of Linc02527 in placenta was positively correlated with the expression of serum TBA, CG, ALT and AST. Error bars represented standard error. Asterisks(*) and ** indicated p < 0.05 and 0.001, respectively Official journal of the Cell Death Differentiation Association Hu et al. Cell Death and Disease (2018)9:979 Page 6 of 14 Table 2 Biochemistry (mean ± SD) Parameter NP (n = 20) ICP (n = 20) P value TBA (µmol/L) 3.06 ± 2.12 25.04 ± 16.19 <0.01 CG (µmol/L) 0.85 ± 0.49 25.71 ± 26.66 <0.01 ALT (U/L) 11.75 ± 7.30 143.25 ± 118.48 AST (U/L) 19.35 ± 7.55 88.4 ± 66.05 Table 3 Subject demographic and characteristics (mean ± SD) Parameter NP (n = 20) ICP (n = 20) P value Age (years) 30.95 ± 3.35 29.20 ± 4.34 NS <0.01 Height (cm) 160.45 ± 4.84 159.70 ± 2.68 NS <0.01 Weight (kg) 70.325 ± 7.87 67.25 ± 8.19 NS 14.275 ± 4.31 13.625 ± 2.86 NS Total bilirubin (µmol/L) 9.10 ± 2.62 11.64 ± 5.39 NS Weight gain (kg) Direct bilirubin (µmol/L) 3.11 ± 1.49 4.92 ± 3.64 <0.05 Gravidity 2.30 ± 1.78 2.45 ± 1.76 NS Indirect bilirubin (µmol/L) 6.00 ± 2.19 6.72 ± 3.15 NS Parity 1.25 ± 0.44 1.3 ± 0.47 NS of ICP (Fig. 2c, d and Fig. S2A-C). The serum TBA, CG, ALT and AST was increased in ICP compared with that normal pregnant (Fig. 2e–h and Table 2). The Birth body length of newborn in ICP group was shorter than that of normal group (Table 3). The serum TBA, CG, ALT and AST was positively correlated with the expression of Linc02527 in placenta (Fig. 2i–l). The serum TBA, CG, ALT and AST was positively correlated with the expression of Linc02527 in serum (Fig. 2 S2D-G). Linc02527 promoted proliferation and autophagy in HTR8 cells We next determined the function of Linc02527. We firstly detected the apoptosis rate after silencing or ectopically expressed Linc02527 in HTR8 cells. The apoptosis rate is not significant difference between HTR8 cells infected with LV3-lnc02527-1 or lnc02527-2 and HTR8 cells infected with LV3-NC (Fig. 3a). Also, the apoptosis rate is not changed in HTR8 cells infected with LV5lnc02527 compared with infected with LV5-NC (Fig. 3c). The expression of BCL-2 and Bax was not changed when silencing or ectopically expressed Linc02527 in HTR8 cells (Fig. 3b, d). This data revealed that Linc02527 did not regulate apoptosis in HTR8 cells. So we detected whether Linc02527 regulated autophagy in HTR8 cells. As we expected, the expression of LC3-II was decreased in HTR8 cells infected LV3-lnc02527-1 or lnc02527-2 compared to that infected LV3-NC (Fig. 3b). The expression of LC3-II was increased in HTR8 cells infected LV5-lnc02527 compared to that infected LV5-NC (Fig. 3d). To investigate whether Linc02527 regulated autophagic flux, we quantified the levels of LC3-II, the autophagosome associated form of the protein, in the absence and presence of the lysosomal inhibitor chloroquine. The ratio between the LC3-II levels with and without chloroquine as an index of overall autophagic flux25. We observed a increase of this ratio in HTR8 cells infected LV5-lnc02527 compared to that infected LV5-NC (Fig. 3e).This data indicated that Linc02527 promoted autophagic flux. We Official journal of the Cell Death Differentiation Association Gestational weeks at delivery 38.39 ± 0.95 37.82 ± 1.03 NS Birthweight (kg) 3.2 ± 0.32 3.04 ± 0.44 NS Birth body length (cm) 50.35 ± 1.27 48.85 ± 1.90 <0.01 determined whether autophagy was activated in the placenta of ICP. We found that the expression of LC3-II, ATG5, ATG7 and Beclin1 was increased in placenta of ICP compared to normal pregnant (Fig. 3f). We also investigated whether Linc02527 was involved in the proliferate ability. We observed that silencing Linc02527 suppressed cellular proliferate; however, ectopically expressed Linc02527 promoted the cellular proliferate ability in HTR8 cells (Fig. 3g–i). To determine the mechanism of Linc02527, the location of Linc02527 was detected. Our results revealed that Linc02527 was located in the cytoplasm and nucleus (Fig. 3k). Linc02527 promoted autophagy by sponging miR-3185 The expression of Linc02527 was increased in HTR8 cells infected LV5-lnc02527 compared to that infected LV5-NC (Fig. 4a). The differential miRNA expression was screened using miRNA array (S2). We verified six most significant down-regulated genes using qPCR. Six miRNAs (miR-1323, miR-3185, miR-3935, miR-3187, miR3663 and miR-515) was decreased in HTR8 cells infected LV5-lnc02527 compared to that infected LV5-NC (Fig. 4b and S2). Bioinformatics prediction (LncBase Predicted v.2) that there was a binding site between miR-3185 and Linc02527 (Fig. 4c). Luciferase reporter gene assay confirmed the binding of miR-3185 with Linc02527 (Fig. 4d). Over-expression of miR-3185 suppressed the expression of Linc02527 (Fig. 4e). However, silencing miR-3185 increased the expression of Linc02527(Fig. 4f). Bioinformatics prediction(microRNA.org) that ATG5 and ATG7 were the potential target of miR-3185. So we detected the expression of ATG5 and ATG7 through silencing or over-expression of miR-3185 in HTR8 cells. The expression of ATG5 and ATG7 was decreased after over-expression of miR-3185. In contrast, the expression of ATG5 and ATG7 was increased after silencing miR-3185 (Fig. 5a–c). The LC3-II also was regulated by Hu et al. Cell Death and Disease (2018)9:979 Page 7 of 14 Fig. 3 Linc02527 regulated autophagy and proliferate in HTR8 cells. a The apoptosis rate was detected using flow cytometry when silencing Linc02527. b The expression of LC3-II, BCL-2 and BAX was detected by western blot after silencing Linc02527.(c)The apoptosis rate was detected using flow cytometry when ectopically expression of Linc02527. d The expression of LC3-II, BCL-2 and BAX was detected by western blot when ectopically expression of Linc02527. e The expression of LC3-II was detected by western blot. LV5-NC: HTR8 cells infected by LV5-NC. LV5-NC + CQ: HTR8 cells infected by LV5-NC, then adding chloroquine at 10uM for 24 h. LV5: HTR8 cells infected by LV5-lnc02527. LV5 + CQ: HTR8 cells infected by LV5- lnc02527, then adding chloroquine at 10 uM for 24 h. f The expression of LC3 in placenta of ICP and normal group was detected by western blot. g–j The cellular proliferation was detected by EdU. k The location of Linc02527 was determined hybridization in situ. Lnc02527 probe was detected the location of Linc02527. NC was negative control. Error bars represented standard error. Asterisks(*) and ** indicated p < 0.05 and 0.001, respectively Official journal of the Cell Death Differentiation Association Hu et al. Cell Death and Disease (2018)9:979 Page 8 of 14 Fig. 4 The expression of miR-3185 was regulated by Linc02527. a The expression of Linc02527 was determined by qPCR. b The miRNAs expression was detected by qPCR in HTR8 cells infected LV5-lnc02527 or LV5-NC. c The potential binding sites between Linc02527 and miR-3185 were predicted. d HTR8 cells were co-transfected with miR-3185 mimic or control RNA (NC) with luciferase reporter plasmids containing either wild-type (pMIRlnc02527) or mutant (pMIR-lnc02527m) of Linc02527 genes. Luciferase expression was measured. The fold changes of the relative luciferase activity in miR-3185 mimic indicated that plasmid transfected cells were normalized to NC, indicating plasmid transfected cells. e The expression of Linc02527 was detected by transfecting miR-3185 mimic or NC(negative control). f The expression of Linc02527 was detected by transfecting miR-3185 inhibitor or NC(negative control). Error bars represented standard error. Asterisks(*) and ** indicated p < 0.05 and 0.001, respectively Official journal of the Cell Death Differentiation Association Hu et al. Cell Death and Disease (2018)9:979 Fig. 5 (See legend on next page.) Official journal of the Cell Death Differentiation Association Page 9 of 14 Hu et al. Cell Death and Disease (2018)9:979 Page 10 of 14 (see figure on previous page) Fig. 5 The expression of ATG5 and ATG7 was regulated by miR-3185. a The expression of ATG5 mRNA was detected when transfecting with miR3185 mimic, miR-3185 mimic control, miR-3185 inhibitor or miR-3185 inhibitor mimic. b The expression of ATG7 mRNA was detected when transfecting with miR-3185 mimic, miR-3185 mimic control, miR-3185 inhibitor or miR-3185 inhibitor mimic. c The protein expression of ATG5, ATG7 and LC3-II was determined when transfecting with miR-3185 mimic, miR-3185 mimic control, miR-3185 inhibitor or miR-3185 inhibitor mimic. d, e The potential binding sites between miR-3185, ATG5 and ATG7 were predicted. f, g HTR8 cells were co-transfected with miR-3185 mimic or control RNA (NC) with luciferase reporter plasmids containing either wild-type (pMIR-ATG5-3’UTR and pMIR-ATG7-3’UTR) or mutant (pMIR-ATG53’UTRm and pMIR-ATG7-3’UTRm) of ATG5 and ATG7 genes. Luciferase expression was measured. The fold changes of the relative luciferase activity in miR-3185 mimic indicated that plasmid transfected cells were normalized to NC, indicating plasmid transfected cells. h, i The expression of ATG5 and ATG7 was determined in HTR8 cells infected with LV5-NC or LV5-lnc02527. j, k The expression of ATG5 and ATG7 was determined in HTR8 cells infected with LV3-NC, LV2-lnc02527-1 or LV2-lnc02527-2. Error bars represented standard error. Asterisks(*) and **indicated p < 0.05 and 0.001, respectively To further explore the mechanism of Linc02527 regulation autophagy. The RNA pull down assay was performed. Then using SDS-PAGE gel electrophoresis and Silver stain six differential bands were obtained(Fig. 6a). Using MALDI-TOF-MS, some protein including YBX1 were identified(S3). It indicated that YBX1 was directly bond to Linc02527(Fig. 6a–c).We confirmed that YBX1 was directly bond to Linc02527 through RNA pull down assay (Fig. 6f). The expression of YBX1 was increased in HTR8 cells infected LV5-lnc02527 compared to that infected LV5-NC(Fig. 6d). The expression of YBX1 was decreased in HTR8 cells infected LV3-lnc02527-1 or LV3-lnc02527-2 compared to that infected LV3-NC(Fig. 6e). Previous study reported that P21 pathway was activated by YBX126. P21 pathway can promotes autophagy27–29. P21 was elevated when over-expression of Linc02527. P21 was downregulated when silencing Linc02527 expression(Fig. 6d, e). So, we guessed Linc02527 bond to and stabilized YBX1, then activated P21 pathway and prompted autophagy. group (Fig. 7d). This data revealed that autophagy was activated in ICP rat. We also observed YBX1 and P21 was elevated in the placenta of ICP rat compared to normal group (Fig. 7d). Then, we explore whether activated autophagy or inhibited autophagy can affect the growth of C57 mouse. We observed that the growth of C57 mouse was retarded when autophagy was activated using rapamycin compared to normal group (Fig. 7E1–2). Compared with the normal group, the growth of C57 mouse is not different when autophagy was inhibited using chloroquine (Fig. 7E3). The inhibition of growth by rapamycin is partly relieved through adding chloroquine (Fig. 7E4). The number of fetuses is no significant difference between normal control group, rapamycin group, chloroquine group and rapamycin + chloroquine group (Fig. 7f).The fetuses birth weight, body length and placenta weight were decreased in rapamycin group than that of normal control group (Fig. 7g–i). The fetuses birth weight, body length and placenta weight were decreased in rapamycin group than that of normal control group (Fig. 7g–i). The fetuses birth weight, body length and placenta weight were decreased in rapamycin + chloroquine group than that of rapamycin group (Fig. 7g–i). There was not significant difference between chloroquine group and normal control group (Fig. 7g–i). These data revealed that the growth of C57 mouse was repressed when autophagy was activated. In normal condition, inhibited autophagy using chloroquine did not affect the growth of C57 mouse. However, in the condition of autophagy was activated, inhibited autophagy using chloroquine can improve the growth of C57 mouse. Rapamycin caused fetal growth retardation, possibly due to inhibition of the mTOR pathway. Autophagy was involved in growth of fetal mouse Discussion We have demonstrated Linc02527 promoted autophagy in vitro. However, we did not find homologous sequence of Linc02527 in rat and mouse. We observed autophagy was activated in ICP. So, we detected whether autophagy was activated in ICP in the rat model. The serum TBA, ALT and AST was increased in ICP in the rat model (Fig. 7a–c). The LC3-II, ATG5 and ATG7 expression was elevated in the placenta of ICP rat compared to normal In the present research, we found that Linc02527 was increased expression in serum and placenta of ICP patients. Linc02527 promoted autophagy in HTR8 cells. Linc02527 regulated ATG5 and ATG7 though sponging miR-3185. Linc02527 directly binding to YBX1 and activated P21, then the autophagy was promoted. We also found that regulation of autophagy was mediated the growth of fetal mouse. miR-3185 (Fig. 5c). We predicted that there was a binding site between miR-3185, ATG5 and ATG7 (Fig. 5d, e). Luciferase reporter gene assay confirmed the binding of miR-3185 with ATG5 and ATG7 (Fig. 5f, g). The expression of ATG5 and ATG7 was increased in HTR8 cells infected LV5-lnc02527 compared to that infected LV5-NC (Fig. 5h, i). The expression of ATG5 and ATG7 was decreased in HTR8 cells infected LV3-lnc02527-1 or LV3-lnc02527-2 compared to that infected LV3-NC (Fig. 5j, k). This data suggested that Linc02527 promoted autophagy by sponging miR-3185. Linc02527 directly binding to YBX1 and activated P21 Official journal of the Cell Death Differentiation Association Hu et al. Cell Death and Disease (2018)9:979 Page 11 of 14 Fig. 6 Linc02527 directly binding to YBX1 and activated P21. a Silver stains for protein gels obtained by Linc02527 RNA Pulldown. Bio-lnc02527 (sense): treatment group; Bio-lnc02527 (antisense). The Linc02527 specific binding protein gels (in red box) was identified by MALDI-TOF-MS. Results show that YBX1 was a direct binding protein to Linc02527. b, c YBX1 was identified by MALDI-TOF-MS. d The expression of LC3-II,YBX1 and P21 was determined in HTR8 cells infected with LV5-NC or LV5-lnc02527. e The expression of LC3-II,YBX1 and P21 was determined in HTR8 cells infected with LV3-NC, LV2-lnc02527-1 or LV2-lnc02527-2. f The Linc02527 specific binding protein gels was identified by RNA pulldown. FL was the biotin Linc02527. NC was the Linc02527 without biotin label Linc02527 was located in 6q21. In 27 different human tissues, it is distinctive high-expression in placenta and testis30. In this study, we observed Linc02527 expression was increased in the placenta and serum of ICP patients. The serum TBA, CG, ALT and AST was elevated in ICP patients. The serum TBA and CG were diagnostic criteria of ICP31–33. The expression of Linc02527 was positively with the serum TBA, CG, ALT and AST. So, Linc02527 is a potential biomarker of Intrahepatic Cholestasis of Pregnancy(ICP). In this research, we noted that Linc02527 was not involved in apoptosis. However, it promoted autophagy and proliferate. Many studies have demonstrated that Official journal of the Cell Death Differentiation Association LncRNA regulated the target genes by sponging miRNAs34–36. ATG5 and ATG7 are two important moleculars to promote autophagy37–41. We found miR-3185 inhibited autophagy by targeting ATG5 and ATG7. Linc02527 directly bond to and down-regulated miR-3185. Our data revealed Linc02527 regulated ATG5 and ATG7 through sponging miR-3185. YBX1 regulates tumor growth via CDC25a pathway in human lung cancer42. lncRNA GAS5 enhances G1 cell cycle arrest via binding to YBX1 to regulate p21 expression in stomach cancer26. Phosphorylation of YBX1 activates NF-κB in colon cancer. We found Linc02527 directly bond to and stabilized YBX1. At the same time, Linc02527 increased the expression of P21. Hu et al. Cell Death and Disease (2018)9:979 Page 12 of 14 Fig. 7 Autophagy was involved in growth of fetal mouse. a–c The serum TBA, ALT and AST were detected in in ICP in the rat model. d The expression of LC3-II was detected in the placenta of ICP rat or NP group(normal control). e–i Forty C57 pregnant mice were randomly divided into four groups: group NC(E1), group RAPA(E2), group CQ(E3) and group CQ + RAPA(E4). From day 12 of gestation of gestation, mice in group NC were injected intraperitoneally with normal saline for 6 days consecutively. Group RAPA were injected intraperitoneally with rapamycin (5 mg/kg). Group CQ were injected intraperitoneally with chloroquine (50 mg/kg). Group RAPA + CQ were injected intraperitoneally with rapamycin (5 mg/kg) and chloroquine (50 mg/kg). On day 21 of gestation, the pregnant mice were sacrificed. Then, the fetal mice was calculated for body weight, fetus number, bodylenth and placenta weight. Error bars represented standard error. Asterisks(*) and **indicated p < 0.05 and 0.001, respectively Official journal of the Cell Death Differentiation Association Hu et al. Cell Death and Disease (2018)9:979 P21 pathway can promotes autophagy27–29. So, we guessed Linc02527 bond to and stabilized YBX1, then activated P21 pathway and prompted autophagy. These results indicated that Linc02527 was a potential target for ICP. Autophagy is an evolutionarily conserved catalytic process by which cytoplasmic components including damaged macromolecules and organelles are degraded43. An increased autophagosome formation was observed in placenta from preeclampsia and FGR44. In this study, we found LC3-II expression was elevated in placenta from ICP. The expression of LC3-II also was increased in the placenta of ICP rat. This result indicated that autophagy was activated in ICP. Autophagy has an important role in early embryo development. Increased embryonic or fetal death caused by inactivation of autophagy related genes in mouse. Atg5 deficient mice were fertilized normally, but manifested embryonic lethality at last45. Beclin1 deficient mice embryos also die as early as embryonic day 7.546. Previous study has demonstrated chloroquine and hydroxychloroquine inhibit bladder cancer cell growth by targeting basal autophagy47. Hydroxychloroquine (HCQ) inhibits autophagy and sensitize some cancer cells to chemotherapy48. However, as an inhibitor of autophagy, HCQ reduces neonatal morbidity in women with SLE by significantly decreasing the rate of prematurity and intrauterine growth restriction49. We found chloroquine (CQ) did not increase the amount of fetal mice. But induced autophagy using rapamycin, the development of fetal mice was retard and the fetuses birth weight, body length and placenta weight were decreased. Chloroquine (CQ) can rescue the retarded development and increase the fetuses birth weight, body length and placenta weight at the condition of activated autophagy. Based on these results, we guessed that activated autophagy impaired the growth of fetal mice. Inhibited autophagy promoted the growth of fetus at the condition of activated autophagy. Chloroquine is a potential drug of ICP. To our knowledge, this is the first time reported that autophagy was activated in ICP. The expression of Linc02527 was increased in placenta and serum in ICP patients. Linc02527 bond to and stabilized YBX1, then activated P21 pathway and prompted autophagy. We also found Chloroquine (CQ) can rescue the retarded development at the condition of activated autophagy. So, Linc02527 was a potential biomarker and target for ICP. Chloroquine may be a drug for treating ICP. Acknowledgements We thank members of our laboratory for technical help and stimulating discussions. This work was supported by the Natural Science Foundation of Chongqing (CSTC 2012JJB10030); the National Science Foundation of China (81172492); the National Science Foundation of China (81501220); and Chongqing Municipal Health and Family Planning Commission Medical Research Fund(2017MSXM031). Official journal of the Cell Death Differentiation Association Page 13 of 14 Conflict of interest The authors declare that they have no conflict of interest. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Information accompanies this paper at (https://doi.org/ 10.1038/s41419-018-1013-z). Received: 25 May 2018 Revised: 28 August 2018 Accepted: 29 August 2018 References 1. Trauner, M., Fickert, P. & Wagner, M. MDR3 (ABCB4) defects: a paradigm for the genetics of adult cholestatic syndromes. Semin. Liver Dis. 27, 77–98 (2007). 2. Allen, A. M. et al. The Epidemiology of Liver Diseases Unique to Pregnancy in a US Community: A Population-Based Study. Clin. Gastroenterol. Hepatol. 14, 287–294.e1 (2016). 2. 3. Ahmed, K. T., Almashhrawi, A. A., Rahman, R. N., Hammoud, G. M. & Ibdah, J. A. Liver diseases in pregnancy: diseases unique to pregnancy. World J. Gastroenterol. 19, 7639–7646 (2013). 4. Almashhrawi, A. A., Ahmed, K. T., Rahman, R. N., Hammoud, G. M. & Ibdah, J. A. Liver diseases in pregnancy: diseases not unique to pregnancy. World J. 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PLOS ONE RESEARCH ARTICLE Late middle Miocene caviomorph rodents from Tarapoto, Peruvian Amazonia Myriam Boivin ID1*, Laurent Marivaux2, Walter Aguirre-Diaz3, Aldo Benites-Palomino ID3,4, Guillaume Billet5, François Pujos ID6, Rodolfo Salas-Gismondi3,7, Narla S. Stutz ID2,8, Julia V. Tejada-Lara2,3, Rafael M. Varas-Malca ID3, Anne H. Walton9, Pierre-Olivier Antoine2 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Boivin M, Marivaux L, Aguirre-Diaz W, Benites-Palomino A, Billet G, Pujos F, et al. (2021) Late middle Miocene caviomorph rodents from Tarapoto, Peruvian Amazonia. PLoS ONE 16(11): e0258455. https://doi.org/10.1371/journal. pone.0258455 Editor: Jürgen Kriwet, University of Vienna, AUSTRIA Received: July 19, 2021 Accepted: September 24, 2021 Published: November 3, 2021 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0258455 Copyright: © 2021 Boivin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files (S1 Table and S2 File). The fossil material is permanently stored in the Vertebrate 1 Laboratorio de Paleontologı́a de Vertebrados, Instituto de Ecorregiones Andinas (INECOA), Universidad Nacional de Jujuy, CONICET, San Salvador de Jujuy, Jujuy, Argentina, 2 Laboratoire de Paléontologie, Institut des Sciences de l’Evolution de Montpellier (ISEM, UMR 5554, CNRS/UM/IRD/EPHE), Université de Montpellier, Montpellier, France, 3 Departamento de Paleontologı́a de Vertebrados, Museo de Historia Natural—Universidad Nacional Mayor San Marcos (UNMSM, DPV-MUSM), Lima, Peru, 4 Paläontologisches Institut und Museum, Universität Zürich, Zürich, Switzerland, 5 Département Origine et Evolution, Muséum national d’Histoire naturelle (MNHN, CR2P-UMR 7207 CNRS/MNHN/Sorbonne Université), Paris, France, 6 Laboratorio de Paleontologı́a, Instituto Argentino de Nivologı́a, Glaciologı́a y Ciencias Ambientales (IANIGLA), CCT–CONICET–Mendoza, Mendoza, Argentina, 7 BioGeoCiencias Lab, Facultad de Ciencias y Filosofı́a/CIDIS, Laboratorios de Investigacion y Desarrollo (LID), Centro de Investigación para el Desarrollo Integral y Sostenible (CIDIS), Universidad Peruana Cayetano Heredia, Lima, Peru, 8 Programa de PósGraduação em Geociências, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil, 9 Springfield Technical Community College, Springfield, Massachusset, United States of America * mboivin@idgym.unju.edu.ar Abstract Miocene deposits of South America have yielded several species-rich assemblages of caviomorph rodents. They are mostly situated at high and mid- latitudes of the continent, except for the exceptional Honda Group of La Venta, Colombia, the faunal composition of which allowed to describe the late middle Miocene Laventan South American Land Mammal Age (SALMA). In this paper, we describe a new caviomorph assemblage from TAR-31 locality, recently discovered near Tarapoto in Peruvian Amazonia (San Martı́n Department). Based on mammalian biostratigraphy, this single-phased locality is unambiguously considered to fall within the Laventan SALMA. TAR-31 yielded rodent species found in La Venta, such as the octodontoid Ricardomys longidens Walton, 1990 (nom. nud.), the chinchilloids Microscleromys paradoxalis Walton, 1990 (nom. nud.) and M. cribriphilus Walton, 1990 (nom. nud.), or closely-related taxa. Given these strong taxonomic affinities, we further seize the opportunity to review the rodent dental material from La Venta described in the Ph.D. volume of Walton in 1990 but referred to as nomina nuda. Here we validate the recognition of these former taxa and provide their formal description. TAR-31 documents nine distinct rodent species documenting the four extant superfamilies of Caviomorpha, including a new erethizontoid: Nuyuyomys chinqaska gen. et sp. nov. These fossils document the most diverse caviomorph fauna for the middle Miocene interval of Peruvian Amazonia to date. This rodent discovery from Peru extends the geographical ranges of Ricardomys longidens, Microscleromys paradoxalis, and M. cribriphilus, 1,100 km to the south. Only one postcranial element of rodent was unearthed in TAR-31 (astragalus). This tiny tarsal bone most likely documents one of the two species of Microscleromys and its morphology indicates terrestrial generalist adaptations for this minute chinchilloid. PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 1 / 80 PLOS ONE Paleontological collection of the “Museo de Historia Natural, Universidad Nacional Mayor de San Marcos” (MUSM), Lima, Peru. Funding: LM and POA received funding from The Leakey Foundation and the LabEx CEBA (ANR-10LABX-0025-01). POA received funding from the National Geographic Society and from the French ‘Agence Nationale de la Recherche’ (ANR) in the framework of the GAARAnti program (ANR-17CE31-0009). LM received funding from the CoopIntEER CNRS-CONICET (n˚ 252540). POA and FP received funding from the ECOS-SUD/FONCyT (n˚ A-14U01) international collaboration programs. MB received funding from the ‘Laboratoire de Planétologie et de Géodynamique de Nantes‘. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Miocene caviomorphs from Tarapoto, Peru Introduction Caviomorphs (Caviomorpha Wood, 1955) are hystricognathous rodents originating from South America, such as guinea pig, New World porcupines, chinchillas, and spiny rats. Nowadays, they show a great taxonomical diversity and a large array of lifestyles (arboreal, fossorial, semi-aquatic, and terrestrial) [1]. Among caviomorphs, two main clades, each divided into two superfamilies, are recognised [2–4]: Erethicavioi Boivin, 2019 (comprising Cavioidea Fischer, 1817 + Erethizontoidea Bonaparte, 1845 + stem-groups) and Octochinchilloi Boivin, 2019 (comprising Chinchilloidea Bennett, 1833 + Octodontoidea Waterhouse, 1839 + stemgroups). Their evolutionary history extends back to the late middle Eocene, with the earliest representatives of the group so far recorded in Peruvian Amazonia [5–8]. The Miocene record of caviomorphs is particularly well documented. Numerous fossilbearing localities yielded abundant specimens and species-rich assemblages of rodents [9]. Most of them are situated at high and middle latitudes, notably in Argentina (e.g., Pinturas, Sarmiento, Santa Cruz, Collón Cura, Cerro Azul and Ituzaingo formations) [9–15] and Chile (e.g., Laguna del Laja and Pampa Castillo) [16, 17]. At low latitudes of South America, two remarkable and very species-rich caviomorph faunas are known, La Venta in Colombia (Honda Group; late middle Miocene) [18] and Acre in Brazil (Solimões Fm. [Formation]; late Miocene; see [19]). Since the early discoveries of fossil-bearing localities in the Honda Group of the upper Magdalena valley in 1923, considerable field efforts led by petroleum companies and several scientific teams were performed in this region of Colombia, and notably in the La Venta area, until 1992 [20, 21]. As a result, 140 localities over 52 different stratigraphic levels, yielding thousands of fossils of many vertebrate groups were discovered in the Honda Group dating from the late middle Miocene [22]. In particular, the recognition in 1945 of the first fossil remains of primates in the Magdalena valley [23, 24] has been one of the most significant discoveries that motivated numerous subsequent field expeditions [20]. The mammal fauna from La Venta encompasses at least 77 species and 80 genera, representing 29 families [25, 26]. Concerning rodents, around 20 taxa documenting the four main caviomorph superfamilies have so far been reported (excluding the indeterminate genera and species) [18, 27–30]. In addition to La Venta, the Peruvian faunas from MD-67 (Madre de Dios) [31], the Fitzcarrald local fauna (near Atalaya, Ucayali) [32, 33], and CTA-45 (near Contamana, Loreto) [34], also middle Miocene in age, document representatives of the four caviomorph superfamilies (4, 8, and 2 taxa, respectively). Finally, a fourth late middle Miocene caviomorph-bearing locality also occurs in low latitudes, from the Socorro Formation in Venezuela, but so far it has only yielded a fragmented distal femur of a giant caviomorph [35]. Over the last decade, our yearly paleontological surveys in the Tarapoto area (San Martı́n Department, Peruvian Amazonia), have allowed the discovery of a new fossil-bearing locality, TAR-31, in the vicinity of the Juan Guerra village. This locality is considered to be late middle Miocene in age (Laventan SALMA) based on mammalian biostratigraphy (see [36]; see Material and Methods section of this paper). TAR-31 yielded a diverse assemblage of aquatic and terrestrial vertebrates, including numerous caviomorph specimens that are the subject of this paper. The primary purpose of the present work is to describe the new caviomorph materials found in TAR-31. This locality shares taxa or closely related taxa with La Venta fauna described by one of us (AHW) in the framework of her PhD [37]: Microsteiromys, Ricardomys, and Microscleromys. Because Microsteiromys, Ricardomys, Microscleromys, and the species included within these genera were not formally described (nomina nuda) [18, 37, 38], here we also revise the material from La Venta attributed to these taxa, in order to clarify their systematics. PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 2 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Material and methods TAR-31, Tarapoto, Peru The rodent fossil material from Peruvian Amazonia described in the present work originates from the TAR-31 locality, situated in the vicinity of the small town Juan Guerra, along the Mayo River (Tarapoto area, San Martı́n Department, Peru; Fig 1A and 1B). TAR-31 consists of a 10–15 cm-thick yellow microconglomerate interbedded within a grey cross-stratified and sandstone-dominated fluvial unit (Fig 2A [36]). The latter is intercalated between thick violingrey variegated paleosols pointing to the existence of a meandering river with sustainable floodplain [36]. These levels were originally mapped as belonging to the lower member of Ipururo Formation (middle Miocene in age in the Huallaga basin) [39–42], an assignation in agreement with the nature of their facies and depositional environment sequence. The fossil content of the TAR-31 encompasses plants, amber clasts, crabs, chondrichthyans, osteichthyans, anurans, chelonians, crocodylomorphs, birds, and mammals (including metatherians, xenarthrans, liptoterns, notungulates, sirenians, chiropterans, primates, and caviomorph rodents). The TAR-31 mammal assemblage includes a didelphid marsupial (currently under study by one of us, NSS), the interatheriine notoungulate Miocochilius sp., the didolodontid Megadolodus sp., the platyrrhine primate Neosaimiri aff. fieldsi [36], and the caviomorph fauna described here (see ‘Age of TAR-31’ section of this present work). This assemblage recalls some of the Laventan SALMA localities: Quebrada Honda in Bolivia (13.1– 12.2 Ma) [43–46], the Fitzcarrald local fauna in SE Peru [33], and especially the lower part of the Villavieja Formation in the La Venta area in Colombia (13.8–11.6 Ma; [47] and see above). Accordingly, the TAR-31 locality most likely documents the late middle Miocene Laventan SALMA [36]. The material from TAR-31 was collected by excavating and wet screening (2 and 1 mm meshes) about 550 kg of sediment during our yearly field expeditions (August 2015, 2016, and Fig 1. Geographic location of TAR-31 (San Martı́n Department, Peru; late middle Miocene, Laventan SALMA). (A) Location map of the Tarapoto area (star symbol) in Peru. (B) Location map of the TAR-31 (star symbol) in the Tarapoto area. SALMA, South American Land Mammal Age. https://doi.org/10.1371/journal.pone.0258455.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 3 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru 2018). The caviomorph material corresponds to about 400 isolated teeth (complete or fragmentary), one fragment of mandible (MUSM 4643), one fragment of maxilla (MUSM 4375), and one astragalus (MUSM 4658). No permits were required for the described study, which complied with all relevant regulations. The field work on TAR-31 was carried out in the framework of the ongoing cooperation agreement between the ‘Museo de Historia Natural de la Universidad Nacional Mayor San Marcos’ (Lima, Peru) and the ‘Institut des Sciences de l’Evolution de Montpellier-Université de Montpellier’ (France). The TAR-31 fossil specimens are permanently stored in the palaeontological collection of the ‘Museo de Historia Natural, Universidad Nacional Mayor de San Marcos’, Lima, Peru (MUSM). La Venta area, Colombia The fossil rodent material from La Venta re-analysed here (originally studied in the framework of the Ph.D. of Anne H. Walton) [18, 37] originates from the badland exposures located in the vicinities of the Villavieja and La Victoria villages, in the upper valley of the Magdalena River (Huila Department, Colombia). Numerous fossil-bearing localities are known in the La Venta area (and the Magdalena upper valley in general) [26]. These are assigned to two successive formations of the Honda Group: La Victoria and Villavieja formations [48]. The studied material from this area was found in: (i) one locality from La Victoria Formation (IGM-DU loc. 075, between the Chunchullo Beds and the Tatcoa Sandstone Beds), and (ii) distinct localities from the Villavieja Formation, including several localities in the Fish Bed (CVP 5, 8, 9, 10, 10A, and 13B), two localities in the Monkey Bed (IGM-DU loc. 006–1 and IGM-DU loc. 022 or UCMP loc. V4536), and one in the El Cardón Red Beds (IGM-DU loc. 032 and its screenwashed locality 032–5). The Unit between the Chunchullo and Tatacoa Sandstone Beds is 149.3 m-thick, and is composed of very fine sandstones and mudstones alternating with metric medium-grained sandstone lenses [48]. The Villavieja Formation is divided into the Baraya Member, including the Fish and Monkey beds, and the Cerro Colorado Member, including the El Cardón Red Beds [48]. The Baraya Member is very fossiliferous and is composed mainly of gray mudstones and sandstones with minor layers of red mudstones, while the Cerro Colorado Member is less fossiliferous and consists of thick horizons of red mudstones with a very small amount of volcanic litharenites and chert litharenites [48]. The La Venta fauna is late middle Miocene in age (13.8–12.05 Ma) based on 40Ar/39Ar datings realised on volcanic units from different localities of the Honda Group (on biotites, hornblendes, and plagioclases; 13.8– 12.2 Ma) and magnetostratigraphic correlations of the Honda Group (13.61 [base of C5ABn chron] –12.05 Ma [top of C5An.1n]) [26, 47, 49]. The La Venta area yielded a very rich vertebrate fauna including chondrichthyans, osteichthyans, amphibians, squamates, chelonians, crocodylomorphs, birds and mammals (metatherians, xenarthrans, astrapotheres, litopterns, notoungulates, cetaceans, chiropterans, sirenians, primates and caviomorph rodents) [21, 22]. The caviomorph remains from La Venta included in this work were formerly studied by one of us [18, 37]. In a doctoral thesis manuscript, Walton [37] described several new genera and species such as Microsteiromys jacobsi, Ricardomys longidens, Microscleromys paradoxalis, and Microscleromys cribriphilus. As the latter taxa were not formally described, they should be considered as not available following the articles 8, 9, 11, 13 of the International Code of Zoological Nomenclature (i.e., nomen nudum; p. 132–143, 246 [38]). As formally recognised for nomina nuda (“A nomen nudum is not an available name, and therefore the same name may be made available later for the same or a different concept; in such a case it would take authorship and date [Arts. 21, 50] from that act of establishment, not from any earlier publication as a nomen nudum” [38], we chose to keep the taxonomic names proposed by Walton [37]. For reasons related to the covid pandemic and confinement, the original material from La Venta PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 4 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru could not be directly observed. The description and comparison of this material in the present work is based on published photos and drawings [18, 37]. Most of these specimens were collected by surface collecting and wet screening (2 and 0.6 or 0.25 mm meshes) in the years 1985 through 1989. This material is stored in the ‘Museo Geológico José Royo y Gómez, Servio Geológico Colombiano’ (IGM; formerly, ‘Instituto Nacional de Investigaciones en Geociencias, Mineria y Quı́mica’) and the ‘Departamento de Geociencias, Universidad Nacional de Colombia’ (UNC), Bogotá, Colombia, as well as in the Field Museum of Natural History, Chicago (FMNH), USA. Some specimens do not have collection number (unnumbered specimens). We used their field number to refer to them (S1 Table). Imaging For the largest specimens (mandibular and maxillary fragments) and high-crowned teeth found at TAR-31, we used X-ray microtomography (μCT-scan) for obtaining three-dimensional digital models (3D surface renderings). The dental/jaw specimens and the astragalus were scanned with a resolution of 6 μm and 5 μm, respectively, using a μCT-scanning station EasyTom 150/Rx Solutions (Montpellier RIO Imaging [MRI], ISEM, Montpellier, France). They have been virtually delimited by manual segmentation under AVIZO 7.1 software (Visualization Sciences Group). The specimens were prepared within a ‘Label Field’ module of AVIZO, using the segmentation threshold selection tool. The other teeth figured in this paper were photographed with a Scanning Electron Microscope (SEM) HITACHI S 4000 (Institut des Neurosciences de Montpellier [INM], France). Some of the pictures of the astragalus are photographs of the original specimen, which are the result of the fusion of multifocus images obtained with an optical stereomicroscope (Leica M 205C) connected to a camera (Leica DFC 420C; ISEM). Nomenclature and comparisons The main terminology for the rodent dentition follows Boivin & Marivaux [50], and the literature cited therein. Some extant caviomorphs and related extinct forms show a highly derived/ specialised dental pattern with respect to that of the oldest representatives of this group (for which the aforementioned nomenclature was based on). This is the case of two taxa described below: (i) Caviidae indet. gen. et sp., for which nomenclatures proposed by several authors [51–55] were followed (Fig 2A); and (ii) the adelphomyine Ricardomys longidens, for which we partly followed the nomenclature proposed by Patterson & Pascual [56] (Fig 2B and 2C). Lower case letters are used for lower dentition (dp, for decidious premolar; p, for premolar; m, for molars) and upper case letters for upper dentition (dP, for decidious premolar; P, for premolar; M, for molars). The astragalar nomenclature is based on Ginot et al. [57], Rose & Chinnery [58], and Wible & Hughes [59]. The caviomorph taxa cited in the text and used for dental comparisons are listed in S2 Table. Three isolated teeth from MD-67 locality in Peru (MUSM 1974, 1975, and 4298; early middle Miocene) were originally attributed to cf. Microsteiromys sp [31]. They are reassigned here in light of the new material from Tarapoto. For the MUSM 4658 astragalus, we used the same comparative sampling as Boivin et al. [60]. In addition, we consulted supplementary astragalar material attributed to the erethizontid Steiromys duplicatus (MACN A 10055–78, 10081 and 10082; see [61]), the caviids Dolicavia minuscula (MMP 10055–78) and Galea leucoblephara (INBIAL-CV 00290), the neoepiblemid Neoepiblema sp. (probably N. acreensis; UFAC 5249 and 61840), the octodontids Abalosia castellanosi (MMP 1439-M) and Pithanotomys columnaris (MACN-Pv 7429–7431), the echimyid Eumysops chapalmalensis (MACN-Pv 17868), and the ctenomyid Actenomys priscus (MMP 367-S and 395-S). When the fossils described in this paper are compared with several taxa, they are PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 5 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Fig 2. Dental nomenclature for cavioid (A) and adelphomyine (B, C) rodent teeth in occlusal view. (A) p4; 1, anterior projection of the anterior lobe; 2, interprismatic furrow; 3, apexes; 4, hypoflexid; 5, enamel; 6, dentine; 7, posterior lobe; 8, longitudinal furrow opposite to the hypoflexid (= primary internal flexid); 9, anterior lobe. (B) Lower molar; 1, metaconid (+ its posterior arm?); 2, first lamella; 3, metalophulid I; 4, protoconid; 5, ectolophid; 6, hypoflexid; 7, anterior outgrowth of the hypoconid; 8, hypoconid; 9, anterior arm of the hypoconid; 10, posterolophid; 11, third lamella; 12, metaflexid; 13, second lamella; 14, entoconid; 15, hypolophid; 16, confluence between the anteroflexid and the mesoflexid. (C) Upper molar; 1, first lamella; 2, anteroloph; 3, posterior arm of the protocone (= lingual protoloph); 4, protocone; 5, posterior outgrowth of the protocone; 6 hypoflexus; 7, mure; 8, anterior arm of the hypocone; 9, hypocone; 10, posteroloph; 11, confluence of the distal mesoflexus with the metaflexus; 12, mesostyle; 13, third lamella; 14, third transverse crest (= mesoloph and/or mesolophule); 15, mesial mesoflexus; 16, second lamella; 17, paracone; 18, labial protoloph; 19, paraflexus; ant., anterior; ling., lingual. (B, C) Note that the position of the fused structures is speculative. The dental terminology is modified after several authors [50–56]. Not to scale. https://doi.org/10.1371/journal.pone.0258455.g002 primarily listed according to their chronostratigraphic order (from the oldest to the most recent) and then alphabetically. Nomenclatural acts The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix “http://zoobank.org/”. The LSID for this publication is: urn:lsid:zoobank.org:pub:ECE79D679203-4929-9CCF-7C8D51C8326E. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: LOCKSS. Dental associations Working on isolated teeth requires the consideration of distinct levels of morphological variation: (i) variation depending on the dental locus; (ii) variation owing to the occlusal wear; (iii) intra-specific or intra-taxonomic variation or polymorphism; and (iv) inter-specific or intertaxonomic variation. In this study, several taxa from TAR-31 are described. The association of the material attributed to these taxa is based on several complementary criteria: (i) a similar occlusal size; (ii) a similar crown height (for a given occlusal size and stage of wear); (iii) a similar proportion of the maximum mesiodistal length and the maximum linguolabial width; (iv) a similar occlusal outline; (v) a similar occlusal pattern (bunolophodonty/lophodonty; taeniodonty/non-taeniodonty; presence/absence, development, position of cusp[-id]s/styl[e/id]s; PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 6 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru presence/absence, continuity/discontinuity, length, height and obliquity of loph[-id]s; and presence/absence and types of fusion of dental structures); and (vi) a compatible occlusal pattern between upper and lower teeth. For teeth at advanced stages of wear, we took into account the potential impact of occlusal wear on characters aforementioned. Based on a wide array of closely- or distantly-related rodent taxa, we prefered to use a size criterion for the recognition of m1s vs m2s and M1s vs M2s (i.e., m1/M1s to be smaller than the m2/M2s in a given species). The recognition of other loci is based on the occlusal shape of teeth (for more details, see descriptions in the Systematic Palaeontology section) and on the presence and/or absence of mesial/distal vertical contact facets due to contiguous teeth. The occlusal shape of M3s is further characterised by a smaller and more labial hypocone with respect to the protocone than on M1–2s. However, the recognition of molar loci is quite ambiguous for several taxa, especially for the first two upper molars. In that case, we determined these loci with uncertainty (e.g., lower molar, m1?, m2?, m3?, upper molar, M1 or M2, M1 or M2?, M3?). Measurements and statistics For each dental specimen of TAR-31, we measured both the maximum mesiodistal length (len) and linguolabial width (wid), as well as the maximum lingual crown height (Hg) and labial crown height (Hb; S1 Table). These measurements were made by M. Boivin with a measuroscope Nikon 10 and a Keyence Digital Microscope VHX-2000F. For Microsteiromys jacobsi and Ricardomys longidens from La Venta, we used the measurements available in Walton [37] (S1 Table). However, we noted some inconsistencies regarding the m3 of the type of Ricardomys, IGM 183847, between the measurements reported in Table 2, p. 33 [37] and the scaled photo of the specimen in Fig 9A, p. 31 [37]. Similarly, the measurements of the type of M. jacobsi (FMNH PM 54672 in Table 6, p. 58 [37]) do not match with its scaled representation (in Fig 16A, p. 56 [37]). For these two specimens, we chose to use the measurements made from their figuration in the Figs 9A and 16A [37]. For the Laventan material attributed to Microscleromys, the measurements made from the figures of the specimens in Walton (Figs 11–13 [37]) and those from Walton (Fig 24.2 [18]) are different. We extracted the measurements from the first study (Ph.D.) because the specimens were photographed (instead of drawn) with a larger scale and more material is represented. The hypsodonty index of a tooth (HI) [62] equals its crown height divided by its anteroposterior length (H/ML); teeth with a HI < 1 are considered as brachydont, those with a HI = 1 are considered as mesodont, and those with a HI > 1 are considered as hypsodont. The latter are either protohypsodont when they still have roots, or euhypsodont if they lack roots [51]. Two hypsodonty indexes were calculated in function to the side of the crown considered: from Hb (HIb) and from Hg (HIg). The HI values mentioned in the descriptions were measured on specimens at the early stages of wear. The MUSM 4658 astragalus from TAR-31 was measured following the protocol of Ginot et al. [57] (Table 1). Table 1. Measurements (mm) of the astragalus from TAR-31, MUSM 4658. ABW AmTL ATL ATW EL EW HH HW LBH 2.265 ~1.220 3.088 2.649 1.447 1.231 0.954 1.316 1.069 LTL MBH MTL NL SL SW TW mTAH 1.860 1.682 2.015 1.382 1.211 1.035 1.832 0.725 ABW, Astragalus Body Width; AmTL, Astragalar-medial Tarsal facet Length; ATL, Astragalus Total Length; ATW, Astragalus Total Width; EL, Ectal facet Length; EW, Ectal facet Width; HH, Head Height; HW, Head Width; LBH, Lateral Body Height; LTL, Lateral Trochlear Length; MBH, Medial Body Height; MTL, Medial Trochlear Length; NL, Neck Length; SL, Sustentacular Facet Length; SW, Sustentacular Facet Width; TW, Trochlear Width; mTAH, medial Trochlear Arc Height. https://doi.org/10.1371/journal.pone.0258455.t001 PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 7 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru In order to help identifying the number of species and discriminating which species of Microscleromys are present in TAR-31 and La Venta, we performed three types of statistical analyses on the maximum mesiodistal length and linguolabial width by locus (i.e., dp4, p4, m1– 2(?), m3(?), dP4, P4, M1–2(?), M3(?)) with R v.4.0.3. [63] (S1 and S2 Files). First, we tested statistical differences between potential taxonomic groups mentioned by Walton (M. paradoxalis nom. nud., M. cribriphilus nom. nud., M. ?paradoxalis, and Microscleromys sp.) [37] and Microscleromys from TAR-31 using Kruskal-Wallis, Permutation Analysis of Variance, Student and Wilcoxon tests, depending to what preconditions were satisfied, with the ‘dunn.test’, ‘RVAideMemoire’, ‘stats’ and ‘trend’ packages [63–66]. Second, partitioning analyses were conducted until five (dp4), nine (m3) or fifteen (other loci) groups depending the number specimens for each loci with the ‘cluster’ package [67]. Finally, we performed several Bayesian models on the most numerous loci including several specimens previously attributed to M. cribriphilus and M. paradoxalis (i.e., p4, m1–2(?), and M1–2(?)) in order to obtain the most probable partioning with the ‘glm’ function of ‘MASS’ and ‘stats’ packages [63, 68]. We tested 39 models for the M1– 2(?) and 51 models for the p4 and m1–2(?) including the partionings following the previous systematic hypotheses and those obtained with the partitioning analyses (see S1 File). Abbreviations Institutional abbreviations. FMNH, Field Museum of Natural History, Chicago, USA; IGM, Servicio Geológico Colombiano (before Instituto Nacional de Investigaciones en Geociencias, Mineria y Quı́mica [INGEOMINAS]), Museo Geológico José Royo y Gómez, Bogotá, Colombia; IGM-DU, Field numbers from expeditions by the INGEOMINAS in cooperation with Duke University. Specimens deposited at the Museo Geológico José Royo y Gómez, Bogotá, Colombia; INBIAL-CV, Instituto de Biologı́a de la Altura, San Salvador de Jujuy, Argentina; ING-KU, Field numbers from expeditions by the INGEOMINAS in cooperation with Kyoto University. Specimens deposited at the Museo Geológico José Royo y Gómez, Bogotá, Colombia; LACM, Los Angeles County Museum, Los Angeles, USA; MCZ, Museum of Comparative Zoology, Cambridge, USA; MACN, Museo Bernardino Rivadavia, Buenos Aires, Argentina; MLP, Museo de La Plata, La Plata, Argentina; MMP, Museo Municipal De Ciencias Naturales Lorenzo Scaglia, Mar del Plata, Argentina; MNHN, Museum National d’Histoire Naturelle, Paris, France; MPEF-PV, Museo Paleontológico Egidio Feruglio, Trelew, Argentina; MUSM, Museo de Historia Natural de la Universidad Nacional Mayor San Marcos, Lima, Peru; UCMP, University of California Museum of Paleontology, Berkeley, USA; UFAC, Coleção de Paleovertebrados do Laboratório de Pesquisas Paleontológicas, Universidade Federal do Acre, Rio Branco-AC, Brazil; UNC, Departamento de Geociencias, Universidad Nacional de Colombia, Bogotá, Colombia. Other abbreviations. CVP, locality belongs to the Fish Bed in La Venta area collected by screenwashing during the expeditions by the INGEOMINAS in cooperation with Duke University; IGM-DU loc., fossiliferous locality found during the expeditions by the INGEOMINAS in cooperation with Duke University in La Venta area; UCMP loc., fossiliferous locality found during the expeditions by the University of California in La Venta area. Systematic paleontology Nomenclatural Remark: The new species and genera described below must be referred to Boivin & Walton, 2021, following the article 50.1 and the “recommendation 50A concerning multiple authors” of the International Code of Zoological Nomenclature (p. 182 [38]). This is of particular interest for nomina nuda that were previously used by Walton [18, 37] and made available here, following the International Code of Zoological Nomenclature [38]. PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 8 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Rodentia Bowdich, 1821 Ctenohystrica Huchon, Catzeflis & Douzery, 2000 Hystricognathi Tullberg, 1899 Caviomorpha Wood, 1955 Erethicavioi Boivin, 2019 in Boivin, Marivaux & Antoine, 2019 Erethizontoidea Bonaparte, 1845 Microsteiromys Boivin & Walton gen. nov. urn:lsid:zoobank.org:act:70F9B9F7-51EB-4FFF913C-2E89EBF5F9DB Type species Microsteiromys jacobsi gen. et sp. nov. Species content Only the type species. Derivation of name Based on the original description of the species [37]: ‘In reference to its small size and general resemblance to the erethizontoid Steiromys Ameghino, 1887’. Geographical and stratigraphical distribution La Venta, Baraya and Cerro Colorado members, Villavieja Fm. (Laventan SALMA, late middle Miocene), Huila Department, Colombia. Generic diagnosis As for the type and only known species. Microsteiromys jacobsi Boivin & Walton sp. nov. urn:lsid:zoobank.org:act:1DF51272-C9494105-BC5C-299F88252206 Fig 3 and S1 Table Nom. nud. Microsteiromys jacobsi Walton, 1990, Fig 16A–16C, p. 56. Nom. nud. Microsteiromys jacobsi Walton, 1997, Figs 24.1B and 24.2B, p. 394, 395. Holotype FMNH PM 54672, right mandibular fragment bearing dp4–m3 (Walton, 1990: Fig 16A; Walton, 1997: Figs 24.1B and 24.2B; Fig 3A). Referred material IGM-DU 89–249, left mandibular fragment bearing p4–m1 (Walton, 1990: Fig 16C; Fig 3B); IGM-DU 88–034, right posterior fragment of lower tooth (Walton, 1990: Fig 16B; Fig 3C). PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 9 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Fig 3. Microsteiromys jacobsi gen. et sp. nov. from La Venta, Colombia (late middle Miocene, Laventan SALMA). (A) FMNH PM 54672��� , right mandibular fragment bearing dp4–m3. (B) IGM-DU 89–249, left mandibular fragment bearing p4–m1 (reversed), (C) IGM-DU 88–034, right posterior fragment of m1 or m2. Occlusal views. The asterisks appoint the holotype material. Based on Walton (Fig 16A-16C [37]). The illustrations of the fossil specimens are computerised schematic line drawings. https://doi.org/10.1371/journal.pone.0258455.g003 Derivation of name Based on the original description of the species [37]: ‘In honour to Dr. Louis L. Jacobs, palaeontologist and educator’. Type locality IGM-DU loc. 022 or UCMP loc. V4536 (only for the type), Monkey Beds, La Venta Badlands, upper Magdalena Valley, Huila Department, Colombia. Other localities CVP 10 (for IGM-DU 88–034), Fish Bed and screewashed locality 032–5 (for IGM-DU 89– 249), El Cardón Red Bed, La Venta Badlands, upper Magdalena Valley, Huila Department, Colombia. Formation and age Baraya and Cerro Colorado members, Villavieja Fm., late middle Miocene (i.e., Laventan SALMA). Diagnosis Rodent characterised by a small size and brachydont teeth. Lower teeth with a tetralophodont and non-taeniodont pattern, complete metalophulid I, and oblique and complete ectolophid. dp4s with a mesiodistally short and linguolabially wide trigonid and a curved metalophulid I. Large p4s with a linguolabially wide trigonid. Mesiodistally elongated lower molars with a narrow mesoflexid and well-extended metaflexid. Differs from Eopululo, Protosteiromys, PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 10 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Branisamyopsis, Hypsosteiromys, Parasteiromys, Eosteiromys, Steiromys, and Neosteiromys in having a smaller size; from Eosteiromys annectens and Steiromys duplicatus in having non-taeniodont lower teeth; from Hypsosteiromys in having brachydont lower teeth with transverse lophids and a complete metalophulid I, and less elongated dp4s; from Shapajamys, Branisamyopsis, Eosteiromys homogidens, Steiromys, and Coendou insidiosus in having tetralophodont dp4s; from Eosteiromys segregatus in having a longer p4 with a long anterior arm of the entonconid; from Shapajamys, Palaeosteiromys, Protosteiromys, Branisamyopsis, Eosteiromys annectens, Steiromys, and Neosteiromys in having more rectangular lower molars; from Palaeosteiromys, Branisamyopsis australis, Branisamyopsis praesigmoides, Steiromys duplicatus, and Neosteiromys in having always tetralopodont lower molars; from Shapajamys, Branisamyopsis praesigmoides and Steiromys duplicatus in having an ectolophid always complete and oblique on lower molars; from Shapajamys in having an ectolophid always oblique on lower molars; from Palaeosteiromys in having lower molars with a metaflexid more extended; from Protosteiromys, Branisamyopsis, Eosteiromys homogidens, Steiromys detentus, and Neosteiromys in having a narrower mesoflexid on lower molars. Description FMNH PM 54672 (Fig 3A) and IGM-DU 89–249 (Fig 3B) are two mandibular fragments bearing dp4–m3 and p4–m1, respectively. The body of the FMNH PM 54672 mandible is anteriorly broken at the level of the anterior part of the lower diastema, which makes visible the lower incisor alveola. Posteriorly, the angular apophysis and most of the ascending ramus, including the mandibular condyle, are missing. The coronoid process is broken at its base. Labially, a mental foramen is located at the midpoint of the body of the mandible height (i.e., dorsoventrally). The notch for the insertion of the tendon of the zygomatico-mandibularis pars infraorbitalis is medium-sized, below dp4–m1, and centrally situated on the labial edge of the mandible. The anterior tip of the masseteric crest ends below the m1, and links the notch for the insertion of the tendon of the zygomatico-mandibularis pars infraorbitalis, at the level of its posteroventral region. The masseteric crest is posteriorly broken. It is posteroventrally directed and prominent in its anterior part. The dp4 of the FMNH PM 54672 mandibular fragment (Fig 3A) is mesially and distolingually broken at the level of the metaconid, metalophulid I, mesostylid, entoconid, and posterolophid. This tooth is more worn and shorter than the molars (m1–3) beared by FMNH PM 54672. It is characterised by a short trigonid, which has an equal width with the talonid. This dp4 is brachydont and non-taeniodont. Despite its damaged state, no supplementary lophids are visible in addition to the metalophulid I, second transverse cristid, hypolophid and posterolophid, which suggests that the FMNH PM 54672 dp4 was tetralophodont. As this tooth is at an advanced stage of wear, the cuspids are not well defined to the lophids. On the lingual margin of the tooth, the anteroflexid and mesoflexid are closed, thereby suggesting that the mesostylid (or its arms) was connected to the metaconid (or its posterior arm) and its posterior arm reaches the entoconid (or its anterior arm). The metalophulid I, mesially convex, seems to be complete and strongly connected to the protoconid labially (hypothesis 1 on Fig 4A1; see the remark below). This cuspid is crestiform and faintly linked to the second cristid. The latter is slightly oblique (i.e., mesiodistally directed). It bears posteriorly a tiny and horrizontal cristulid, which joins the mesial margin of the hypolophid, dividing the mesoflexid into two small and round fossettids. The labiodistal aspect of the second cristid links to a transverse hypolophid via a very short and oblique ectolophid. The hypoconid is mesiolingually-distolabially compressed. It displays strong anterior arm, anterior outgrowth and posterolophid. The anterior outgrowth is mesiolabially oriented and PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 11 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Fig 4. Hypotheses regarding the homology of structures on dp4 of the erethizontoid Microsteiromys jacobsi (A) and Nuyuyomys chinqaska (B). (A1) Hypothesis 1: protoconid in anterior position, aligned with the metalophulid I (followed here). (A2) Hypothesis 2: protoconid in posterior position, aligned with the second transverse cristid. (B1–9) Equivalent transversal cristids (and their associated cuspids/stylids) indicated by numbers (1–6) on all dp4s of N. chinqaska. (B10) Different hypotheses regarding the position of the anterior cuspids/stylids based on the morphology of MUSM 4333. For the transversal cristids 1 and 2, the position and number of the structures can slightly vary depending the considering specimen (e.g., MUSM 4332). In the case of the hypotheses 1–4, the neoconid(s) can be present on the cristid 1 or not (correspond solely to thickenings of this cristid). The hypotheses 1 and 3 are the most consistent with the morphology and development of the dental structures. (A) IGM-DU 89–249, (B1) MUSM 4326, (B2) MUSM 4327, (B3) MUSM 4328, (B4) MUSM 4329, (B5) MUSM 4325, (B6) MUSM 4332, (B7) MUSM 4331, (B8) MUSM 4330, and (B9) MUSM 4333. Ed, entoconid; Hd, hypoconid; Md, metaconid; Mld, mesostylid; Nd, neoconid; Pd, protoconid. The illustrations of the specimens are computerised schematic line drawings. https://doi.org/10.1371/journal.pone.0258455.g004 PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 12 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru delimits with the anterior arm of the hypoconid, hypolophid, ectolophid, second transverse cristid and protoconid, a triangular-shaped hypoflexid, oriented distolingually, and showing a wide labial opening. Although the lingual part of the posterolophid is missing, the metaflexid appears lingually closed as is the case as well for the anteroflexid and the mesoflexid. The p4 of the IGM-DU 89–249 mandibular fragment (Fig 3B) is much longer and moderately worn with respect to the associated m1, which shows a very advanced state of wear characterised by a fusion all lophids and cuspids/stylids. As the FMNH PM 54672 dp4, it is brachydont, tetralophodont and non-taeniodont. However, it is much longer than the dp4 and has a mesial margin more straight. The protoconid corresponds to a large dentine plateform on the mesiolabial corner of the tooth. It is mesially connected to a metalophulid I, reaching the metaconid on the lingual margin, and distally to the second cristid and to an oblique and long ectolophid. The labial part of the metalophulid I is mesiolingually directed, while its lingual part is slightly directed distolingually. The metaconid is faintly linked to the mesostylid, which appears to have an equal size to the former. Due to the moderate state of wear of the tooth, the presence of a posterior arm of the metaconid cannot be assessed, but if it would be present, the latter would be short. The mesostylid bears short anterior and posterior arms. Comparatively, the entoconid has longer arms, especially its anterior arm, reaching the posterior arm of the mesostylid. The second cristid is complete and slightly mesiolingually directed, whereas the hypolophid and the posterolophid are transverse. There is a connection between the latter and the posterior arm of the entoconid, which lingually closes the metaflexid. Thus, all the lingual flexids are closed and form round-oval fossettids. The anterior arm of the hypoconid is strong and mesiolingually oriented, whereas its anterior outgrowth is long and mesiolabially oriented. These cristids, with the ectolophid and protoconid, delimit a widely open and retroverse hypoflexid (triangular-shaped and distolingually directed). The m1–3 of the FMNH PM 54672 specimen (Fig 3A) have a similar/compatible size with the unique and very worn molar (m1) borne by the IGM-DU 89–249 mandibular fragment (Fig 3B). On FMNH PM 54672, all molars are roughly equal in length. The m1 and m2 have a similar width, whereas m3 is narrower. The latter is characterised by a shorter posterolophid due to a linguolabial compression of its posterior part. The morphology of lower molars has the same pattern than that of dp4 and p4. As dp4 and p4, molars are brachydont, tetralophodont, non-taeniodont with a strong anterior arm of the hypoconid, and they have an oblique ectolophid and a triangular-shaped hypoflexid showing a wide labial opening. This flexid has a general distolingual direction, but the latter varies depending on the locus: it is slight on m3, more oblique on dp4 and m1, and the most oblique on p4 and m2. As dp4 and p4, m1–2 display all their lingual flexids closed, whereas m3 has a mesoflexid opened by a shallow and thin furrow, and a posterolophid well-separated from the entoconid. On m1–2, this cuspid appears to have a long posterior arm On m1–3, the metalophulid I is complete, transverse to slightly arched. The metaconid bears a long posterior arm strongly connected to the mesostylid (or its anterior arm). The second cristid and hypolophid are close and separated by a narrow mesoflexid (i.e., furrow-like flexid). These two transverse cristids are straight and roughly parallel on m1 and m3. On m2, most part of the second cristid (i.e., lingual part) is slightly distolingually directed. Because of a thinning on its central part on m1 and its orientation change on m2, the second cristid may be composed of two cristids on these lower molars: a neomesolophid lingually and a posterior arm of the protoconid labially. On all molars, the posterolophid is curved, especially on m3. The metaflexid/metafossettid is mesiodistally well extended. PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 13 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Remark Considering the pattern of dp4s of some erethizontoids (Steiromys detentus, MLP 15−339; Erethizon dorsatum, MCZ 51367) [69], another hypothesis can be proposed with respect to the position of the protoconid on the FMNH PM 54672 dp4 (hypothesis 2 on Fig 4A2): this cuspid could be situated more posterolingually than in hypothesis 1 and aligned with the second transverse cristid. The discovery of a pristine dp4 of this species would make it possible to decide between these two proposed hypotheses. Comparisons Brachydonty, non-taeniodonty, extended and moderately narrow flexids, weak obliquity of lophids, and p4 with a wide trigonid suggest erethizontoid affinities for these specimens from La Venta. They are markedly smaller than most of erethizontoid species, except for Kichkasteiromys raimondii, Shapajamys labocensis [70], Palaeosteiromys amazonensis [71], Noamys hypsodonta [72] and the material from the early middle Miocene of Madre de Dios, Peru formerly attributed to cf. Microsteiromys sp. (MD-67; [31] and see below). Kichkasteiromys raimondii, N. hypsodonta and the taxon from MD-67 being documented only by upper teeth, direct morphological comparisons with the material of La Venta cannot be made. The lower molars from La Venta differ from those of Shapajamys and Palaeosteiromys in being more rectangular. They are characterised by an ectolophid always complete and oblique, contrary to Shapajamys, and by a metaflexid more extended than in Palaeosteiromys. These specimens from La Venta are always tetralophodont, contrary to those of Palaeosteiromys, Branisamyopsis australis, Branisamyopsis praesigmoides, Steiromys duplicatus and Neosteiromys, which can exhibit a pentalophodont pattern by the addition of a mesial neolophid [71, 73–77]. By its rounded shape as well as its short and wide trigonid, the FMNH PM 54672 dp4 is close to the dp4s of the extant species Coendou insidiosus. The FMNH PM 54672 dp4 is tetralophodont, as dp4s of Hypsoteiromys [74, 78], but the latter are clearly more elongated, with a longer trigonid and more extended lingual flexids. Contrary to FMNH PM 54672, dp4s of other erethizontoids are elongated with a pentalophodont (Eosteiromys homogidens, Eosteiromys annectens and C. insidiosus) or hexalophodont (Shapajamys, Branisamyopsis, ?Eosteiromys sp. nov. sensu Candela [69], and Steiromys) pattern. MNHN 1903-3-18 (Fig 32 [79]) was originally determined as a m3 of Protosteiromys asmodeophilus. However, its trigonid is clearly narrower than its talonid, a feature better characterising a premolar (p4 or dp4). MNHN 1903-3-18 is tetralophodont and non-taeniodont as p4s and dp4s from La Venta, but has lingual flexids much more extended. The material from La Venta differs from Eopululo, Protosteiromys, Branisamyopsis, Hypsosteiromys, Parasteiromys, Eosteiromys, Steiromys and Neosteiromys [73–76, 78–82] in having a smaller size; from Hypsosteiromys in having brachydont lower teeth with transverse lophids and a complete metalophulid I; from E. annectens and S. duplicatus in having non-taeniodont lower teeth; from Branisamyopsis, S. duplicatus, Steiromys verzii sensu Candela [74], and some p4s of S. detentus in having a p4s with tetralophodont pattern; and from Eosteiromys segregatus in having a longer p4 with a long anterior arm of the entonconid; from Protosteiromys, Branisamyopsis, E. annectens, Steiromys and Neosteiromys in having more rectangular lower molars; from Protosteiromys, Branisamyopsis, E. homogidens, Steiromys detentus and Neosteiromys in having a narrower mesoflexid on lower molars; from B. praesigmoides and S. duplicatus in having an ectolophid always complete on lower molars. To sum up, these few specimens from La Venta show a set of distinctions (i.e., small size; brachydonty; tetralophodont and non-taeniodont lower teeth; a complete metalophulid I, and an oblique and complete ectolophid on lower teeth; round dp4s with a short and wide trigonid; mesiodistally elongated lower molars with narrow mesoflexid and well-extended metaflexid) that allows us to refer PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 14 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru them to a new genus and new species. Thus, we validate Microsteiromys jacobsi Walton, 1990, which had remained until now as a nomen nudum. Nuyuyomys Boivin & Walton gen. nov. urn:lsid:zoobank.org:act:360EBF95-C067-4C81-81F48FAD41067E7E Type species Nuyuyomys chinqaska gen. et sp. nov. Species content Only the type species. Derivation of name From Nuyuy, flooding in Quechua language, referring to the location of both fossil localities yielding this genus (at the water level only during the driest season and below it most of the year) and -mys, mouse in Greek, a classical suffix for rodents. Geographical and stratigraphical distribution Pilcopata (MD-67), unnamed Fm. (Colloncuran-Laventan, middle Miocene), Madre de Dios Department, Peru; Tarapoto/Juan Guerra (TAR-31), lower member, Ipururo Fm. (Laventan, late middle Miocene), San Martı́n Department, Peru. Generic diagnosis As for the type and only known species. Nuyuyomys chinqaska Boivin & Walton sp. nov. urn:lsid:zoobank.org:act:353264FD-EB4C428F-9F6D-EFC6DC1D7A04 Fig 5 and S1 Table 2013 cf. Microsteiromys sp. Antoine et al., Fig 3M, 3N, p. 94. Holotype MUSM 4308 (Fig 5R), right M1?. Referred material MUSM 4325–4329 (Fig 5F), left dp4s; MUSM 4330–4333 (Fig 5A), right dp4s; MUSM 4334 (Fig 5B), left p4; MUSM 4335–4339, left lower molars; MUSM 4340–4342, right lower molars; MUSM 4343, 4344, left m1s; MUSM 4345–4348 (Fig 5C and 5G), right m1s; MUSM 4349– 4357 (Fig 5H, 5J and 5K), left m2s; MUSM 4358–4363 (Fig 5D and 5L–5N), right m2s; MUSM 4364 (Fig 5I), left m3; MUSM 4365, 4366 (Fig 5E and 5O), right m3s; MUSM 4284–4288 (Fig 5Z), left dP4s; MUSM 4289–4294 (Fig 5P, 5U, 5C’ and 5D’), right dP4s; MUSM 4295 (Fig 5Q), left P4; MUSM 4296, 4297, right upper molars; MUSM 4299, right M1 or M2?; MUSM 4300– 4306, left M1?s; MUSM 4307–4314 (Fig 5A’ and 5B’), right M1?s; MUSM 4315, right M2 (or M3)?; MUSM 4316–4320 (Fig 5S and 5W), left M2?s; MUSM 4321, 4322, right M2?s; MUSM 4323 (Fig 5X), left M3; MUSM 4324 (Fig 5T), right M3. PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 15 / 80 PLOS ONE PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 Miocene caviomorphs from Tarapoto, Peru 16 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Fig 5. Erethizontoid rodents from TAR-31, Peru (late middle Miocene, Laventan SALMA). (A-E’) Nuyuyomys chinqaska gen. et sp. nov. (F’) Erethizontoidea gen. et sp. indet. 1. (G’) Erethizontoidea gen. et sp. indet. 2. (A) MUSM 4332, right dp4 (reversed). (B) MUSM 4334, left p4. (C) MUSM 4347, right m1 (reversed). (D) MUSM 4359, right m2 (reversed). (E) MUSM 4366, right m3 (reversed). (F) MUSM 4329, left dp4. (G) MUSM 4348, right m1 (reversed). (H) MUSM 4355, left m2. (I) MUSM 4364, left m3. (J) MUSM 4356, left m2. (K) MUSM 4352, left m2. (L) MUSM 4362, right m2 (reversed). (M) MUSM 4363, right m2 (reversed). (N) MUSM 4360, right m2 (reversed). (O) MUSM 4365, right m3 (reversed). (P) MUSM 4290, right dP4 (reversed). (Q) MUSM 4295, left P4. (R) MUSM 4308��� , right M1? (reversed). (S) MUSM 4319, left M2?. (T) MUSM 4324, right M3 (reversed). (U) MUSM 4292, right dP4 (reversed). (V) MUSM 4306, left M1?. (W) MUSM 4320, left M2?. (X) MUSM 4323, left M3. (Y) MUSM 4303, left M1?. (Z) MUSM 4285, left dP4. (A’) MUSM 4309, right M1? (reversed). (B’) MUSM 4314, right M1? (reversed). (C’) MUSM 4293, right dP4 (reversed). (D’) MUSM 4294, right dP4 (reversed). (E’) MUSM 4298, M1 or 2?. (F’) MUSM 4367, left p4. (G’) MUSM 4368, right M1 or M2 (reversed). The asterisks appoint the holotype material. The illustrations of the fossil specimens are scanning electron photomicrographs. https://doi.org/10.1371/journal.pone.0258455.g005 Tentitavely referred material MUSM 1974 (Antoine et al., 2013: Fig 3M, p. 94), left P4; MUSM 1975 (Antoine et al., 2013: Fig 3N, p. 94), right upper molar; MUSM 4298 (Fig 5E’), left M1 or M2?. Derivation of name From chinqaska, vanished in Quechua language, referring to the disappearance of both MD67 and TAR-31 outcrops since their discovery, due to water erosion and paleontological exploitation, respectively. Type locality TAR-31, Tarapoto/Juan Guerra, Mayo River, Shapaja road, San Martı́n Department, Western Amazonia, Peru. Other locality MD-67, Pilcopata, Rinconadera and Madre de Dios rivers, Madre de Dios Department, Western Amazonia, Peru. Formation and age Unnamed Fm., middle Miocene (i.e., Colloncuran-Laventan SALMA); lower member, Ipururo Fm., late middle Miocene (i.e., Laventan SALMA). Diagnosis Rodent characterised by small size, brachydont lower teeth and dP4s, brachydont-mesodont P4s and upper molars, and a weak obliquity of loph(-id)s. dp4s, p4s and upper teeth with a non-taeniodont pattern. Lower molars with a non-taeniodont or pseudo-taeniodont pattern at early stages of wear, the former pattern being the most frequent. Elongated dp4s with a hexalophodont pattern. p4s are longer than wide, with a large trigonid and tetralophodont pattern. Lower molars with a tetralophodont or pentalophodont pattern, the latter corresponding to the addition of a neolophid between the metalophulid I and the second transverse cristid. dP4s with a pentalophodont or tetralophodont pattern, the former pattern being the most frequent. The metaloph (when present) can be lingually free or connected to the posteroloph or to both the third crest and posteroloph on dP4s. Round and tetralophodont P4s with an anterior arm of the protocone thicker and mesiolabially directed connected to an anteroloph lower, thinner and roughly transverse, and without endoloph. Pentalophodont upper molars with a metaloph always long and joining the metacone to the posteroloph on M2?s and complete or reduced on M1?s and M3?s. Nuyuyomys is markedly smaller than all erethizontoid species except PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 17 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Kichkasteiromys, Shapajamys, Palaeosteiromys, Microsteiromys and Noamys. Differs from Eosteiromys homogidens, Eosteiromys annectens, Hypsosteiromys, Microsteiromys and Coendou insidiosus in having hexalophodont dp4s; from Shapajamys, Protosteiromys, Eosteiromys and Microsteiromys in having no neolophid on the anteroflexid on lower molars; from Shapajamys, Branisamyopsis praesigmoides and Steiromys duplicatus in having an ectolophid always complete. Differs from Kichkasteiromys, Shapajamys and Palaeosteiromys in having a metaloph disconnected labially to the metacone on some upper molars; from Shapajamys in having no distinct metaloph on P4s, metaloph more developed on ?M2s and an ectolophid always oblique on lower molars; from Kichkasteiromys in having a lingual protoloph less lingually situated and a posterior outgrowth of the protocone more oblique on upper molars; from Palaeosteiromys in having a mesoflexid more extended on m3s; from Microsteiromys in having more elongated dp4s with a narrower trigonid, the mesoflexid still lingually open at advanced stage of wear on m1–2s and less rectangular lower molars. Differs from Hypsosteiromys in having loph(-id)s more transverse, round P4s, pentalophodont M1–2s, and a complete metalophulid I on lower teeth; from Noamys in having pentalophodont and sligthly smaller dP4 and M1 without oval-shaped occlusal contour and flexi less extended. Differs from Eosteiromys annectens and Steiromys duplicatus in having non-taeniodont lower teeth; from Neosteiromys and Parasteiromys in having always a complete mure on dP4s and upper molars; from Eopululo, Protosteiromys pattersoni and Neosteiromys in having a P4 without endoloph; from Eosteiromys homogidens in having a mesial mesoflexus less mesiodistally extended on P4s; from Eosteiromys annectens, Branisamyopsis and Neosteiromys in having non-taeniodont dP4s and upper molars; from Eopululo and Protosteiromys pattersoni in having upper molars always with a metaloph (which is not reduced, far from being a relictual structure); from Protosteiromys, Parasteiromys, Branisamyopsis, Eosteiromys, Steiromys and Neosteiromys in having some elongated upper molars (potential M1s) and an incomplete metaloph on some upper molars; from Protosteiromys medianus in having a complete third crest on M3s. Description dp4s are brachydont and much longer than wide, with a talonid slightly wider than the trigonid. They are non-taeniodont and hexalophodont. In addition to six complete transverse lophids, one dp4 (MUSM 4332; Fig 5A) has a tiny low cristulid stemming from the metalophulid I on the anteroflexid. Owing to its low elevation and shortness, it is interpreted here as a neolophid. The entoconid and hypoconid and their associated lophids (i.e., hypolophid [cristid 5 in Fig 4B1–4B9], anterior outgrowth of the hypoconid, anterior arm of the hypoconid, and posterior arm of hypoconid/posterolophid [cristid 6 in Fig 4B1–4B9]), distally positioned on the tooth, are well recognisable. The entoconid is the largest lingual cuspid. It is clearly isolated on almost all dp4s. On MUSM 4331, its isolation is less important because it is separated from the posterolophid by a thinner and shallower notch. The hypolophid is transverse or slightly arcuate (i.e., mesially concave). The crestiform hypoconid displays an anterior arm, which is mesiolingually oriented, thin and linked to the labiodistal extremity of the hypolophid. On MUSM 4332 (Fig 5A), the mesialmost part of the anterior arm of the hypoconid is lower than its distal part, but it is connected to the hypolophid and clearly separates the hypoflexid from the metaflexid (i.e., non-taeniodont pattern). The hypoconid exhibits a strong and short anterior outgrowth. The posterolophid is particularly long and curved, notably in its lingual part. On the less worn dp4s, it is composed of two (MUSM 4329; Fig 5F) or three (MUSM 4327 and 4332; Fig 5A) interconnected parts, a posterior arm of the hypoconid and posterolophid included. Mesially, the recognition of the main cuspids (i.e., protoconid, metaconid, and mesostylid) and cristids (i.e., posterior arm of the protoconid, metaconid cristid, mesolophid, PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 18 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru and neomesolophid) is ambiguous by the addition of some neostructures (i.e., neoconids and neolophids). Several hypotheses of homology can be proposed (Fig 4B10). The mesialmost cristid (cristid 1 in Fig 4B1–4B9) is curved. It (mainly) corresponds to the metalophulid I. It shows one or several cuspate thickenings in its central part and/or extremities (MUSM 4325, 4326, 4329, and 4330, 4332, 4333; Figs 4B1, 4B4–4B6, 4B8, 4B9 and 5A and 5F). They can correspond to the main cuspids (i.e., protoconid, metaconid, mesostylid) and/or neoconids (Fig 4B10). The second cristid (cristid 2 in Fig 4B1–4B9) is highly variable. It can be constituted of one or two linked cristulids, which can be associated to the thickenings of the metalophulid I. They can be connected to the central part of the cristid 1 (MUSM 4329 and 4333; Fig 4B4 and 4B9) and/or to one (MUSM 4325; Fig 4B5) or two of its extremities (MUSM 4326, 4330, and 4332; Fig 4B1, 4B6 and 4B8). The next transverse cristid (cristid 3 in Fig 4B1–4B9) is thick and long, stemming from a cuspate structure on the lingual margin of the tooth to connect to a large another cuspid-shaped structure on the labial margin. These cuspid-like structures are clearly isolated at early stages of wear (MUSM 4326, 4330, and 4332; Figs 4B1, 4B6, 4B8 and 5A) and can link the structures located mesially (metalophulid I and annexes) at more advanced staged of wear (MUSM 4329 and 4333; Figs 4B4, 4B9 and 5F). On MUSM 4326 and 4329, this transverse cristid has two parts: a lingual cristid connected to a labial one, equally sized (MUSM 4326; Fig 4B1) or shorter (MUSM 4329; Figs 4B4 and 5F). Owing to the important development of this cristid and of its cuspate structures on all dp4s, they are probably not neostructures (hypotheses 1 and 3 in Fig 4B10). The transverse cristid appearing in fourth position (cristid 4 in Fig 4B1–4B9) is labially connected to the hypolophid via the distal ectolophid on all dp4s. This cristid can be continuous (MUSM 4327, 4328, and 4330–4333; Fig 4B2, 4B3, 4B6–4B9) or divided into two parts, which are joined (4326 and 4329; Fig 4B1 and 4B4). In addition, it can be high (MUSM 4326 and 4330–4333; Fig 4B1, 4B6–4B9), entirely low (MUSM 4327; Fig 4B2) or only its labial part can be low (MUSM 4329; Figs 4B4 and 5F). On MUSM 4327 (Fig 4B2), it is slightly connected to a bulbous cuspid clearly distinct on the lingual margin of the tooth. MUSM 4334 is an almost pristine p4 (Fig 5B). It is longer than wide, with a trigonid narrower than the talonid. It is brachydont and tetralophodont. On its mesiolingual corner, the crestiform metaconid is convex forward. Distally, this cuspid displays a very short posterior arm. There is a tiny spur-like cristulid on the anteroflexid, labially free and stemming from the distal extremity of the posterior arm of the metaconid. Mesially, the metaconid has a transverse and long anterior arm. From its central part, a short cristulid runs distally on the anteroflexid. The anterior arm of the metaconid is linked to a faintly curved anterior arm of the protoconid. The latter cristid is clearly sloped with a high distal part stemming from the triangular-shaped protoconid. The second transverse cristid is essentially composed of a long neomesolophid joining a very short posterior arm of the protoconid. The ectolophid is mainly oblique and Sshaped. It reaches the distal slope of the protoconid. The entoconid is high and it has a sharp posterior arm. Labially, this cuspid is connected to a hypolophid, strong and distolingually oblique. On the labiodistal margin of the ectolophid-hypolophid junction, a tiny cristulid is present. Its top is separate from the anterior arm of the hypoconid by a very shallow notch. However, the contact between these structures remains high despite the presence of this notch (i.e., non-taeniodont pattern). The anterior outgrowth of the hypoconid is short and directed mesiolabially. The posterolophid is transverse with curved lingual and labial extremities. On the lingual margin, all structures (i.e., metaconid, mesostylid, entoconid and lingual extremity of the posterolophid) are merged together at their base, but they remain disconnected at their apex. The mesostylid is discreet and isolated from the posterior arm of the metaconid and the entoconid by wide and deep furrows. The notch separating the entoconid from the lingual extremity of the posterolophid is thinner and shallower. The anteroflexid and metaflexid are PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 19 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru more expanded than the mesoflexid. The hypoflexid is triangular-shaped with a wider labial openning. The lower molars are brachydont and rectangular-shaped. m3s (MUSM 4364–4366; Fig 5E, 5I and 5O) show a talonid slightly narrower than the trigonid, notably lingually. m1s and m2s usually have a trigonid and a talonid roughly of same width and m2s are larger than m1s and m3s. On all lower molars, the metaconid, protoconid and hypoconid are crestiform, whereas the mesostylid and entoconid are more bulbous. The metalophulid I is transverse and complete, joining the metaconid to the protoconid. On the lower molars at the earliest stage of wear (MUSM 4342, 4347, and 4359; Fig 5C and 5D), this cristid corresponds to a long anterior arm of the metaconid, whose lingual extremity is low and linked a short anterior arm of the protoconid. On most lower molars, the mesostylid is linked to the posterior arm of the metaconid, this connection can be slight or tight. These two structures can be separated by a wide furrow (e.g., MUSM 4336, 4357, and 4365; Fig 5O) or a thin notch (e.g., MUSM 4356, 4347, and 4348; Fig 5C, 5G and 5J). Several lower molars exhibit two short cristulids on their mesiolingual area: the first cristulid is situated on the distolingual part of the metalophulid I, and the second is located distally to the metaconid, on its posterior arm (MUSM 4344, 4352, 4355, 4364, and 4362; Fig 5H, 5I, 5K and 5L). On MUSM 4344, 4364 and 4362 (Fig 5I and 5L), these two cristulids are joined, and form a mesial neolophid on the anteroflexid (i.e., pentalophodont pattern). On MUSM 4352 and 4355, they remain separate (Fig 5H and 5K). On MUSM 4351, there is only the cristulid of the metalophulid I, whereas on MUSM 4348 (Fig 5G), there is only the cristulid distally to the metaconid. The second transverse cristid is a long neomesolophid directly connected to the protoconid or via its very short posterior arm. It can be slightly oblique, transverse, or sometimes sinuous (S-shaped). The ectolophid is always oblique, either aligned with the protoconid or a little more longitudinal. The entoconid can display strong anterior and posterior arms. On MUSM 4336 and 4351, the posterior arm is particularly long. The latter can be connected (e.g., MUSM 4336, 4352, 4364, and 4359; Fig 5D, 5I and 5K) or disconnected (e.g., MUSM 4351, 4354, 4356, 4357, 4347, and 4365; Fig 5C, 5J and 5O) to the posterolophid. The hypolophid is well marked and often parallel to the second transverse cristid. The anterior arm of the hypoconid is complete and high in most cases (i.e., nontaeniodont pattern). Two lower molars, MUSM 4356 and 4347 (Fig 5C and 5J), show a pseudo-taeniodont pattern. On MUSM 4356 (Fig 5J), the anterior arm of the hypoconid is complete, but moderately low. On MUSM 4347 (Fig 5C), there is a very low cristulid stemming from the hypolophid-ectolophid junction and joining the mesial slope of the hypoconid, thereby involving a partial confluence between the metaflexid and the hypoflexid. dP4s are longer than wide with a rectangular or slightly trapezoidal occlusal outline. On all dP4s (except on MUSM 4293; Fig 5C’; see below), the protocone is somewhat bulbous, a morphology accentuated by a thinning at the level of its connection with the anteroloph. In addition, the lingual extremity of the latter is sometimes thickened (MUSM 4284, 4287, and 4289– 4292; Fig 5P and 5U). The anteroloph is mesially convex, and can have an enamel thickening on its labial part, that we interpret as a parastyle, associated with a spur-like crestule (MUSM 4285, 4287, 4289, 4290, and 4292; Fig 5P, 5U and 5Z). This crestule remains lingually free on the paraflexus. On the labial margin of dP4s, the paracone is the largest cusp. It is clearly separated mesially from the labial extremity of the anteroloph and distally from the mesostyle by wide and deep furrows in most cases. On MUSM 4290 (Fig 5P), the paracone is separate from the anteroloph by a shallower notch. The labial protoloph can be transverse (MUSM 4291), oblique (i.e., mesiolabially directed; MUSM 4293; Fig 5C’) or with an oblique lingual part and a labial part more transverse (on the other dP4s). On MUSM 4287, in its middle, the labial protoloph displays a tiny crestule on its mesial part. Its ends close to the distal extremity of the crestule stemming from the parastyle, but does not connect to it. The mure is oblique (e.g., PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 20 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru MUSM 4290 and 4293; Fig 5P and 5C’) or more longitudinal (i.e., parallel to the mesiodistal long axis of the tooth; e.g., MUSM 4291 and 4292; Fig 5U). Its junction with the labial protoloph is linked to the protocone via its thin anterior arm (i.e., non-taeniodont pattern). Labially, the mesostyle is large, whereas the metacone is faintly visible from the posteroloph. On MUSM 4290–4292 and 4294 (Fig 5P, 5U and 5D’), the mesostyle shows a long posterior arm. The mesostyle or its posterior arm is either linked to the metacone-posteroloph complex (MUSM 4287, 4290, 4291, and 4293; Fig 5P and 5C’) or is separated from this cusp by a thin and shallow notch (MUSM 4285, 4286, 4292, and 4294; Fig 5U, 5Z and 5D’). The third crest is complete and S-shaped. On MUSM 4285 and 4292 (Fig 5U and 5Z), this crest consists of a long mesoloph stemming from the mesostyle and joining lingually the mure-anterior arm of the hypocone junction directly (MUSM 4292; Fig 5U) or via a short mesolophule (MUSM 4285; Fig 5Z). The posteroloph is mesially concave and links the metacone to the hypocone. The metaloph shows a variable length, direction and lingual connection. On MUSM 4285, 4290 and 4292 (Fig 5P, 5U and 5Z), the metaloph is long, turned backwardly (either curved or L-shaped), and linked to the posteroloph via (or not) a posteroloph spur. On MUSM 4286, the metaloph is short, mesiolingually directed and lingually free. On that tooth, there is also a crestule, close to the metaloph and connected lingually to the posteroloph spur and labially to the posteroloph. Due to its position and configuration, it is probably a rest of metaloph. On some worn dP4s (MUSM 4287, 4291, and 4293; Fig 5C’), the metacone and metaloph are likely subsumed within the posteroloph (+ its spur), thereby forming a thick platform distally. Neocrestules connect the third transverse crest to the metacone or to the distal platform on MUSM 4289–4291 (Fig 5P). On MUSM 4294 (Fig 5D’), there is no metaloph or distinct metacone (which is probably fused with the posteroloph). On that tooth, the thin posteroloph bears a long spur, which runs mesiolabially on the posterior flexus and remains free. The hypoflexus forms a U-shaped structure, mesiolabially directed. P4s are round, MUSM 4295 shows an oval outline (Fig 5Q), whereas MUSM 1974 is longer and more circular-shaped (Fig 3M [31]). MUSM 4295 is higher-crowned with respect of the other teeth attributed to Nuyuyomys chinqaska (Hling = 0.89; Hlab = 0.62; Fig 5Q). On MUSM 4295, the area of the protocone and that of the hypocone are large and conical, corresponding to oval regions of dentine delimited by a very thick exterior enamel layer (Fig 5Q). On the two P4s, the mesialmost crest is divided into two parts, which are connected together. Its lingual part, the anterior arm of the protocone, is thicker, mesiolabially directed, longer on MUSM 4295 (Fig 5Q), but shorter on MUSM 1974 (Fig 3M [31]). This crest is in the continuity with the protocone and its short posterior outgrowth. The labial part of the mesialmost crest, the anteroloph, is thinner, roughly transverse, shorter on MUSM 4295, but longer on MUSM 1974. It should be lower than the anterior arm of the protocone (and the other occlusal structures) at early stage of wear, because it is barely worn contrary to all other structures of the occlusal surface. The anteroloph is separate from a strong paracone (or the mesial extremity of its sloped anterior arm on MUSM 4295) by a thin furrow. The labial protoloph is oblique. It is connected, with the mure, to a strong and complete posterior arm of the protocone. On MUSM 4295 (Fig 5Q), the mure is very short and longitudinal (i.e., parallel to the mesiodistal axis of the tooth). On MUSM 1974 (Fig 3M [31]), the mure is absent or reduced to a very tiny crestule from the third crest-anterior arm of the hypocone jonction that remains mesially free. The third crest is almost parallel to the oblique labial protoloph, and is composed of a long mesolophule sligthly linked to a short mesoloph. The mesostyle is strongly connected to a curved and thick posteroloph. There is no clear distinct metacone or metaloph. Hence, the posterior flexus is fully closed. However, the posteroloph appears to have a thickenning, which could correspond to a relictual metaloph, on MUSM 4295 (Fig 5Q). The latter and paraflexus are long, narrow mesiolabially-distolingually extended and oriented on MUSM 4295 (Fig 5Q), PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 21 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru whereas they are more mesiodistally expanded on MUSM 1974 (Fig 3M [31]), due to the higher length of that tooth. The mesial mesoflexus and hypoflexus are clearly open. M3s of Nuyuyomys chinqaska (MUSM 4323 and 4324; Fig 5T and 5X) are well recognisable by their distal part, which is labiolingually pinched (notably labially), and their hypocone, being clearly smaller and with a lingual margin occupying a more labial position than the protocone. Among other upper molars attributed to this taxon, some are small-sized and subsquare or slightly mesiodistally elongated, and other larger can be also subsquare but they are usually more transverse, being wider than long. We interpret them as possibly M1s and M2s, respectively (i.e., M1? and M2?). The height of crown is widely variable. Some upper molars at early stages of wear have a higher crown (e.g., the MUSM 4303 M1? and 4320 M2?; Hling = 0.74–0.89) than other, which are less worn (e.g., the MUSM 4313 and 4314 M2?s; Hling = 0.60–0.69). Because there are teeth with intermediary crown height between these two extremes and no morphological character allowing to differentiate them, we consider that all the material reported here documents a single taxon. On all upper molars, the crests are thin. The paracone and the mesostyle are well differentiated, whereas the protocone and hypocone are more crestiform, as the metacone in most cases. The latter is more cuspate on two upper molars (MUSM 4309 and 4320; Fig 5W and 5A’). The protocone and hypocone are mesiolingually-distolabially and mesiodistally compressed, respectively. As on the MUSM 4295 P4, the anterior arm of the protocone is mesiolabially directed and in continuity with the protocone, its posterior outgrowth and sometimes a part of the anteroloph, like on MUSM 4314 (Fig 5B’). The anteroloph (or its labial part) is more transverse. At early stages of wear, labially the paraflexus can be either closed (MUSM 4298 and 4303; Fig 5Y and 5E’) or opened (e.g., MUSM 1975, 4306, 4308, 4309, 4311, and 4320; Fig 3N [31]; Fig 5R, 5V, 5W and 5A’), separate from the paracone by a thin notch (e.g., MUSM 4308, 4309, and 4320; Fig 5R, 5W and 5A’) or a wider furrow (MUSM 1975, 4306 and 4311; Fig 3N [31]; Fig 5V). The posterior outgrowth of the protocone can be very short, particularly on M1?s, or moderately longer. The posterior arm of the protocone is complete and thin. On MUSM 4303, 4307 and 4314 (Fig 5Y and 5B’), this crest is slightly lower than the protocone, but remains high (i.e., non-taeniodont pattern). Although the paracone and mesostyle are close in position, they do not share connection in most cases. On MUSM 4298, 4320 and 4323 (Fig 5W, 5X and 5E’), however they are linked via their arms (i.e., posterior arm of the paracone and anterior arm of the mesostyle). The labial protoloph is usually straight and mesiolabially directed, but it can have a lingual part oblique, whereas its labial part is more transverse such as on MUSM 4298, 4318 and 4322 (Fig 5E’). Two upper molars (MUSM 4311 and 4317) have one crestule on the mesial margin of the labial protoloph, which is mesially free. MUSM 4323 does not have that crestule, but it shows a tiny enamel swelling on the labial part of the paraflexus (Fig 5X). The third crest is often parallel to the labial protoloph, and is usually composed of a long mesoloph linked to a mesolophular spur. The mure is short but complete, oblique and linked to a strong and long anterior arm of the hypocone. As dP4s, some upper molars have a long posterior arm of the mesostyle (MUSM 4303, 4307, 4319, and 4323; Fig 5S, 5X and 5Y). On other upper molars, this arm is usually short, but it can also be undeveloped (MUSM 1975 and 4314; Fig 3N [31]; Fig 5B’). The metacone can be connected to the mesostyle or its posterior arm (e.g., MUSM 4303, 4308, and 4319; Fig 3N [31]; Fig 5R, 5S and 5Y), or separated from these structures by a very tiny notch (e.g., MUSM 4306, 4307 and 4320; Fig 5V and 5W), except on MUSM 1975 and 4314, where the notch is thicker (Fig 3N [31] and Fig 5B’). On all upper molars, a metaloph is present, either long or short. It is often connected lingually to the posteroloph (via a posteroloph spur on MUSM 4315 and 4320; Fig 5W), but it can also be lingually free (MUSM 4298, 4303, and 4312; Fig 5Y and 5E’). On MUSM 1975, 4308 and 4307 (Fig 3N [31] and Fig 5R), it is reduced to a crestule labially disconnected to the metacone, and lingually linked to the PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 22 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru posteroloph spur. Specifically, the metaloph is always long and joining the metacone to the posteroloph on teeth identified as M2?s, whereas it is complete or reduced on M1?s and M3?s. The MUSM 4323 M3 displays a neocrest stemming from the anterior arm of the metacone, and connecting lingually to the third crest (Fig 5X). The hypocone has a short posterior arm slightly connected to the posteroloph (MUSM 4308, 4309, 4311, 4315, 4318, 4319, and 4324; Fig 5R, 5S, 5T and 5A’) or separate from it by a minute and shallow notch (MUSM 4298, 4303, 4306, 4313, 4320, and 4323; Fig 5V, 5W, 5X, 5Y and 5E’). This notch is wider on MUSM 4314 (Fig 5B’). Some upper molars have a mesiodistal constriction at the base of the hypoflexus (MUSM 4303, 4307, 4312, 4319, and 4324; Fig 5S, 5T and 5Y). Remark 1 The MUSM 4293 and 4294 (Fig 5C’ and 5D’) dP4s have size, a crown height and global occlusal morphology similar than the rest of dP4s attributed to Nuyuyomys chinqaska (i.e., rectangular or slightly trapezoidal occlusal outline, pentalophodonty-tetralophodonty, nontaeniodonty, and U-shaped hypoflexus). Nevertheless, they differ from other dP4s attributed to this taxon in some characters. MUSM 4293 (Fig 5C’) has a protocone strongly linked with the anteroloph without any thinning of this crest. On MUSM 4294 (Fig 5D’), the protocone is less bulbous than on other dP4s of N. chinqaska, and the anteroloph shows a more sligthly thinning. Finally, there is no distinct metacone and metaloph on MUSM 4294 (Fig 5D’). We interpret all these differences between MUSM 4293, 4294, and the rest of dP4s attributed to N. chinqaska as documenting intra-specific variation. Remark 2 We tentatively assigned the MUSM 4298 upper molar (Fig 5E’) to Nuyuyomys chinqaska because it shows a particular pattern, not really shared with other upper molars attributed to N. chinqaska. On that tooth, the apices of the paracone and mesostyle appear to be lingually displaced and they bear very long arms with unusual orientations due to a kind of unusual invagination of the labial crown margin: the anterior arm of the paracone is mesiolabially directed, its posterior arm is very slightly distolabially directed, and the anterior arm of the mesostyle is mesiolingually directed. Owing to the orientation of these arms and the connections anteroloph-anterior arm of the paracone, posterior arm of the paracone-anterior arm of the mesostyle, and mesostyle-metacone, the dental structures of the tooth labially form a “M”. In addition, the extremity of the posterior outgrowth of the protocone is bulged appearing as a cusp-like structure. Nevertheless, MUSM 4298 matches with the morphology of the other upper molars of N. chinqaska by other characters (i.e., size, crown-height, pentalophodonty, non-taeniodonty, crestules on the paraflexus, oblique mure, long mesoloph connected to a mesolophular spur, and tiny and shallow furrow separating the posterior arm of the hypocone from the posteroloph). As far we know, such M-shaped pattern for the labial dental structures on upper molars is not found in caviomorphs or very rare. Consequently, the peculiar characters of MUSM 4298 may result to some abnomalies or extreme intra-specific variation of this taxon. Remark 3 In addition, the specimens of TAR-31 have a small size and general pattern (i.e., non-taeniodonty; complete mure on upper teeth; presence of a metaloph on dP4s and upper molars) similar to those of the material from the early middle Miocene of Madre de Dios formerly attributed to cf. Microsteiromys sp. (MD-67, Peru) [31]. The latter is documented by only two upper teeth: MUSM 1974 P4 and MUSM 1975 upper molar (Fig 3M and 3N, p. 94 [31]). By PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 23 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru their mesiodistally-elongated shape, ?M1s from TAR-31 are close to MUSM 1975. Particularly, MUSM 4307 and 4308 (Fig 5R) from TAR-31 show a labial protoloph and a third crest slightly oblique and a short metaloph labially unconnected to the metacone and lingually linked to the posteroloph spur, as on MUSM 1975. Due to the absence of lower teeth attributed to the taxon from MD-67 and differences on P4 (i.e., longer; absence or strong reduction of the mure; flexus shape), we prefer to tentatively refer this material (MUSM 1974 and 1975) to as Nuyuyomys chinqaska for the time being. Comparisons By its non-taeniodonty, extended flexi(-ds), weak obliquity of loph(-id)s, p4 with a large trigonid, rounded P4, pentalophodont upper molars, and a well-defined, and slightly oblique (faintly backwardly oriented) metaloph on upper molars, the material from TAR-31 exhibits strong erethizontoid affinities. Moreover, like on some lower molars from TAR-31, a subpentalophodont pattern (i.e., presence of a short neolophid in the anteroflexid) is otherwise found in some erethizontoids (e.g., Palaeosteiromys amazonensis, Branisamyopsis australis, Branisamyopsis praesigmoides, Steiromys duplicatus and Neosteiromys pattoni) [71, 74–77]. The material from TAR-31 shows brachydont lower teeth and submesodont-mesodont upper teeth, a rather rare crown condition found among the erethizontoids, which are usually brachydont. The only extinct erethizontoids so far showing high tooth crowns are Hypsosteiromys (Sarmiento Fm., Argentina; early Miocene) [74, 78, 83] and Noamys (site 3 of Rı́o Chico Locality, Guanaco Fm., Argentina; late Miocene and/or early Pliocene) [72]. However, Nuyuyomys is clearly distinct from the latter taxa. Hypsosteiromys has a larger size, loph(-id)s more oblique, square P4s, tetralophodont M1–2s, tetralophodont-pentalophodont dp4s, and a metalophulid I reduced to an anterior arm of the metaconid disconnected to the protoconid on lower teeth. Noamys has tetralophodont and sligthly longer dP4 and M1 with oval-shaped occlusal contour and flexi more extended, especially anteroposteriorly. The specimens from TAR-31 are markedly smaller than all erethizontoid species except Kichkasteiromys raimondii, Shapajamys labocensis, Palaeosteiromys amazonensis, Microsteiromys jacobsi gen. et sp. nov., and Noamys [18, 37, 70–72] (this work). Contrary to Shapajamys, the P4s from TAR-31 and MD-67 (MUSM 4295 and MUSM 1974) do not have any distinct metaloph. Unlike the material from MD-67 and TAR-31, Kichkasteiromys, Shapajamys and Palaeosteiromys do not display a metaloph disconnected labially to the metacone on upper molars. One M1 of Shapajamys (MUSM 2993) has a reduced metaloph that is lingually free, as on some M1?s from TAR-31. However, ? M2s from TAR-31 have a metaloph more developed than in Shapajamys. Upper molars from TAR-31 differ from the MUSM 2925 M1 or M2 of Kichkasteiromys (Fig 2D, p. 92 [70]) in having a lingual protoloph less lingually situated and a posterior outgrowth of the protocone more oblique. For lower teeth, like on the material from TAR-31, some erethizontoids (Shapajamys, Branisamyopsis, ?Eosteiromys sp. nov. sensu Candela [69], and Steiromys) show hexalophodont dp4s with accessory structures. By its elongated shape and tetralophodont pattern, the MUSM 4334 p4 is particularly close to the p4 of M. jacobsi (IGM-DU 89–249 in Fig 16C, p. 56 [37]; Fig 3B), whereas dp4s from TAR-31 are much more elongated, and with a narrower trigonid than in M. jacobsi (FMNH PM 54672 in Fig 24.2B, p. 395 [18]; Fig 3A). In addition, dp4s from TAR-31 are hexalophodont, while the FMNH PM 54672 dp4 appears tetralophodont. The lower molars of M. jacobsi (FMNH PM 54672) and of Shapajamys do not have any neolophid on their anteroflexid, contrary to several specimens from TAR-31 and Palaeosteiromys. The material from TAR-31 differs from M. jacobsi in having the mesoflexid still lingually open at advanced stage of wear on m1–2s and less rectangular lower molars; and from Shapajamys in having an ectolophid always complete and oblique on lower molars. Compared to the MUSM PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 24 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru 2861 m3 of Palaeosteiromys (Fig 4L, p. 77 [71]), m3s from TAR-31 have no neolophid or have a shorter one, and show a mesoflexid more extended. The material from TAR-31 differs from Eosteiromys annectens and Steiromys duplicatus in having non-taeniodont lower teeth; Eosteiromys homogidens, Eosteiromys annectens, and Coendou insidiosus in having hexalophodont dp4s; from Protosteiromys and Eosteiromys in having no neolophid on the anteroflexid on lower molars; from Branisamyopsis praesigmoides and Steiromys duplicatus in having an ectolophid always complete; from Neosteiromys and Parasteiromys in always having a complete mure on dP4s and upper molars; from Eopululo, Protosteiromys pattersoni and Neosteiromys in having a P4 without endoloph; from Eosteiromys homogidens in having a mesial mesoflexus less mesiodistally extended on P4s; from Eosteiromys annectens, Eosteiromys kramarzi sensu Candela [74], Branisamyopsis and Neosteiromys in having non-taeniodont dP4s and upper molars; from Eopululo, Protosteiromys pattersoni and the three Erethizonthoidea indet. from Acre region (Brazil; Late Miocene) [84, 85] in having upper molars always with a metaloph (which is not reduced, far from being a relictual structure); from Protosteiromys, Parasteiromys, Branisamyopsis, Eosteiromys, Steiromys and Neosteiromys in having some elongated upper molars (potential M1s) and an incomplete metaloph on some upper molars; from Branisamyopsis pardinas sensu Candela [74] in having a complete third crest on M1–2s; from Protosteiromys medianus in having a complete third crest on M3s [74–77, 80–83]. To summarise, the erethizontoid teeth from TAR-31 are morphologically close to those of Kichkasteiromys, Shapajamys laboensis, Palaeosteiromys, and M. jacobsi. In having elongated dp4s, with a hexalophodont pattern, the material from TAR-31 clearly differs from that of M. jacobsi. Hence, we consider the dental material from TAR-31 as documenting a new genus and species: Nuyuyomys chinqaska. Erethizontoidea gen. et sp. indet. 1 Fig 5F’ and S1 Table Referred material MUSM 4367, left p4 (Fig 5F’). Locality TAR-31, Tarapoto/Juan Guerra, Mayo River, Shapaja road, San Martı́n Department, Western Amazonia, Peru. Formation and age Lower member, Ipururo Fm., late middle Miocene (i.e., Laventan SALMA). Description MUSM 4367 is a pristine p4, with rounded corners and trigonid slightly narrower than the talonid (Fig 5F’). It is brachydont, tetralophodont and non-taeniodont. The entoconid is the highest and largest cuspid. The metaconid and hypoconid are mesiodistally compressed, whereas the mesostylid is more bulbous. On the mesiolabial corner of MUSM 4367, the metalophulid I is connected to a cuspate structure with a size similar to the mesostylid, that we interpret as the protoconid due to its position and connection with the metalophulid I. The latter is complete and continuous. It has a transverse and long lingual part connecting to the poorly distinct metaconid, whereas its labial one is shorter and curved. On its distal slope, the metaconid has a short and quite transverse posterior arm, which is disconnected to the PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 25 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru mesostylid. The second transverse cristid is straight and well-marked. It appears to be formed by a long neomesolophid, connecting the mesostylid to a shorter and sloping posterior arm of the protoconid. The latter is labially linked to a labial cuspid-shaped structure. This structure is elongated, mesiolabially directed. It is aligned with a cristid stemming from the hypolophidanterior arm of the hypoconid junction (= the ectolophid or a part of the ectolophid), but remains separate from it, as well as from the protoconid mesiolabially, by notches. This elongated cuspid-shaped structure could correspond to a part of the ectolophid fused with the protoconid cristid (see [4]). The entoconid is somewhat bulbous and displays thick, but faintly defined, arms. Its anterior arm is clearly separate from the mesostylid, whereas its posterior arm almost connects to the lingual extremity of the posterolophid, a very thin and shallow notch separating both cristids. The hypolophid is slightly oblique and delimits, with the entoconid, the curved posterolophid and the strong and high anterior arm of the hypoconid, a well-extended and oval metafossettid. The anteroflexid and mesoflexid are rectangular-oval and have a similar extension, smaller than that of the metafossettid. The U-shaped hypoflexid with a distolingual direction is linguolabially well-expanded. Comparisons MUSM 4367 is brachydont with a large trigonid, non-taeniodont pattern, extended flexi(-ds), and a weak obliquity of loph(-id)s, which are characters found in erethizontoids. In addition to its size, which is similar to that of p4s of Microsteiromys jacobsi (IGM-DU 89–249) and Nuyuyomys chinqaska (MUSM 4334), MUSM 4367 has a similar morphology (i.e., brachydonty, tetralophodonty, non-taeniodonty, and oblique ectolophid). However, contrary to p4s of M. jacobsi and N. chinqaska, as well as the MNHN 1903-3-18 premolar of Protosteiromys asmodeophilus (Fig 32 [79] and see above), MUSM 4367 is more round shaped and less elongated, with a shorter trigonid and a straight mesial crown margin. In particular, MUSM 4367 differs from MUSM 4334 of N. chinqaska in having a wider trigonid comparing to the width of the talonid, a higher labial part of the metalophulid I, and a protoconid more mesially canted; from MNHN 1903-3-18 of P. asmodeophilus in having smaller lingual flexid/fossettids and a larger hypoflexid. Unlike p4s of Hypsosteiromys, MUSM 4367 has more straight lophids and a complete metalophulid I connected to the protoconid [74, 83]. The MLP 93-XI-18-1 p4 of Eosteiromys homogenidens [74] appears to have a similar mesiolabial morphology to MUSM 4367 in having a second cristid labially connected to an elongated and oblique structure, which may correspond to a part of the ectolophid + protoconid cristid, disconnected to the rest of the ectolophid. This structure is slightly connected to the protoconid on MLP 93-XI-18-1, contrary to MUSM 4367, but it was probably disconnected to this cuspid at an earlier stage of wear. MUSM 4367 differs from p4s of E. homogenidens, Eosteiromys annectens [77], Branisamyopsis australis [74, 75], Branisamyopsis praesigmoides [77], and Steiromys detentus [74] in being more square-shaped; of E. homogenidens in having a straight metalophulid I and less extended anteroflexid; of E. annectens in having a non-taeniodont pattern and a less oblique hypolophid and posterolophid; of Eosteiromys segregatus [74] in having no posterior arm of the mesostylid and a more extended metaflexid; of Branisamyopsis, Steiromys duplicatus [74], Steiromys verzii sensu Candela [74], and some p4s of S. detentus in having a tetralophodont pattern; of S. detentus in having a less extended anteroflexid and more oblique ectolophid; and of S. duplicatus in having a straight metalophulid I. MUSM 4367 is smaller than Hypsosteiromys, Branisamyopsis, Eosteiromys and Steiromys. Given the paucity of the material, we provisionally assign MUSM 4367 to Erethizontoidea gen. et sp. indet. 1. Erethizontoidea gen. et sp. indet. 2 Fig 5G’ and S1 Table PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 26 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Referred material MUSM 4368, left M1 or M2 (Fig 5G’). Locality TAR-31, Tarapoto/Juan Guerra, Mayo River, Shapaja road, San Martı́n Department, Western Amazonia, Peru. Formation and age Lower member, Ipururo Fm., late middle Miocene (i.e., Laventan SALMA). Description The unique M1 or 2 (MUSM 4368; Fig 5G’) documenting this taxon is large, brachydont, and non-taeniodont. It has a subsquare to rounded occlusal outline. The main cusps/styles (paracone, mesostyle, protocone, and hypocone) are large and well defined. The labial cusps/styles are also well defined, whereas the lingual ones are somewhat compressed labiodistally-linguomesially, especially the protocone, which has a strongly crestiform aspect. The worn part of the posterior outgrowth of the protocone is very short at this early stage of wear of the tooth. The anteroloph is long and curved. It is lingually connected to the protocone and labially separated from the paracone by a thin notch. The lingual protoloph, stemming from the protocone, is high and reaches the labial protoloph–mure junction (i.e., non-taeniodont pattern). The labial protoloph and the third crest are faintly oblique. The mure, linking these two transverse crests lingually, is longitudinal (i.e., on the mesiodistal axis of the tooth). Labially, a large furrow separates the mesostyle from the paracone. The metacone is visible because it does not display tight connections with the other labial dental structures. A thin and shallow groove separates it from the mesostyle, and a very slight connection is present between the tiny posterior arm of the metacone and the posteroloph. The third transverse crest is complete, but labially thinner and shorter, thereby indicating that it could be formed primarily by a long mesolophule and a short labial mesoloph. The metacone displays a straight and very short metaloph. At the level of this lingual part, the metaloph exhibits: (i) a very short crestule mesially, which connects to the labial extremity of the mesolophule via a longer crestule; and (ii) an alignement of crestules distally, which remain free. This connection between the metaloph and the third crest generates a small and narrow flexus labially located. The hypocone has mesially a long anterior arm, which is strongly linked to the mure-third crest junction, and distally a posterior arm slightly curved and connected to the straight posteroloph. In addition, a third short crest runs distolingually stemming from the hypocone, and can be recognised as a posterior outgrowth of this cusp. In its middle part, the posteroloph bears a spur, labially directed towards the distal crestules of the metaloph but without connection with them. All the main flexi are well extended and deep. Comparisons Among the four superfamilies of caviomorphs, MUSM 4368 exhibits strong erethizontoid affinities in being brachydont, non-taeniodont, pentalophodont, and in showing extended flexi and straight lophs. Erethizontoid molars have usually a better-defined metaloph than MUSM 4368, but some species can also be characterised by a reduced metaloph (e.g., Protosteiromys pattersoni, Eosteiromys kramarzi, Parasteiromys uniformis, and Steiromys detentus) or the absence of this crest (e.g., Eopululo wigmorei, P. pattersoni, Hypsosteiromys, Eosteiromys segregatus, Steiromys detentus, and Noamys hypsodonta [72, 74, 78, 80–83, 86]. However, these species with a reduced metaloph do not show a configuration similar to that of MUSM 4368, with crestules on the PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 27 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru lingual extremity of the metaloph, some of which are connected to the third transverse crest. MUSM 4368 is smaller than the upper molars of P. pattersoni, Hypsosteiromys, P. uniformis, E. kramarzi, E. segregatus, and Steiromys. Moreover, MUSM 4368 differs from the upper molar attributed to E. wigmorei (LACM 143269 in p. 111 [81]) in having a more quadrate shape; from upper molars of Hypsosteiromys in having lower crown and lophs less oblique; of P. uniformis in having a complete mure; and of E. kramarzi in having a lingual protoloph. By its size larger than MUSM 4367 and the material attributed to N. chinqaska, MUSM 4368 probably documents another erethizontoid at TAR-31, provisionally identified as Erethizontoidea gen. et sp. indet. 2. Cavioidea Fischer, 1817 Caviidae Fischer, 1817 Caviidae gen. et sp. indet. Fig 6 and S1 Table Referred material MUSM 4283, right p4 (Fig 6). Locality TAR-31, Tarapoto/Juan Guerra, Mayo River, Shapaja road, San Martı́n Department, Western Amazonia, Peru. Fig 6. Caviidae gen. et sp. indet. from TAR-31, Peru, MUSM 4283, right p4. (A) Occlusal, (B) occluso-labial, (C) crosssectional, (D) posterior, (E) ventral, (F) labial, (G) lingual, (H) anterior views. The illustrations of the fossil specimen are 3D digital models obtained by X-ray μCT surface reconstruction. The image in C is a 2D orthoslice of the tooth (crosssection). https://doi.org/10.1371/journal.pone.0258455.g006 PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 28 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Formation and age Lower member, Ipururo Fm., late middle Miocene (i.e., Laventan SALMA). Description The MUSM 4283 p4 is slightly damaged at the level of its mesiolingual margin (Fig 6A, 6G and 6H). It also shows a crack on its labiodistal part. The enamel and dentine are damaged and eroded in several regions of the crown and occlusal surface. MUSM 4283 is clearly high crowned (Hling = 1.15; Hlab = 1.27; Fig 6B, 6D, 6F and 6G) and, although the presence of roots is not directly visible, the closing of the hypostriid suggests the closing of the crown too (Fig 6E and 6F). Hence, the tooth was seemingly protohypsodont. The cuspids/stylids and lophids are not visible because they are subsumed within three enlarged lobes, corresponding (from anterior to posterior) to the anterior projection, anterior lobe and posterior lobe. The dentine is predominating, surrounded by a continuous and thin enamel layer. The tooth has a triangular mesial margin. The anterior projection is narrower than the anterior lobe, which itself is narrower than the posterior lobe. The anterior projection is roughly tear-drop shaped, with a labial extremity short and distolabially directed. In its lingual part, the projection has an oval structure, which is hollow and shallow (Fig 6A and 6B). Nevertheless, it is not a fossettid because it is not surrounded by enamel layer. It is probably a dentine scar owing to an external impact during the mastication or a taphonomic process. The limit between the anterior projection of p4 and the anterior lobe is marked by the presence of: (i) labially, a flexid almost labially closed; and (ii) lingually, an isolated fossettid (Fig 6A). This fossettid is small, round and shallow. It has a thinner enamel layer than that of the margins of the tooth. As the mesialmost lobe of the tooth is determined as the anterior projection, the flexid almost closed would be the homologous of the interprismatic furrow sensu Pérez [51] separating the anterior projection from the anterior lobe in some stem-Caviidae (Guiomys and “Prodolichotis pridiana”) [51, 87– 90], and which is variably present in Caviidae [1, 55, 91–98]. The enamel layer of the distal margin of this flexid is thinner than that of its mesial margin and of the rest of the exterior enamel layer of the tooth. On the labial margin, the striid corresponding to the opening of the interprismatic furrow is mesiodistally very thin. Its depth represents less than 20% of the crown height (Fig 6B and 6C). Due to their position, the fossettid and/or the interprismatic furrow could be rests of the anteroflexid. The anterior lobe is quite oval and labially separated from the posterior lobe by a triangular hypoflexid. This flexid is linguolabially extended and its lingual extremity reaches more than the half of the width of the tooth. It displays a continuous and homogenous enamel layer. The hypostriid is mesiodistally large and almost reaches the neck of MUSM 4283. On the lingual margin of the tooth, there is an inward fold faintly developed at the transition between the anterior and posterior lobes. It could correspond to a very slight longitudinal furrow opposite to the hypoflexid (= primary internal flexid [hpi]). The posterior lobe is tear-drop shaped, with its labial apex short and mesiolabially directed. In its mesiolingual part, there is a fossettid linguolabially extended, with a thin enamel layer. Its depth is slightly shallower than the striid corresponding to the opening of the interprismatic furrow (Fig 6C). This fossettid probably corresponds to the metafossettid. Because the hypoflexid and metafossettid are not confluent, the tooth is non-taeniodont. The lingual fossettids and the hypoflexid (at its base) show a white deposit, which by its aspect, does not seem to be coronary cement, but more probably sediments that filled them after the death of the animal. Comparisons The protohypsodonty, formation of lobes, linguolabial extension of the hypoflexid, and nontaeniodonty primarily suggest caviid affinities. The very slight development of a possible PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 29 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru longitudinal furrow opposite to the hypoflexid (= hpi), a slight development of the interprismatic furrow (= secondary external flexid [hse]), and a relatively narrow anterior projection exclude hydrochoerine affinities for this material from TAR-31. Moreover, MUSM 4283 does not have any additional flexids as observed in capybaras (i.e., internal flexids, including secondary internal flexids and supernumerary internal flexid; see [99]). MUSM 4283 shares characters, which would be ancestral during the evolution of the caviid family [89, 100], with stemCaviidae: protohypsodonty (Chubutomys, Luantus minor, Luantus propheticus, Luantus toldensis, Phanomys, and Eocardia), presence of lingual fossettids on worn teeth (Asteromys, Chubutomys, Luantus, Phanomys, Eocardia fissa, and Schistomys erro), and a hypoflexid that does not reach the lingual margin of the tooth (Asteromys, Chubutomys, and Luantus) [52, 73, 79, 86, 100–104]. The presence of an anterior projection, which can be associated with an interprismatic furrow, is otherwise found in some derived stem-Caviidae (Guiomys and “Prodolichotis” pridiana) [51, 87–90] and crown-Caviidae [1, 55, 91–98]. However, Guiomys, “Prodolichotis” and the crown caviids do not have any lingual fossettids on worn teeth contrary to MUSM 4283. p4s of L. toldensis (MACN Pv SC4033 in Fig 5F [101]) and Phanomys [100] have a lingual thickening of the metalophulid I/their mesial margin, which could correspond to an incipient anterior projection. If the latter is really present in these taxa, it is less developed than on MUSM 4283. In all Caviidea with an interprismatic furrow associated with an anterior projection on p4s, the interprismatic furrow is never almost labially close, unlike on MUSM 4283. This tooth differs from p4s of Asteromys in having a hypoflexid more linguolabially extended; of Asteromys and Luantus initialis in having a higher crown; of Asteromys, Chubutomys, Eocardia, and L. propheticus in having an anterior projection; of Phanomys in having a thinner enamel; and of Eocardia in having a hypoflexid less linguolabially extended. The stemcaviid Guiomys is euhypsodont unlike MUSM 4283, and shows a p4 with a hypoflexid more linguolabially extended, a more developed anterior projection and longitudinal furrow opposite to the hypoflexid [51, 105]. MUSM 4283 differs from p4s of Schistomys erro and Microcardiodon in being protohypsodont, and in having an anterior projection and a well-marked hypoflexid; of Microcardiodon in having fossettids; and of Schistomys erro in having two persistent lobes [52, 87]. The size of MUSM 4283 is similar with that of Chubutomys simpsoni, Chubutomys leucoreios, Luantus minor, and Guimoys unica. As Dolichotinae gen. 2 large from La Venta (IGM 183425 in Fig 24.7A, p. 401 [18]), MUSM 4283 shows a very slight longitudinal furrow opposite to the hypoflexid, and a lingual extremity of the hypoflexid exceeding the half of the width of the tooth, but without reaching its lingual margin. However, MUSM 4283 is characterised by the presence of fossettids and an anterior projection, contrary to Dolichotinae gen. 2 large from La Venta. MUSM 4283 differs from p4s of “P.” pridiana, Neocavia, Prodolichotis prisca, Dolicavia, Orthomyctera, and Dolichotis in having a less developed longitudinal furrow opposite to the hypoflexid; of Paleocavia, Cavia, Galea and Microcavia in having a clearly less developed longitudinal furrow, which is opposite to the hypoflexid; of Cavia, Galea and Microcavia in having a hypoflexid without cement; of “P.” pridiana, Neocavia, Paleocavia? mawka, Paleocavia impar, P. prisca, Dolicavia, Orthomyctera, Cavia, Dolichotis, and Galea in having a linguolabially narrower hypoflexid; of Cavia in having a more developed anterior projection [28, 54, 55, 90, 94, 95, 106–113]. In conclusion, the morphology of MUSM 4283 appears particular by a mosaic association of characters, combining primitive characters (i.e., protohypsodonty, presence of fossettids on worn teeth, and a hypoflexid, which do not reach the lingual margin of the tooth) and more derived ones (i.e., development of a relatively large anterior projection); and also by an unique configuration of the fossettids and the interprismatic furrow. Thus, it is possible that MUSM 4283 documents a new species among the stemCaviidae, but given the scarce available material, we provisionally identified it as Caviidea gen. et sp. indet. PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 30 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Octochinchilloi Boivin, 2019 in Boivin, Marivaux & Antoine, 2019 Octodontoidea Waterhouse, 1839 Adelphomyidae Patterson & Pascual, 1968, nov. stat. Ricardomys Boivin & Walton gen. nov. urn:lsid:zoobank.org:act:46B7B00E-E14D-4123-BF6D9AB7AFF4AAD4 Type species Ricardomys longidens gen. et sp. nov. Species content Only the type species. Derivation of name Based on the original description of the species [37]: ‘In honor of Richard Kay, professor of Evolutionary Anthropology at Duke University, who discovered the holotype of this species’. Geographic and stratigraphic distribution La Venta (IGM-DU loc. 032), Cerro Colorado Member, Villavieja Fm. (Laventan, late middle Miocene), Huila Department, Colombia; Tarapoto/Juan Guerra (TAR-31), lower member, Ipururo Fm. (Laventan, late middle Miocene), San Martı́n Department, Peru. Generic diagnosis As for the type and only known species. Ricardomys longidens Boivn & Walton sp. nov. urn:lsid:zoobank.org:act:83062BD3-90064C73-BB02-37BF66BF6663 Fig 7 and S1 Table Nom. nud. Ricardomys longidens Walton, 1990, Fig 9A, 9B, p. 31. Nom. nud. Ricardomys longidens Walton, 1997, Figs 24.1C and 24.2D, p. 394, 395. Holotype IGM 183847, left mandibular fragment bearing dp4–m2 (Walton, 1990: Fig 9A, 9B; Walton, 1997: Figs 24.1C and 24.2D; Fig 7A). Referred material MUSM 4397–4401 (Fig 7B and 7F), left dp4s; MUSM 4402–4406 (Fig 7J), right dp4s; MUSM 4407, right lower molar; MUSM 4408, left m1 or m2?; MUSM 4409 (Fig 7C), left m1?; MUSM 4410 (Fig 7G), right m1?; MUSM 4411–4413 (Fig 7D and 7H), left m2?s; MUSM 4414, 4415 (Fig 7K), right m2?s; MUSM 4416–4419 (Fig 7E and 7I), left m3?s; MUSM 4370–4373, 4391– 4394 (Fig 7U and 7V), left upper teeth; MUSM 4374, 4395, 4396 right upper teeth; MUSM 4375 (Fig 7P–7R), left fragment of maxillary bearing dP4 (or M1)? and M1 (or M2)?; MUSM PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 31 / 80 PLOS ONE PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 Miocene caviomorphs from Tarapoto, Peru 32 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Fig 7. Ricardomys longidens gen. et sp. nov. (A) Material from La Venta, Colombia. (B–V) Material from TAR-31, Peru. (A) IGM 183847��� , left mandibular fragment bearing dp4–m2. (B) MUSM 4401, left dp4. (C) MUSM 4409, left m1?. (D) MUSM 4411, left m2?. (E) MUSM 4416, left m3?. (F) MUSM 4400, left dp4. (G) MUSM 4410, right m1? (reversed). (H) MUSM 4413, left m2?. (I) MUMS 4418, left m3?. (J) MUSM 4406, right dp4 (reversed). (K) MUSM 4415, right m2? (reversed). (L) MUSM 4377, left dP4 (or M1)?, morph 1. (M) MUSM 4380, right dP4 (or M1)?, morph 1 (reversed). (N) MUSM 4376, left dP4 (or M1)?, morph 1. (O) MUMS 4384, left M1 or M2?, morph 2. (P–R) MUSM 4375, left maxilla fragment bearing dP4–M1 (or M1–2)?, morph 1 and 2. (S) MUSM 4388, right M1 or M2?, morph 2 (reversed). (T) MUSM 4390, right M3?, morph 3. (U) MUSM 4392, left fragmentary upper molar, morph 4. (V) MUSM 4393, left fragmentary upper molar, morph 4. (A-P, S-V) Occlusal, (Q) labial, and (R) lingual views. The asterisks appoint the holotype material. The illustrations of the fossil specimens are computerized schematic line drawings based on Walton (Fig 9A [37]). (A), scanning electron photomicrographs (B-O and S-V), and 3D digital models obtained by X-ray μCT surface reconstruction (P-R). https://doi.org/10.1371/journal.pone.0258455.g007 4376, 4377 (Fig 7L and 7N), left dP4s (or M1s)?; MUSM 4378–4380 (Fig 7M), right dP4s (or M1s)?; MUSM 4381, right dP4?; MUSM 4382–4384 (Fig 7O), left M1 or M2?s; MUSM 4385– 4388 (Fig 7S), right M1 or M2?s; MUSM 4389, left M2?; MUSM 4390 (Fig 7T), right M3?. Derivation of name Based on the original description of the species [37]: ‘from the latin longidens, in reference to the length and slenderness of the tooth row of the holotype of this species’. Type locality IGM-DU loc. 032 (only for the holotype), El Cardón Red Bed, La Venta Badlands, upper Magdalena Valley, Huila Department, Colombia. Other locality TAR-31, Tarapoto/Juan Guerra, Rı́o Mayo, Shapaja road, San Martı́n Department, Western Amazonia, Peru. Formation and age Cerro Colorado Member, Villavieja Fm.; lower member, Ipururo Fm.; late middle Miocene (i.e., Laventan SALMA). Diagnosis Octodontoid rodent characterised by a small size and non-taeniodont teeth with thick and oblique lamellae. Lower teeth with a trilophodont pattern characterised by the absence of metaconid cristid, posterior arm of the protoconid, neomesolophid and mesolophid. Thick lingual parts of the first and second lamellae on lower teeth, and a first lamella divided into two parts by a thin and shallow furrow on lower molars. Upper teeth with a transversal extension of the hypoflexus, prompt loss of the third transverse crest with wear and the absence of metaloph. Differs from Eodelphomys in being smaller and in having lower molars with a thinner furrow dividing the first lamella, more expanded lingual flexids, metaflexid always open at a more advanced stage of wear, less transversely extended hypoflexid, an entoconid area with thickening and without discernable entoconid arms. Differs from Xylechimys in having less transversely extended hypoflexid, an ectolophid which is not discernable because fused with the hypolophid, hypoconid and the protoconid, an indistinct posterior arm of the metaconid, and no posterior arm of the protoconid on lower molars. Differs from Ethelomys, Paradelphomys, Quebradahondomys, Deseadomys, Adelphomys, Prostichomys, Stichomys, Prospaniomys, and Spaniomys in having a thickening of the lingual parts of the two first lamellae on PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 33 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru lower teeth; from Ethelomys in having thicker cristids and a less-transversely extended hypoflexid on lower molars; from Paradelphomys in having a non-taeniodont and trilophodont dp4s with a lingual part of the first lamella less distally extended and the first and second lamellae very close in position, and no furrow-shaped flexids on lower teeth; from Quebradahondomys in having more oblique cristids on lower molars; from Deseadomys, Adelphomys, Prostichomys, Stichomys, Prospaniomys, and Spaniomys in always having trilophodont dp4s, m1s, and m2s; from Deseadomys and Prospaniomys in having no recognisable cusp(-id)s and more oblique and thicker lamellae; from Deseadomys and Spaniomys in having a less transversely extended hypoflexid on lower teeth; from Prospaniomys in having a more transversely extended hypoflexus on upper teeth; from Adelphomys in having posterior fossette probably less persistent on upper teeth, a metaflexid always open at a more advanced stage of wear on lower teeth, and a first lamella which can be divided into two parts on lower molars; from Spaniomys in having more oblique lamellae, and a straight and distolingually directed second lamella on m3s; and from Prostichomys in having thicker lamellae, posterior fossette less persistent on upper teeth, and no mesostylid and an indistinct posterior arm of the metaconid on lower molars. Description IGM 183847 is a left mandibular fragment preserving dp4–m2 (Fig 7A). The body of the mandible is anteriorly broken at the level of the anterior root of the dp4. Posteriorly, the angular apophysis and all the ascending ramus, including the mandibular condyle, are missing. The coronoid process is broken at its base. Labially, the notch for the insertion of the tendon of the zygomatico-mandibularis pars infraorbitalis is small, below m1, and situated ventrally on the labial edge of the mandible. The anterior tip of the masseteric crest and that of the lateral crest end below the m1, and they appear to be not linked to the notch for the insertion of the tendon of the zygomatico-mandibularis pars infraorbitalis, at the level of its posteroventral and posterodorsal regions, respectively. The masseteric crest is posteriorly broken. It is posteroventrally directed and poorly prominent in its anterior part. The lateral crest is posterodorsally directed and almost undistinct. The teeth referred to this taxon are brachydont. The less worn tooth from TAR-31 (MUSM 4377) presents a fairly high crown, but its hypsodonty indexes stay below 1 (Hling = 0.62; Hlab = 0.63). This tooth was probably submesodont at earlier wear stages. All lower and upper teeth are non-taeniodont. The cusp(-id)s and styles (stylids) are not visible even on the tooth at the earliest stage of wear (MUSM 4377). They are subsumed within loph(-id)s, thereby forming flat-topped and thick crestiform structures (lamellae sensu Patterson & Pascual [56]). Each lamella displays a continuous enamel layer, coating a dentine layer, which is increasingly thicker with the dental wear. The dp4s are longer than wide (Fig 7A, 7B, 7F and 7J). Their trigonid is wide but narrower than the talonid. These teeth are characterised by two roots and a trilophodont pattern with oblique lamellae. Mesiolabially, the area of the protoconid is well expanded and usually triangular-shaped. From this area, emerge mesially the first lamella and distally the second lamella. The first lamella is mesially convex. Its lingual part is short and would correspond to the metaconid area (without its posterior arm?). It can be thick with respect of the thin central part of the first lamella (metalophulid I; IGM 183847, MUSM 4400, 4401, 4405, and 4406; Fig 7A, 7B, 7F and 7J). On the worn MUSM 4397 dp4, the protoconid area is not recognisable from the rest of the first lamella, these structures are fully fused, forming a round and large structure of dentine at the mesial part of the tooth. On all dp4s, the second lamella is a central cristid distolingually directed. It would result of the fusion between the entoconid + the hypolophid + the PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 34 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru mesial and distal ectolophids + a part of the protoconid. As the lingual part of the first lamella on some specimens, the lingual part of the second lamella (the entoconid area), by its thickness, is well discernable from the other structures. There would be neither metaconid cristid, neomesolophid, posterior arm of the protoconid, nor mesolophid. The third lamella is cristiform on the dp4 at the earliest stage of wear (MUSM 4401; Fig 7B) and diamond/parallelogram-shaped on other dp4s. It would include the hypoconid + its anterior outgrowth + the posterolophid. On MUSM 4400 and 4401, it bears a small spur in its mesiolabial aspect (i.e., anterior outgrowth; Fig 7B and 7F). It is linked to the second lamella by a complete and thin anterior arm of the hypoconid. On IGM 183847, the anterior arm of the hypoconid reaches a short cristulid stemming from the second lamella (Fig 7A). The anterior arm of the hypoconid is mesiolingually directed, strongly oblique on IGM 183847 and MUSM 4399–4401 (Fig 7B and 7F), and more longitudinal on MUSM 4404–4406 (Fig 7J). Owing to the absence of the metaconid cristid, posterior arm of the protoconid, neomesolophid and mesolophid, the anterolingual flexid corresponds to the confluence of the anteroflexid with the mesial and distal mesoflexids. This flexid and the metaflexid are widely open lingually and more extended than the U-shaped hypoflexid. Lower molars have a pattern similar to that characterising dp4s (i.e., non-taeniodonty, trilophodonty, and obliquity, global shape and composition of lamellae; Fig 7A, 7C–7E, 7G–7I and 7K). Comparatively to dp4s, lower molars have three roots (i.e., two mesial and one distal) and a more rectangular shape with a transverse mesial margin. Four lower molars (MUSM 4416– 4419; Fig 7E and 7I) show a distal part of the talonid labiolingually pinched, and as such they are probably m3s (determined as m3?s). MUSM 4416 (Fig 7E) also has a posterior root directed distally, a character typically found on m3s. Among the isolated lower molars without distal pinch, some are small-sized, and as such could be m1s (determined as m1?s; MUSM 4409, and 4410; Fig 7C and 7G). On IGM 183847 and MUSM 4417, 4419 and 4415 (Fig 7K), the lingual part of the first lamella is separated from the protoconid area by a thin and shallow furrow open on the mesial margin of the tooth (MUSM 4415 is broken at the level of the furrow giving the impression that it is larger and deeper; Fig 7K). On MUSM 4417, the margins of the furrow are composed of two sloped cristulids, one lingual and one labial. The lingual cristulid stems from the metaconid area (the ?anterior arm of the metaconid), while the labial one stems from the protoconid area (the ?anterior arm of the protoconid). They are connected at the base of the furrow. On molars with a more advanced stage of wear (MUSM 4408–4414 and 4418; Fig 7C–7E and 7G–7I), this furrow is fully closed. The morphology of the second and third lamellae is very similar of that of dp4s, except they are longer. Regarding upper teeth (Fig 7L–7V), the absence of complete tooth row and a documentation of the whole stages of wear make it difficult to recognise the dental intra-specific variation related to the difference of loci, wear, or polymorphism. Four morphs can be distinct. The unique fragment of maxillary found at TAR-31 and attributed to this species, MUSM 4375 (Fig 7P–7R), bears two upper teeth. This specimen and the comparison with upper teeth of other adelphomyines or closely related forms (e.g., Deseadomys, Adelphomys, Prostichomys, and Spaniomys) allow us to propose hypotheses regarding the locus determination for these morphs: • Morph 1: small upper teeth longer than wide or subsquare, and with a quadrangular occlusal outline and three transverse lophs (MUSM 4375–4381; Fig 7L–7N and 7P–7R). MUSM 4381 seems to have three roots (i.e., two labial and one large lingual), whereas other teeth have not conserved their roots. This morph could correspond to the morphology of dP4s (or M1s); • Morph 2: upper teeth larger than those of the morph 1, either subsquare or rectangular, and with quadrangular occlusal outlines, three transverse lophs and four roots (MUSM 4375 and PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 35 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru 4382–4389; Fig 7O–7S). This morph could correspond to the morphology of M1s or M2s. MUSM 4389 has a very short posterior crest as MUSM 4390 (see below morph 3). This shortness may be related to a strong labial displacement of the hypocone. Nevertheless, MUSM 4389 shows typical characters of morph 2: large size, quadrangular occlusal outline with a straight distal margin and four roots. It could correspond to a M2; • Morph 3: this morph is represented by one small upper tooth (MUSM 4390; Fig 7T) characterised by three roots, a more round outline than teeth of the morphs 1 and 2, distal pinch, and three transverse lophs. This morph could correspond to the morphology of M3s; • Morph 4: this morph is represented by six mesially-broken upper teeth (MUSM 4391–4396; Fig 7U and 7V). These fragments of teeth have different size, some are small-sized (MUSM 4391 and 4392; Fig 7U), medium-sized (MUSM 4393 and 4395; Fig 7V), while other are larger (MUSM 4396). They have a more rounded occlusal outline than teeth of morphs 1 and 2, and a distal part slightly labiolingually narrower than the mesial part. They seem to be mesiodistally elongated. Because they are broken, the hypsodonty index cannot be calculated. However, they have thinner crests and deeper flexi than teeth of other morphs, and three (MUSM 4392 and 4395; Fig 7U) or four (MUSM 4391, 4393, 4394, and 4396; Fig 7V) transverse lophs. Owing to the latter characters, this morph likely clusters upper molars at less advanced wear stages than those of morphs 2 and 3. The first lamella is diamond/parallelogram-shaped with a large and rounded apex at the mesiolingual corner of the tooth. Two other apices, the first at the mesiolabial corner of the tooth and the second in the center of its lingual edge, are usually narrower and tapered. This lamella would include the protocone + the posterior outgrowth of the protocone + the anteroloph. The second lamella, which would correspond to the paracone + the labial protoloph, is either transverse or oblique (i.e., mesiolabially directed) or lingually oblique and labially transverse. It is short due to a strong transversal extension of the hypoflexus. The lingual protoloph is thin, distolabially directed and complete, thus linking the first lamella to the second (i.e., non-taeniodont pattern). On the trilophodont upper teeth (MUSM 4373, 4375–4389, 4390, and 4395; Fig 7L–7U), the third lamella is usually bean-shaped, but can be suboval (MUSM 4389 and 4390; Fig 7T). On the tetralophodont MUSM 4394, this third lamella presents a round flexus still distolabially open. On other tetralophodont upper teeth (MUSM 4391–4393 and 4396; Fig 7V), this flexus is fully closed, thereby forming a round fossette. This flexus/fossette separates the third transverse crest from the posteroloph. On all tetralophodont upper teeth, these two transverse lophs are very thin and short, except on MUSM 4394, in which the third transverse crest is thicker. On MUSM 4391 and 4393 (Fig 7V), the third crest is composed of a very short mesoloph, whose lingual extremity is connected to a mesolophular spur. The third lamella on trilophodont upper teeth would include the mesostyle and/or the metacone (see [50]) + the third transverse crest + the posteroloph + the hypocone + its anterior arm. Near the center of the tooth, the second lamella and the third lamella are connected via a mure mesiolabially directed (strongly oblique). Comparisons A small size, non-taeniodont teeth with thick and oblique lamellae, and a transverse extension of the hypoflexus, the prompt loss of the third transverse crest with wear and the absence of metaloph on upper teeth are dental traits found in adelphomyine octodontoids [4, 56], thereby suggesting adelphomyine affinities for this material from La Venta and TAR-31. By their size and morphology, the lower teeth from TAR-31 are close to those of IGM 183847 from La Venta, previously attributed to Ricardomys longidens nom. nud. (Figs 24.1C and 24.2D [18]; PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 36 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Fig 9A, 9B [37]; Fig 7A). As lower teeth from TAR-31, the IGM 183847 lower teeth have a trilophodont pattern characterised by the absence of metaconid cristid, posterior arm of the protoconid, neomesolophid and mesolophid. These lower teeth also show oblique lamellae, thick lingual parts of the first and second lamellae, and a first lamella divided into two parts by a thin and shallow furrow on lower molars. However, the lower molars from TAR-31 slightly differ from IGM 183847 in having the first lamella more oblique. A similar pattern to dp4s from La Venta and TAR-31 is also found on the MUSM 2856 dp4 attributed to Adelphomyinae indet. 2 from CTA-61 (Peru, late Oligocene) [71]. Unlike dp4s from La Venta and TAR31, MUSM 2856 does not have a hypoflexid transversely oriented, but the latter is distolingually oriented. Other adelphomyines or closely related forms have strictly trilophodont lower molars: Eodelphomys (Santa Rosa, Peru; ?late Eocene/early Oligocene) [81], Ethelomys (Cabeza Blanca, Argentina; late Oligocene) [114], Paradelphomys (Sarmiento Fm., Argentina; early Miocene) [56], and Quebradahondomys (Quebrada Honda, Bolivia; late middle Miocene) [105]. Eodelphomys is only known from a single m3 (LACM 144298 in p. 130 [81]), which shows a first lamella divided into two parts by a furrow. However, the latter is larger than that on lower molars from La Venta and TAR-31 (when the furrow is present). The lower molars from La Venta and TAR-31 also differ from LACM 144298 in having lingual flexids more expanded mesiodistally, always open at a more advanced stage of wear, less transversely extended hypoflexid, and an entoconid area with thickening and without discernable entoconid arms. In comparison with the material form La Venta and TAR-31, Ethelomys, Paradelphomys, and Quebradahondomys do not have any thickening of the lingual parts of the two first lamellae (or at least of the second lamella) on lower teeth; Ethelomys has thinner cristids and a more transversely extended hypoflexid on lower molars; Paradelphomys has furrowshaped flexids on lower teeth, a taeniodont and tetralophodont dp4 with a lingual part of the first lamella more distally extended (with posterior arm of the metaconid?) and the first and second lamellae very close in position; and Quebradahondomys has less oblique lamellae on lower molars. As the lower teeth from La Venta and TAR-31, Xylechimys (Laguna de los Machos, Argentina; late Oligocene) [56] appears to develop a lingual thickening of the second lamella on lower molars with the dental wear (visible on the m1 of the holotype). Nevertheless, Xylechimys has a trigonid of lower molars clearly different characterised by: (i) a posterior arm of the protoconid linked to the metaconid area, thus delimiting a small and rounded anterofossettid, (ii) discernable ectolophid and posterior arm of the metaconid, and (iii) a more transversely extended hypoflexid. The general pattern of the upper teeth from TAR-31 is similar to that of adelphomyines (or closely-related forms) documented by upper teeth: Deseadomys (Cabeza Blanca and La Flecha, Argentina; late Oligocene) [79], Adelphomys (Pinturas Fm. and Santa Cruz Fm., Argentina; early Miocene) [73, 77], Prostichomys (Sarmiento Fm. and Pinturas Fm., Argentina; early Miocene) [77, 115, 116], Stichomys (Santa Cruz Fm.; late early Miocene) [73], Prospaniomys (Sarmiento Fm.; early Miocene) [11, 78, 117], and Spaniomys (Pinturas Fm. and Santa Cruz Fm.; Pampa Castillo, Chile; late early Miocene) [16, 73, 77, 118, 119]. Contrary to the material from La Venta and TAR-31, all these taxa display no thickening of the lingual parts of the two first lamellae (or at least of the second lamella) on lower teeth, and can have tetralophodont lower teeth (usually dp4s, m1s or m2s). The tetralophodont pattern of these lower teeth is characterised by the presence of a posterior arm of the protoconid + metaconid cristid and/or neomesolophid (and mesolophid on some dp4 of Prostichomys). The material from La Venta and TAR-31 also differs from Deseadomys and Prospaniomys in having no recognisable cusp(-id)s and more oblique and thicker lamellae; from Deseadomys and Spaniomys in having a less transversely extended hypoflexid on lower teeth; from Prospaniomys in having a more transversely extended hypoflexus on upper teeth; from Adelphomys in having a posterior fossette probably less persistent on upper teeth, always open at a more PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 37 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Fig 8. Octodontoidea gen. et sp. indet. from TAR-31, Peru (A–C) and Microscleromys sp. 1 from La Venta, Colombia (D, E). (A) MUSM 4662, left m1 or m2. (B) MUSM 4660, left dP4. (C) MUSM 4661, right M3 (reversed). (D) UNC unnumbered specimen, left P4. (E) IGM-DU 85–411, left P4. Occlusal views. The illustrations of the fossil specimens are scanning electron photomicrographs (A-C) and computerized schematic line drawings based on Walton (Fig 15 [37]) (D-E). https://doi.org/10.1371/journal.pone.0258455.g008 advanced stage of wear on lower teeth, and a first lamella which can be divided into two parts on lower molars; from Spaniomys in having more oblique lamellae, and a straight and distolingually directed second lamella on m3s; and from Prostichomys in having thicker lamellae, a posterior fossette less persistent on upper teeth, and no mesostylid and no discernable posterior arm of the metaconid on lower molars. To summarize, due to the morphological similarity between the lower teeth from La Venta and TAR-31, we assign them to the same taxon. By its particular set of characters, the entire material from these both areas is determinable as a new species, hence we validate Ricardomys longidens. Octodontoidea gen. et sp. indet. Fig 8 and S1 Table Referred material MUSM 4662, left m1 or m2 (Fig 8A); MUSM 4660, left dP4 (Fig 8B); MUSM 4661, right M3 (Fig 8C). Locality TAR-31, Tarapoto/Juan Guerra, Mayo River, Shapaja road, San Martı́n Department, Western Amazonia, Peru. PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 38 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Formation and age Lower member, Ipururo Fm., late middle Miocene (i.e., Laventan SALMA). Description Due to the very advanced stage of wear of the MUSM 4662 m1 or m2 (Fig 8A), its occlusal pattern is faintly discernable, especially on the mesial area of the tooth, where a broad dentine platform is present. There is a thick, continuous enamel layer on the entire periphery of the crown. It is thinner at the level of the lingual parts of the hypoflexid and of the mesoflexid. Only three transverse lophids are visible, delimited by a very thin enamel layer, which is absent in some regions. The mesialmost lophid is transverse and connects the area of the posterior arm of the metaconid/mesostylid to the junction of the protoconid (or the protoconid cristid) and the ectolophid. This lophid is recognised as a second transverse cristid (i.e., neomesolophid and/or posterior arm of the protoconid). Mesially, it is partially fused with the mesial dentine platform, which probably corresponds to the fusion at least of the metalophulid I with the anteroflexid. The transverse lophid that is second in position, is the hypolophid, running slightly labiomesially from the area of the entoconid to link the posterior part of the ectolophid. The hypolophid is almost parallel to the second cristid. The ectolophid is short and gently oblique. The transverse lophid appearing in third position is a curved posterolophid, lingually connected to a strong posterior arm of the entoconid. The area of the hypoconid is enlarged and oval, formed by the fusion of the hypoconid, its anterior outgrowth and its posterior arm (if present) and/or the labial part of the posterolophid. There is a thick and complete anterior arm of the hypoconid (i.e., non-taeniodont pattern). The mesoflexid and metaflexid are lingually closed, whereas the hypoflexid remains labially open. The metafossettid is oval and smaller than the mesofossettid. The MUSM 4660 dP4 (Fig 8B) has a trapezoidal crown outline in occlusal view (i.e., labial marging longer than the lingual margin). This tooth shows an advanced stage of wear. Labially, the dental structures have a thick enamel layer, while the lingual structures are surrounded by a clearly thinner enamel layer. The posteroloph area is very enlarged due to the wear, and forms a dentine platform, in which a metacone, a metaloph or a rest of this loph may be included. The hypoflexus is triangular and poorly extended linguolabially. It separates lingually the area of the protocone from that of the hypocone. The areas of the protocone, mure and of the hypocone are also strongly enlarged and broadly merged together, as well as with the platform of the posteroloph, forming a S-shape dentine structure on the distolingual part of the tooth. From this dentine structure, three main lophs run labially: the anteroloph, the labial protoloph and the third transverse crest. The anteroloph is curved with a lingual part thicker than its labial part. The labial protoloph and third crest are transverse and parallel. The paraflexus is still open labially, separating the anteroloph from the labial protoloph. In contrast, the other transverse lophs are connected labially, forming a small oval mesial mesofosette and a more linguolabially extended distal mesofosette. As MUSM 4662, the MUSM 4661 M3 (Fig 8C) is much worn, but it documents a slightly earlier stage of wear. A thick enamel layer surrounds the tooth as well, but the latter is internal at the level of the mesial mesoflexus, which is still opened labially contrary to MUSM 4662. Moreover, all the transverse lophs and flexi are still discernable. The hypocone is clearly more labially situated than the protocone, which indicates that the specimen would be a M3. MUSM 4661 is tetralophodont and non-taeniodont. The area of the protocone corresponds to a broad dentine platform, including the protocone, its posterior outgrowth and its anterior arm (if present) and/or the lingual part of the anteroloph. From this platform, the anteroloph runs labially and links the paracone. The lingual part of the anteroloph is transverse, whereas its PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 39 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru labial part is curved. Due to the connection between the anteroloph and the paracone, a parafossette is formed and reduced to a thin and straight structure. The enamel layer that surrounds it is thick, notably on its mesial margin. On the labial margin of the tooth, the paracone and mesostyle are strong and of similar size. The posterior arm of the protocone is thick and oblique. It connects to the mure, which is linguodistally oriented and in the continuity with the anterior arm of the hypocone. The labial protoloph and the third transverse crest (i.e., mesoloph and/or mesolophule) are roughly straight and parallel. There is neither metaloph nor discernable metacone. A well-curved posteroloph is labially connected to the mesostyle and lingually to the area of the hypocone, which is triangular-shaped. The mesial mesoflexus is very thin. The posterior fossette (i.e., confluence of the distal mesoflexus and the metaflexus) is reduced to a very small and rounded structure. As the parafossette, the mesial mesoflexus and the posterior fossette are surrounded by a thick enamel layer. Comparisons By its small size, weak loph(-id) obliquity, non-taeniodonty, tetralophodont upper molars (i.e., absence of metaloph), and a strong mesostyle on upper molars, these three teeth from TAR-31 likely document a single and distinctive octodontoid. Indeed, many Palaeogene and Neogene octodontoids also display these characters. They are usually characterised by a reduction or the absence of the second transverse cristid, and a reduction of the posterior arm of the metaconid on lower molars, and a metacone more distally positioned on upper molars (e.g., Acarechimys, Deseadomys, Dudumus, and Caviocricetus) [30, 79, 120, 121], characters absent on the specimens from TAR-31. However, Paulacoutomys and some lower molars of Platypittamys, Sciamys principalis, Prostichomys and Plesiacarechimys show a complete second transverse cristid and a well-developed posterior arm of the metaconid [77, 115, 116, 122–126], as it is observed on the material from TAR-31. Among the latter taxa, the material from TAR-31 is closer to Paulacoutomys and Plesiacarechimys in having transverse loph(-id)s and a similar extension of the flexi(-ds). In addition, these genera can have an ectolophid slightly oblique on lower molars, as it does on MUSM 4662. The material from TAR-31 differs from Paulacoutomys and Plesiacarechimys in having a smaller size; from Platypittamys and Prostichomys in having a more longitudinal ectolophid on lower molars; from Platypittamys in having M3 with third crest that is not curved, a mesial mesoflexus less extended, a posterior flexus/fossette more extended; from Prostichomys and S. principalis in having a mesostyle less connected to the posteroloph on dP4s and upper molars; from Prostichomys in having a more transverse hypolophid on lower molars, a more transverse labial protoloph on dP4s and upper molars, and more transverse M3 with a wider distal part; and from S. principalis in having a mesial part of the lower molars (= fusion of the metalophulid I with the second cristid) more mesiodistally long at its lingual and labial extremities, a hypoflexid less extended labiolingually, and a mesoflexid which is lingually closed although a metafossettid is still present on lower molars, and a hypoflexus less extended labiolingually and a mesial mesoflexus which is strongly reduced although the posterior fossette is still present on upper molars. In sum, given the condition and scarcity of the material, we provisionally refer these teeth to an octodontoid indet. Chinchilloidea Bennett, 1833 Microscleromys Boivin & Walton gen. nov. urn:lsid:zoobank.org:act:785C5F6A-DCCD4C02-B136-A2B49702E6DA Type species Microscleromys paradoxalis gen. et sp. nov. PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 40 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Species content The type species and Microscleromys cribriphilus gen. et sp. nov. Derivation of name Based on the original description of the species [37]: ‘In reference to its small size and general resemblance to Scleromys Ameghino, 1887’. Geographic and stratigraphic distribution La Victoria Fm. and Baraya Member of the Villavieja Fm. (Laventan SALMA, late middle Miocene), Huila Department, Colombia; Tarapoto/Juan Guerra (TAR-31), lower member, Ipururo Fm., (Laventan SALMA, late middle Miocene), San Martı́n Department, Peru. Generic diagnosis Chinchilloid rodent characterised by a small or moderate size. Relative size of m3s/M3s smaller in average than that of m1–2s/M1–2s. Brachyodont to mesodont teeth. P4s and upper molars with noticeable high crowns. Upper teeth with a higher lingual crown and lower molars with a higher distolabially crown. Absence of cement or in small quantity. Homogeneous thickness of the enamel layer of dental strutures. Poorly distinct cusp(-id)s from the loph(-id)s, which do not form lamina. At early stages of wear, lower teeth with pseudo-taeniodont or taeniodont pattern, lower molars with a pseudo-taeniodont pattern more frequently present, taeniodont P4s, dP4s and upper molars with a taeniodont pattern more frequently present. dp4s with a metalophulid I not connected to the metaconid and protoconid at early stages of wear, a posterolophid well-separated from the entoconid, a mesostylid mesially and distally isolated in most cases, and a tendency to the reduction or absence of the third crisitid. Lower molars with usually a groove on the metalophulid I at early stages of wear. Suboval dP4s, with well-arcuate anteroloph and posteroloph as well as a bulbous protocone slightly or strongly separate from the anteroloph. Round/oval P4s with a connection between the posterior outgrowth of the protocone and the hypocone. Upper molars with usually a mure oblique and aligned with the anterior arm of the hypocone. Important variation in terms of development, configuration and orientation of the second transverse cristid on p4s and lower molars, and of the third transverse crest and metaloph on upper teeth, but with a tendency to their reduction and absence. Trilophodont lower molars in most cases. Among upper teeth, P4s with stronger reduction of the third crest and metaloph. Upper molars with a third crest more frequently reduced to a mesoloph and without metaloph. Persistent flexids/fossettids on lower molars and flexi/fossettes except the posterior ones (the distal mesoflexus and metaflexus if present) on upper molars. Differs from Chambiramys in having slightly higher crowns, and more quadrangular upper molars; from Eoincamys, Incamys and Scleromys/“Scleromys” in having more square-shaped upper molars and a more important reduction of the third transverse crest on upper molars (i.e., more frequent trilophodont pattern than in these genera); from Eoincamys ameghinoi, Eoincamys pascuali and Incamys in having a second transverse cristid usually more reduced on lower molars; from Eoincamys pascuali in having a thinner furrow (when it is present) on the metalophulid I, which can be situated at the middle of this lophid; from Saremmys in having lower-crowned teeth, thinner loph(-id)s, a metaconid postionned more distally and strongly connected to the protoconid by a thick second cristid (metaconid cristid and/or posterior arm of the protoconid) on dp4s, a less oblique hypolophid on p4s and lower molars, a more bulbous protocone and a hypocone mesiolabially-distolingually compressed on dP4s, and some upper molars with a distinct metacone and a long metaloph and PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 41 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru mesoloph; from Saremmys, Scleromys/“Scleromys”, Garridomys and Drytomomys in having an anterior arm of the hypoconid, which can be present on p4s and lower molars, and in a more curved posterolophid on lower molars; from Incamys, Saremmys, Scleromys/“Scleromys”, Garridomys and Drytomomys in having a smaller size; from Garridomys and Drytomomys in having a second transverse cristid usually more reduced on lower molars; from Garridomys in having a homogeneous thickness of the enamel layer, thinner loph(-id)s, and strong and high protoconid and ectolophid on p4s; from “Scleromys” colombianus and Drytomomys aequatorialis in having a separation between the hypocone and the posteroloph thinner and less persistent at early wear stages on upper molars; and from Drytomomys in having a less oblique hypolophid on lower molars. Microscleromys paradoxalis Boivin & Walton sp. nov. urn:lsid:zoobank.org:act:38631BE932E6-4E5D-B5CE-469C37B98D8C Fig 9 and S1 Table Nom. nud. Microscleromys cribriphilus Walton, 1990, Fig 12F, 12G-12O, p. 45. Nom. nud. Microscleromys paradoxalis Walton, 1990, Fig 11A-11J, p. 39. Nom. nud. Microscleromys ?paradoxalis Walton, 1990, Fig 13B, 13C p. 49. Microscleromys sp. Walton, 1990, Fig 13A, p. 49. Nom. nud. Microscleromys cribriphilus Walton, 1997, Fig 24.2.N, p. 295. Nom. nud. Microscleromys paradoxalis Walton, 1997, Fig 24.2.G-I, p. 295. Holotype IGM 250321, right M1 or M2? (Fig 9P). Referred material MUSM 4643 (Fig 9A–9E), left fragment of mandible bearing incisor and dp4–m1; ING-KU 8604, right fragment of mandible bearing p4–m2; MUSM 4640–4642 (Fig 9F), left dp4s; IGM 250306 (Fig 9G), left p4; IGM 250305 and 250308 (Fig 9K), right p4s; MUSM 4644, left lower molar; IGM 250265, MUSM 4645, right lower molar; IGM 250288, 250307, IGM-DU 88–814, 89–675, MUSM 4646–4650 (Fig 9H, 9I, 9M and 9N), left m1 or m2; IGM 250319, MUSM 4651–4656 (Fig 9L), right m1 or m2s; IGM 250274, 250285, 250304, MUSM 4657 (Fig 9J), left m3s; IGM 251020 (Fig 9O), right m3; IGM 250302, IGM-DU 88–815 (Fig 9Z), ING-KU unnumbered specimen (Fig 9U), MUSM 4623 (Fig 9Q), left dP4s; MUSM 4624 (Fig 9A’), left P4; MUSM 4625 (Fig 9V), right P4; MUSM 4626, left upper molar; MUSM 4627, right upper molar; IGM 250283, IGM-DU 89–673, MUSM 4628–4631 (Fig 9X, 9B’ and 9C’), left M1 or M2?s; MUSM 4632–4636 (Fig 9R, 9S and 9W), right M1 or M2?s; IGM 250284, MUSM 4637 (Fig 9T), left M3?s; IGM-DU 85–410, MUSM 4638, 4639 (Fig 9Y and 9D’), right M3?s. Derivation of name Based on the original description of the species [37]: ‘from the latin paradoxalis, in reference to the strange and confusing morphology of this species’. In light of the last discoveries of caviomorph faunas in Peru, the morphology of this species does not appear so strange or confusing, but we prefer maintaining this name for the sake of both usage and courtesy. PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 42 / 80 PLOS ONE PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 Miocene caviomorphs from Tarapoto, Peru 43 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Fig 9. Microscleromys paradoxalis gen. et sp. nov. Mateial from TAR-31, Peru (black background) and from La Venta, Colombia (white background). (A–E) MUSM 4643, left mandibular fragment bearing dp4–m1. (F) MUSM 4641, left dp4. (G) IGM 250306, left p4. (H) MUSM 4647, left m1 or m2. (I) MUSM 4648, left m1 or m2. (J) MUSM 4657, m3. (K) IGM 250308, right p4 (reversed). (L) MUSM 4654, right m1 or m2 (reversed). (M) MUSM 4650, left m1 or m2. (N) MUSM 4649, left m1 or m2. (O) IGM 251020, right m3 (reversed). (P) IGM 250321��� , right M1 or M2? (reversed). (Q) MUSM 4623, left dP4. (R) MUSM 4634, right M1 or M2? (reversed). (S) MUSM 4633, right M1 or M2? (reversed). (T) MUSM 4637, left M3?. (U) ING-KU unnumbered specimen, left dP4. (V) MUSM 4625, right P4 (reversed). (W) MUSM 4632, right M1 or M2? (reversed). (X) MUSM 4631, left M1 or M2?. (Y) MUSM 4638, right M3? (reversed). (Z) IGM-DU 88–815, left dP4. (A’) MUSM 4624, left P4. (B’) MUSM 4630, left M1 or M2?. (C’) MUSM 4629, left M1 or M2?. (D’) MUSM 4639, right M3? (reversed). (A, F-D’) Occlusal, (B) anterior, (C) posterior, (D) lingual, and (E) labial views. The asterisks appoint the holotype material. The illustrations of the fossil specimens are 3D digital models obtained by X-ray μCT surface reconstruction (A-E), scanning electron photomicrographs (F, H-J, L-N, Q-T, V-Y, and A’-D’), and computerized schematic line drawings based on Walton (Fig 24.2 [18]; Fig 13 [37]) (G, K, O, P, U, and Z). https://doi.org/10.1371/journal.pone.0258455.g009 Type locality CVP 13B locality, Fish Bed near Casa Cuzco, La Venta Badlands, upper Magdalena Valley, Huila Department, Colombia. Other localities localities unknown (for ING-KU unnumbered specimen and ING-KU 8604), IGM-DU loc. 075, between the Chunchullo and the Tatcoa Sandstone Beds (only for IGM 250265), and localities of the Fish Bed (including CVP 5, 8, 9, 10, and 10A) and IGM-DU loc. 006–1, Monkey Beds (only for IGM-DU 88–814), La Venta Badlands, upper Magdalena Valley, Huila Department, Colombia; TAR-31, Tarapoto/Juan Guerra, Rı́o Mayo, Shapaja road, San Martı́n Department, Western Amazonia, Peru. Formation and age La Victoria Fm. and Baraya Member of the Villavieja Fm.; lower member, Ipururo Fm.; late middle Miocene (i.e., Laventan SALMA). Diagnosis Species with teeth approximately 25–30 per cent larger than those of M. cribriphilus. dP4s can have a pseudo-taeniodont or taeniodont pattern. Description MUSM 4643 is a right fragment of mandible bearing a part of the lower incisor and dp4–m1 (Fig 9A–9E). The incisor is mesially and distally broken, but the preserved portion testifies to a long radius of curvature of this tooth. The preserved part of the dentary corresponds to the ventrally broken body below the dp4–m1. It is very damaged, showing the base of the roots of the cheek teeth and the entire surface of the preserved fragment of incisor. The anterior root of dp4 is mesiodistally large, perpendicular to the plan of the neck, but clearly oriented mesioventrally in the dentary. Conversely, the posterior root of m1 is slightly oriented distoventrally. The morphology of the notch for the insertion of the tendon of the zygomatico-mandibularis pars infraorbitalis, masseteric crest or lateral crest is not accessible. In addition to the dp4 preserved on MUSM 4643 (Fig 9A), three other dp4s are attributed to Microscleromys paradoxalis (Fig 9F). MUSM 4641 is complete (Fig 9F), whereas MUSM 4642 is a posterior fragment of dp4, in which the metaconid and metalophulid I are missing. MUSM 4640 is worn and abraded especially at the level of the metalophulid I, which is reduced to its distal part. The two complete dp4s are longer than wide, with a trigonid narrower than PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 44 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru the talonid. The metalophulid I is distally well arcued, forming the mesial edge of the tooth. It is separate from the metaconid and protoconid by narrow notches on the MUSM 4643 dp4 (Fig 9A). On MUSM 4641 (Fig 9F), a similar configuration is present for its lingual extremity, whereas its labial extremity reaches the base of the protoconid. A continuous second cristid links the metaconid to the protoconid. Hence, it would be formed by a metaconid cristid and/ or posterior arm of the protoconid. It is mesiolingually directed and faintly curved on MUSM 4641 (Fig 9F) and MUSM 4643 dp4 (Fig 9A). It appears more transverse on MUSM 4640. On the MUSM 4643 dp4, MUSM 4641 and 4642 (Fig 9A and 9F), a mesostylid is present on the lingual margin. It can be mesially and distally isolated (MUSM 4641; Fig 9F), mesially isolated (MUSM 4643 dp4; Fig 9A) or connected to both the metaconid and entoconid (MUSM 4642). From that stylid, a neomesolophid runs labially and connects to a mesolophid reduced to a low spur. The two complete dp4s show neither neomesolophid nor mesolophid. The mesial and distal ectolophids are distolingually directed and aligned. They are not discernable from each other on the MUSM 4640, 4642 and 4643 dp4 (Fig 9A). On MUSM 4641 (Fig 9F), they are constituted of a short and thin mesial part (?mesial ectolophid) connecting to a longer and thicker distal part (?distal ectolophid). The labial part of the hypolophid is slightly oblique (i.e., distolingually directed), whereas its lingual part is more transverse. At early stages of wear (MUSM 4641; Fig 9F), the hypoconid displays a very short anterior outgrowth, which becomes longer with wear (MUSM 4642 and 4643 dp4; Fig 9A). The dp4s at advanced stages of wear have a non-taeniodont pattern, whereas MUSM 4641 shows a low and thin anterior arm of the hypoconid (i.e., pseudo-taeniodont pattern). The posterolophid is strong, well curved and separate from the entoconid by a large furrow. The hypoflexid is V-shaped with an anterior margin longer than the distal one. The p4s are slightly longer than wide with the trigonid smaller than the talonid (Fig 9G and 9K). They are tetralophodont. Due to the moderate-advanced stage of wear of all p4s, the conids and stylids are not discernable from the lophids. The mesial margin of p4s is constituted by a transverse metalophulid I. Although it cannot be confirmed, a mesostylid may be present because the area of the posterior arm of the metaconid is long, especially on IGM 250306 (Fig 9G) and 250308 (Fig 9K), and could be fused with this stylid distally. The second cristid is complete on IGM 250306 (Fig 9G) and 250308 (Fig 9K), transverse on the former and mesiolingually directed on the latter. Moreover, IGM 250306 has a tiny spur on its lingual margin between the second cristid and the entonconid (Fig 9G). This spur could be a very short neomesolophid. On IGM 250305, the second cristid is reduced to a lingual cristid, labially free. It woud be a metaconid cristid or a neomesolophid depending on if it is stemming from the metaconid or the mesostylid, respectively. The metaconid area is thick on the mesiolingual corner of the p4 of the mandibular fragment ING-KU 8604, and could include a metaconid cristid. On that tooth, the protoconid has a very short posterior arm, and posteriorly to the metaconid area, there is a spur similar to that of IGM 250306 (Fig 9G), which is probably a rest of neomesolophid. The posterior arm of the metaconid (or mesostylid) is slightly connected to the entoconid on IGM 250305, 250306 (Fig 9G) and 250308 (Fig 9K). It is strongly linked to this cuspid on the most worn p4 (ING-KU 8604 p4). The ectolophid is oblique and connected mesially to the protoconid on all p4s. It connects distally to the hypolophid on IGM 250305, 250306 (Fig 9G) and 250308 (Fig 9K). Contrary to the other specimens, the ING-KU 8604 p4 shows the labial extremity of the hypolophid linked to the posterolophid instead of the ectolophid. The hypolophid is roughly transverse on all p4s. The hypoconid is fused with its anterior outgrowth, and the posterolophid is curved, with its lingual extremity reaching the entoconid. The anterior arm of the hypoconid is strong and complete on all p4s, except on IGM 250308 in which this arm is absent and the hypoflexid is fully confluent with the metaflexid (Fig 9K). PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 45 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Lower molars are brachydont (Hling = 0.47–0.57; Hlab = 0.49–0.72; Fig 9A, 9H–9J and 9L– 9O). The talonid of m3s is slightly narrower than the trigonid and with a distal part pinched labiolingually. The cuspids are very cristiform and thus, faintly differentiated from the cristids. At early stages of wear, the lower molars display a groove at the level of the metalophulid I separating it into an anterior arm of the metaconid and an anterior arm of the protoconid. The position of this groove varies: either in the middle part of the metalophulid I (MUSM 4647 and 4648; Fig 9H and 9I), lingual to the protoconid (IGM 250319, IGM-DU 88–814, 89–675, MUSM 4650 and 4655; Fig 9M), or closer to the metaconid (MUSM 4654; Fig 9L). On some other lower molars, the anterior arms of the metaconid and protoconid are slightly linked, either equally-sized (MUSM 4649–4653; Fig 9N) or the anterior arm of the protoconid can be shorter (MUSM 4657; Fig 9J). The posterior arm of the metaconid is strong and long (MUSM 4647, 4650, and 4655; Fig 9H and 9M), except on MUSM 4654 in which it is short (Fig 9L). On that tooth, there is a short posterior arm of the protoconid and metaconid cristid, which are both labially free. IGM 250304 has a metaconid cristid, which runs distolabially and remains free. On some lower molars at an advanced stage of wear (e.g., IGM 250274, 250319, 251020, IGM-DU 88–814, 89–675, ING-KU 8604, MUSM 4652, and 4653; Fig 9O), there is a dentine plateform at their mesiolingual corner, formed by the fusion of the metaconid, its arms, and possibly a metaconid cristid (+ ?neomesolophid + ?mesostylid). On MUSM 4644 and 4646, the ectolophid shows a small expansion, which could be a relic of the posterior arm of the protoconid. The ectolophid is oblique, aligned with the crestiform protoconid in most cases, and sometimes with the labial part of the hypolophid, its lingual part being more transverse (IGM-DU 88–814, 89–675, IGM 250319, 251020, and MUSM 4657; Fig 9J and 9O). The entoconid occupies a mesial position with respect to the hypoconid. The former usually has long and thick anterior and posterior arms, which are separated mesially to the posterior arm of the metaconid and distally to the posterolophid by tiny furrows (MUSM 4647, 4652, and 4653; Fig 9H). On MUSM 4650, the entoconid, having shorter and thinner arms, is mesially and distally isolated from these structures by larger notches (Fig 9M). The anterior arm of the hypoconid is either incomplete (MUSM 4654 and 4657; Fig 9J and 9L), notched (MUSM 4651 and 4653) or absent (e.g., IGM 250274, 250285, 251020, ING-KU 8604, MUSM 4643, and 4655; Fig 9A and 9O). On some lower molars at advanced stage of wear, the anterior arm of the hypoconid is, however, connected to the hypolophid-ectolophid junction (i.e., non-taeniodont pattern). The anterior outgrowth of the hypoconid is thin and becomes longer with wear. In addition, the hypoconid is strongly connected to the posterolophid. The latter is thick and C-shaped or hook-shaped. The dP4s have a suboval/subtrapezoidal occlusal outline, with rounded corners (Fig 9Q, 9U and 9Z). The cusps are quite bulbous. Mesially, the anteroloph is curved and separate labially from the paracone. Lingually, it is strongly (IGM-DU 88–815 and ING-KU unnumbered specimen; Fig 9U and 9Z) or slightly (IGM 250302 and MUSM 4623; Fig 9Q) connected to a massive protocone or its anterior arm. The posterior outgrowth of the protocone is very short. On IGM 250302 and ING-KU unnumbered specimen (Fig 9U), a short crestule (i.e., neoformation) runs distally from the anteroloph and remains free. On all dP4s, the lingual protoloph is absent (i.e., taeniodont pattern). The labial protoloph is strong and either transverse (IGM 250302, ING-KU unnumbered specimen, and MUSM 4623; Fig 9Q and 9U) or oblique (IGM-DU 88–815; Fig 9Z). The mure is complete, oblique, and tends to be aligned with the anterior arm of the hypocone. On the labial margin, the paracone and mesostyle have a similar size, whereas the metacone remains poorly distinct (not well-defined) as it is fused with the curved posterolophid. The mesostyle is connected to the metacone solely (or its anterior arm) on IGM-DU 88–815 (Fig 9Z), whereas it joins the paracone and metacone or its anterior arm on ING-KU unnumbered specimen and MUSM 4623 (Fig 9Q and 9U). On IGM 250302, the PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 46 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru mesostyle is mesially and distally isolated. The third transverse crest corresponds to a mesoloph, which is lingually free (IGM 250302) or slightly connected to a mesolophular spur stemming from the anterior arm of the hypocone (MUSM 4623; Fig 9Q), or directly to the anterior arm of the hypocone (ING-KU unnumbered specimen and IGM-DU 88–815; Fig 9U and 9Z). Most of dP4s have a short metaloph backwardly connected to the posteroloph via a longer posteroloph spur or not, delimiting a small and oval metafossette. However, it can be more slightly connected to the posteroloph spur (MUSM 4623; Fig 9Q). On IGM-DU 88–815, the metacone does not seem to bear a metaloph (Fig 9Z). On that tooth, a long crest slightly linked to the posteroloph in its lingual part is present, which could be a rest of metaloph and/or the posteroloph spur. In addition, there is a tiny crestule stemming from the labial part of the posteroloph, which connects to the labial extremity of the metaloph/posteroloph spur, thus delimiting a small and oval fossette on IGM-DU 88–815 (Fig 9Z). MUSM 4624 (Fig 9A’) and 4625 (Fig 9V) are two complete P4s at moderate stages of wear. They preserve a part of their roots: a lingual one very massive and two labial much smaller, which externally diverge. These two specimens are oval and high-crowned, especially at the level of their lingual margin, their labial margin being lower (Hling = 0.80–0.87; Hlab = 0.62– 0.70). On the labial margin, the paracone is large and connected to a strong and oblique labial protoloph. On MUSM 4625 (Fig 9V), the mure strongly links the labial protoloph to the short anterior arm of the hypocone. On that tooth, the mure is aligned with the anterior arm of the hypocone. On MUSM 4624 (Fig 9A’), the mure is slightly connected to the anterior arm of the hypocone by a thin and short crestule. In addition to the paracone, the rest of the occlusal outline is composed of a curved and long crest formed by the fusion of the anteroloph, protocone, its anterior arm and its posterior outgrowth, the mesostyle, posteroloph, and hypocone. On MUSM 4624, the paracone is mesially and distally separated from this long crest by a thin furrow (Fig 9A’), while the anteroloph reaches this cusp on MUSM 4625 (Fig 9V). On MUSM 4625, there are two connected crestules, one stemming from the anteroloph and the other from the labial protoloph (Fig 9V). The lingual protoloph is absent (i.e., taeniodont pattern). On MUSM 4624, the mesostyle is smaller than the paracone, whithout any mesoloph, and it joins the posteroloph (Fig 9A’). On that tooth, a metaloph stemming from a metacone entirely subsumed within the posteroloph, runs mesiolingually. Its lingual extremity is distally connected to a spur of the posteroloph, and ends close to a longer mesolophule, without connection to the latter. On MUSM 4625, the posteroloph exhibits a thickening in its labial part, which may correspond to its fusion with the mesostyle/mesoloph and/or metacone/metaloph (Fig 9V). The upper molars determined as M1 or M2?s are clearly mesiodistally elongated (IGM 250283, MUSM 4633, and 4634; (Fig 9R and 9S) or subsquare (IGM 250321, IGM-DU 89–673, MUSM 4629–4632, and 4635; Fig 9W, 9X, 9B’ and 9C’). It seems that these upper molars show a tendency of shape variation, ranging from mesiodistally elongated at early stages of wear to squarer configuration at later stages of wear. On these upper molars, the hypocone is either more labially situated than the protocone, or situated at the same level of this cusp. Other upper molars have a hypocone clearly positioned labially with respect to the protocone, and thus they could be M3s (determined as M3?s). All upper molars have a taeniodont pattern characterised by a paraflexus and hypoflexus fully confluent. On the two upper molars at the earliest stage of wear (MUSM 4634 and 4637; Fig 9R and 9T), there is a short anteroloph (+ parastyle?), which is separate from the long and curved anterior arm of the protocone by a thin (MUSM 4634; Fig 9R) or wider (MUSM 4637; Fig 9T) furrow. The anteroloph is either not connected to the paracone (e.g., IGM-DU 89–673, MUSM 4629, 4632, 4634; Fig 9R, 9W and 9C’), slightly (MUSM 4633, 4638; Fig 9S and 9Y) or more strongly (MUSM 4637, 4639; Fig 9T and 9D’) connected to this cuspid. The strong labial protoloph is usually oblique but it PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 47 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru can also be transverse (IGM 250321 and MUSM 4629; Fig 9C’). The anterior arm of the hypocone is oriented mesiolabially, except on MUSM 4634, in which it is more longitudinal. It tends to be aligned with a short mure. The labial protoloph can be in continuity to both the mure and anterior arm of the hypocone with roughly the same orientation (MUSM 4632, 4635, 4637, and 4639; Fig 9T, 9W and 9D’). On MUSM 4635, the labial protoloph bears on its mesial part a short crestule, which remains free on the paraflexus. It likely corresponds to a neocrest. Some upper molars have a well-defined mesostyle (MUSM 4626, 4632, 4634, 4637, and 4639; Fig 9R, 9T, 9W and 9D’). At early stages of wear, a thin and shallow notch separates the paracone from the mesostyle. A mesoloph, stemming from this style, is usually presents with a medium or short length, but it can also be more reduced as on MUSM 4637, in which it is incipient and poorly distinct from the mesostyle (Fig 9T). It can be transverse, either lingually free (IGM 250321 and MUSM 4637; Fig 9T), connected to a short mesolophule (MUSM 4633; Fig 9S), or directly connected to the anterior arm of the hypocone (MUSM 4631; Fig 9X). Some upper molars (IGM 250283, 250284, IGM-DU 89–673, MUSM 4630, and 4634; Fig 9R and 9B’) have a mesoloph backarldly directed and linked to the posteroloph (or its spur), thereby delimiting an oval and small posterior fossette. On MUSM 4626, 4632, and 4639 (Fig 9W and 9D’), the third crest is absent (neither mesoloph nor mesolophule). There is no clear metacone on all upper molars, but a metaloph connected lingually to the posteroloph is present on MUSM 4631 (Fig 9X). It is faintly linked to the posterior arm of the mesostyle on that tooth. One upper molar at an early stage of wear, MUSM 4639 (Fig 9D’), has a trilophodont pattern without neither third crest nor metaloph. At more advanced stages of wear, some molars are trilophodont but with a thick posteroloph, which may include a mesoloph and/or a metaloph and their associated cusps/styles (MUSM 4627–4629, 4632, 4635, and 4638; Fig 9W, 9Y and 9C’). Microscleromys cribriphilus Boivin & Walton sp. nov. urn:lsid:zoobank.org:act:74B60D5F-A8B3-4A2C-9483-D22739400EC8 Fig 10 and S1 Table Nom. nud. Microscleromys cribriphilus Walton, 1990, Fig 12A-12E, p. 45. Nom. nud. Microscleromys cribriphilus Walton, 1997, Fig 24.2.K-M,O, p. 295. Nom. nud. Microscleromys paradoxalis Walton, 1997, Fig 24.2.J, p. 295. Holotype MUSM 4491, left M1 or M2? (Fig 10H’). Referred material MUSM 4545, right fragmentary dp4 or p4; MUSM 4532–4541 (Fig 10A, 10F and 10K), left dp4s; MUSM 4542–4544, right dp4s; IGM-DU 88–818, MUSM 4546–4550 (Fig 10G, 10L and 10P), left p4s; IGM 251018 (Fig 10Q), MUSM 4551–4558 (Fig 10B), right p4s; IGM-DU 89– 676, left m1 or m2 (or p4); MUSM 4558–4561, left lower molars; MUSM 4562–4565, right lower molars; MUSM 4566, left m1 or m2?; IGM 250309, right m1 or m2?; MUSM 4567–4588 (Fig 10H and 10M), left m1?s; MUSM 4589–4600 (Fig 10C), right m1?s; MUSM 4601–4608 (Fig 10D, 10I, 10N and 10S), left m2?s; IGM 250303 (Fig 10R), MUSM 4609–4615, right m2s; MUSM 4621, left m3?; MUSM 4616–4620 (Fig 10E, 10O and 10T), left m3s; MUSM 4622 (Fig 10J), right m3; MUSM 4420–4433 (Fig 10U and 10E’), left dP4s; IGM 250320 (Fig 10J’), PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 48 / 80 PLOS ONE PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 Miocene caviomorphs from Tarapoto, Peru 49 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Fig 10. Microscleromys cribriphilus gen. et sp. nov. Material from TAR-31, Peru (black background) and from La Venta, Colombia (white background). (A) MUSM 4532, left dp4. (B) MUSM 4553, right p4 (reversed). (C) MUSM 4599, right m1? (reversed). (D) MUSM 4603, left m2?. (E) MUSM 4619, left m3. (F) MUSM 4540, left dp4. (G) MUSM 4548, left p4. (H) MUSM 4581, left m1?. (I) MUSM 4606, left m2?. (J) MUSM 4622, right m3 (reversed). (K) MUSM 4536, left dp4. (L) MUSM 4546, left p4. (M) MUSM 4583, left m1?. (N) MUSM 4605, left m2?. (O) MUSM 4616, left m3. (P) MUSM 4550, left p4. (Q) IGM 251018, right p4 (reversed). (R) IGM 250303, right m2? (reversed). (S) MUSM 4602, left m2?. (T) MUSM 4620, left m3. (U) MUSM 4425, left dP4. (V) MUSM 4453, left P4. (W) MUSM 4505, right M1 or M2? (reversed). (X) MUSM 4514, right M2? (reversed). (Y) MUSM 4521, left M3?. (Z) MUSM 4445, right dP4 (reversed). (A’) MUSM 4466, right P4 (reversed). (B’) MUSM 4489, left M1 or M2?. (C’) MUSM 4511, left M2?. (D’) MUSM 4530, right M3? (reversed). (E’) MUSM 4428, left dP4. (F’) MUSM 4452, left P4. (G’) MUSM 4502, right M1 or M2? (reversed). (H’) MUSM 4491��� , left M1 or M2?. (I’) MUSM 4525, right M3? (reversed). (J’) IGM 250320, right dP4 (reversed). (K’) IGM 251040, right P4 (reversed). (L’) MUSM 4508, right M1 or M2? (reversed). (M’) MUSM 4513, left M2?. (N’) MUSM 4517, left M3?. Occlusal views. The asterisks appoint the holotype material. The illustrations of the fossil specimens are scanning electron photomicrographs (A-P, S-I’, and L’-N’) and computerized schematic line drawings based on Walton (Fig 24.2 [18]; Fig 14 [37]) (Q, R, J’, and K’). https://doi.org/10.1371/journal.pone.0258455.g010 MUSM 4434–4447 (Fig 10Z), right dP4s; MUSM 4469, right P4 or M3?; MUSM 4448–4458 (Fig 10V and 10F’), left P4s; IGM 251040 (Fig 10K’), MUSM 4459–4468 (Fig 10A’), right P4s; MUSM 4470, 4471, left upper molars; MUSM 4472–4478, right upper molars; MUSM 4479– 4497 (Fig 10B’ and 10H’), left M1 or M2?s; MUSM 4498–4510 (Fig 10W, 10G’ and 10L’), right M1 or M2?s; MUSM 4511–4513 (Fig 10C’ and 10M’), left M2?s; MUSM 4514–4516 (Fig 10X), right M2?s; MUSM 4517–4524 (Fig 10Y and 10N’), left M3?s; MUSM 4525–4531 (Fig 10D’ and 10I’), right M3?s. Derivation of name Based on the original description of the species [37]: from the latin cribri (sieve) and philus (liking to or attracted to) ‘because this rodent is so far only known from isolated teeth recovered by screenwashing’. Type locality TAR-31, Tarapoto/Juan Guerra, Mayo River, Shapaja road, San Martı́n Department, Western Amazonia, Peru. Other localities Localities of the Fish Bed (including CVP 9, 10A, and 13B), La Venta Badlands, upper Magdalena Valley, Huila Department, Colombia. Formation and age Lower member, Ipururo Fm.; Baraya Member of the Villavieja Fm.; late middle Miocene (i.e., Laventan SALMA). Diagnosis Species with teeth approximately 25–30 per cent smaller than those of M. paradoxalis. dP4s can have a non-taeniodont pattern characterised by a complete and high lingual protoloph in continuity with the labial protoloph. Description The pattern of teeth usually follows that of M. paradoxalis described above, with few exceptions, but mostly the smaller size allows for a clear distinction of the two species (see S1 File). dp4s are longer than wide, with a trigonid narrower than the talonid. The protoconid appears to be the largest cuspid. On the dp4 at the earliest stage of wear (MUSM 4532; Fig PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 50 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru 10A), the metaconid is the most cristiform cuspid, which is linguolabially pinched. On worn dp4s (MUSM 4539, 4540, and 4544; Fig 10F), the protoconid displays three connections: (i) a mesiolingual one with a curved metalophulid I, which extends lingually to the metaconid; (ii) a distolingual one with the second transverse cristid, which connects labially to the metaconid; and (iii) a distal one with the mesial extremity of the mesial ectolophid. On the dp4s at the earliest stage of wear (MUSM 4532 and 4536; Fig 10A and 10K), the protoconid is separated from the metalophulid I and from the mesial ectolophid by two notches. The notch between the protoconid and the metalophulid I is the deepest and thus, it closes at a latter stage of wear than that between the protoconid and the mesial ectolophid (MUSM 4534). Conservely to MUSM 4532, the protoconid is also isolated from the second transverse cristid, which is reduced to a medium-sized metaconid cristid, on MUSM 4536 (Fig 10K). However, both structures are connected at their base. On MUSM 4532, 4534, and 4536 (Fig 10K), the metalophulid I is not connected to the metaconid (Fig 10A). On all dp4s, the second transverse cristid is slightly directed mesiolingually. The mesial and distal ectolophids are thin, distolingually directed, aligned and often not discernable from each other. A small mesostylid is often present (MUSM 4534, 4536, 4539, 4540, and 4541; Fig 10F and 10K). It is isolated from the entoconid on all dp4s. It is connected to the metaconid on MUSM 4534, 4539 and 4540 (Fig 10F). On MUSM 4532 (Fig 10A), a mesostyle may be present, fused with the long posterior arm of the metaconid. On MUSM 4539, a complete third transverse cristid links the mesostylid with the ectolophids. The other dp4s do not show any development of the third cristid. On all dp4s, a well-marked hypolophid links the large entoconid to the distal ectolophid. In most cases, the labial part of the hypolophid is slightly oblique (i.e., distolingually directed), whereas its lingual part is more transverse. The hypoconid always displays a short anterior outgrowth and a low, but complete anterior arm, reaching the hypolophid-distal ectolophid junction (i.e., pseudotaeniodont pattern). Besides, the hypoconid has lingually a very short posterior arm, connecting to a strong and well-curved posterolophid on MUSM 4532 and 4534 (Fig 10A). At early stages of wear, the posterolophid is separate from the entoconid or its posterior arm by a large furrow. The three lingual flexids show a similar large expansion. The hypoflexid is less extended, distolingually oriented and V- or U- shaped. Its anterior margin is longer than its distal one, except on MUSM 4533. p4s are brachydont-submesodont (Hling = 0.45–0.77; Hlab = 0.54–0.81; Fig 10B, 10G, 10L, 10P and 10Q). They are slightly longer than wide, with a talonid clearly wider than the trigonid. By their high elevation and large surface, the protoconid and hypoconid are the most distinct cuspids of the crown at the earliest stage of wear (MUSM 4548; Fig 10G). The protoconid is labiolingually pinched, whereas the hypoconid is mesiodistally compressed. At the mesiolingual corner, the metaconid is well defined during the first stages of wear (MUSM 4548 and 4553; Fig 10B and 10G). It displays a short or moderate posterior arm (MUSM 4548, 4550, and 4553; Fig 10B, 10G and 10P). On MUSM 4548 (Fig 10G), the metalophulid I is continuous and high, joining the protoconid to the metaconid. On MUSM 4546 and 4553 (Fig 10B), the anterior arms of the protoconid and metaconid are short, strongly sloped and separated by a large furrow, which is thinner on MUSM 4547 and 4550 (Fig 10P). The protoconid frequently has a short posterior arm, which can be very short (MUSM 4546–4548; Fig 10G and 10L) or reduced to a spur (IGM-DU 88–818, MUSM 4550, 4551, and 4557; Fig 10P). On IGM-DU 88–818, MUSM 4547 and 4557, it is lingually free. The posterior arm of the protoconid is absent on IGM 251018, MUSM 4553 and 4558 (Fig 10B). A thin and short metaconid cristid can be present (MUSM 4546 and 4548; Fig 10G and 10L). On MUSM 4546 (Fig 10L), it is slightly connected to the posterior arm of the protoconid. On MUSM 4548 (Fig 10G), these two cristids are linked at their base but not connected at their apex. The worn MUSM 4554 p4 shows an oblique second transverse cristid, linking the metaconid to the protoconid. Thus, this cristid PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 51 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru may be formed here by the fusion of a metaconid cristid with a posterior arm of the protoconid. On MUSM 4551, a round and small mesostylid is clearly distinct, separated from the metaconid (or its posterior arm) and entoconid by tiny notches. On that tooth, a cristid, probably a neomesolophid, is faintly connected to the mesostylid and to a very tiny posterior arm of protoconid. In addition, it shares a very slight connection mesially with the mesialmost lophid and distally with the hypolophid. On MUSM 4546, the distal extremity of the posterior arm of the metaconid displays a tiny spur that may be a neomesolophid that would be connected to a mesostylid fused with the posterior arm of the metaconid (Fig 10L). At some advanced stage of wear, the metaconid, its anterior and posterior arms, metaconid cristid, as well as possibly the mesostylid and neomesolophid (if present), fuse and form a small platform of dentine on the mesiolingual corner of the teeth. If the entoconid has anterior and posterior arms, the latter is longer than the former (MUSM 4547, 4548, 4550, and 4556; Fig 10G and 10P), except on IGM 251018 and MUSM 4546, in which they are equally-sized (Fig 10L and 10Q). The entoconid (or its anterior arm) is separated from the mesostylid-posterior arm of the metaconid by a large furrow (MUSM 4548 and 4553; Fig 10B and 10G) or a very thin notch (MUSM 4546, 4547, 4550, and 4556; Fig 10L and 10P). On p4s at a more advanced stage of wear, these structures are connected. Stemming from the entoconid, a hypolophid runs labiomesially (i.e., oblique) and slightly connects to the ectolophid (IGM-DU 88–818, MUSM 4546, and 4548; Fig 10L). The anterior arm of the hypoconid usually reaches the hypolophidectolophid junction (i.e., non-taeniodont pattern). On p4s at early stages of wear (MUSM 4546, 4548, and 4553; Fig 10B, 10G and 10L), this cristid is more or less notched but remains elevated. However, on MUSM 4550 (Fig 10P), the notch of the anterior arm of the hypoconid is more pronounced, involving a partial confluence between the metaflexid and the retroverse hypoflexid (i.e., pseudo-taeniodont pattern). IGM 251018 appears to have a taeniodont pattern (Fig 10Q). The posterior arm of the hypoconid is fused with the strong and curved posterolophid, the latter extending lingually and ending directly distal to the entoconid, connecting to its base. The hypoconid displays a long and thin anterior outgrowth. Lower molars are brachydont-mesodont (Hling = 0.50–0.79; Hlab = 0.54–0.91; Fig 10C– 10E, 10H–10J, 10M–10O and 10R–10T). The m3s have a distal part of the talonid labiolingually pinched, and thus are more recognisable than m1s and m2s. Some lower molars appear smaller, and could be m1s (identified as m1?s). The m3s are either roughly equal in size to the lower molars determined as m1?s or smaller. Lower molars differ from p4s in having a wider trigonid comparatively to the talonid. The cristids are thick and incorporate in their ends the cuspids, which are thus faintly differentiated (except for the entoconid). The metalophulid I can show a thin interruption (groove) either in its middle part, separating equally-sized anterior arms of the metaconid and of the protoconid (MUSM 4611), or directly lingual to the protoconid, separating a long anterior arm of the metaconid from a very short and low anterior arm of the protoconid (MUSM 4588 and 4616; Fig 10O). On some other lower molars, both arms are slightly connected (e.g., IGM 250303, MUSM 4573, 4597, 4599, and 4620; Fig 10C, 10R and 10T). MUSM 4586 shows an additional cristid stemming from the metalophulid I, running distally and ending freely. The metaconid has a strong posterior arm, which can be long (e.g., MUSM 4592) or short (e.g., MUSM 4580, 4588, and 4599; Fig 10C). This cristid connects to a distinct mesostylid on some lower molars (MUSM 4580, 4582, 4584, 4588, 4597, 4599, 4601, and 4605; Fig 10C and 10N). A neomesolophid stemming from the mesostylid can be present (e.g., MUSM 4580, 4588, and 4605; Fig 10N) or absent (e.g., MUSM 4582, 4584, 4597, 4599, and 4601; Fig 10C). The neomesolophid is either limited to a very short enamel spur or short cristulid. It is labially free on all specimens. Two lower molars (MUSM 4597 and 4613) exhibit a short lingual cristulid from the metaconid, recognised as a metaconid cristid, which remains labially free. As p4s, some lower molars at an advanced stage of wear have a PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 52 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru mesiolingual dentine platform, formed by the fusion of the metaconid, its arms, and possibly the metaconid cristid and/or neomesolophid + mesostylid. On several lower molars (e.g., MUSM 4560, 4563, 4568, 4571, 4588, 4613, and 4622; Fig 10J), there is a small lingual expansion on the ectolophid that is likely to be a relic of the posterior arm of the protoconid. With the reduction or absence of the neomesolophid and the posterior arm of the protoconid, the mesoflexid is partly or entirely confluent with the anteroflexid. The ectolophid is oblique, frequently aligned with the crestiform protoconid and the hypolophid, thereby forming a long mesiocentral (diagonal) cristid. Lingually, the entoconid is the most differentiated cuspid. It is frequently mesiodistally elongated by developing strong anterior and posterior arms, which are particularly long on MUSM 4582. They tend to be mesially connected with wear to the mesostylid-posterior arm of the metaconid, and distally to the posterolophid. On lower molars at earliest stages of wear, thin furrows usually isolate the entoconid from these other structures mesially and distally. A short but strong hypolophid extends from the entoconid to the distal extremity of the ectolophid. As on p4s, the hypoconid on lower molars is mesiodistally compressed and displays a thin anterior outgrowth, which becomes long with wear. The hypoconid is distal in position to the entoconid. The anterior arm of the hypoconid can be present or absent (e.g., IGM 250303, MUSM 4600, and 4616; Fig 10O and 10R). When it is present, it is faintly developed and runs to the ectolophid–hypolophid junction. It is grooved (e.g., MUSM 4579, 4605, and 4620; Fig 10N and 10T) or deeply notched (e.g., MUSM 4569 and 4602; Fig 10S), showing a suite of intermediate sizes of the notch. Hence, the metaflexid is more or less confluent with the hypoflexid (i.e., from pseudo-taeniodont pattern to taeniodont pattern). However, many lower molars at an advanced stage of wear show a complete anterior arm of the hypoconid, which can be thick (i.e., non-taeniodont pattern; e.g., MUSM 4581, 4606, and 4622; Fig 10H–10J). A massive and hook-shaped posterolophid strongly connects to the hypoconid, both structures being fused and in perfect continuity. dP4s are longer than wide and with rounded corners (Fig 10U, 10Z, 10E’ and 10J’). They have a suboval occlusal outline, with a well-arcuate anteroloph (i.e., mesially convex) and posteroloph (i.e., distally convex). All dP4s are brachydont. The cusps are usually moderately inflated. The anteroloph is curved, short, and located far mesially. At early stages of wear, two notchs separate the anteroloph labially from the paracone (except on MUSM 4445; Fig 10Z) and lingually from the strong and bulbous protocone or its anterior arm. The first notch is wide, while the second is highly variable in terms of development. It corresponds to a very large notch on some dP4s (MUSM 4425, 4427, 4428, 4431, 4432, and 4435; Fig 10U and 10E’), whereas, on other teeth, it is a thinner and shallower furrow (MUSM 4429, 4439, 4440, and 4443) or a tiny groove (MUSM 4430, 4437, 4442, 4445, and 4446; Fig 10Z). On MUSM 4438, the protocone is mesiolingually-distolabially pinched, developing a short anterior arm, which is strongly connected to the anteroloph. The posterior outgrowth of the protocone is very short on the whole dP4s. On MUSM 4422 and 4428 (Fig 10E’), a well-cuspate parastyle is discernable on the mesiolabial corner of the tooth. This style bears short, but strong, anterior and posterior arms, separate by deep furrows from the anteroloph and paracone, respectively. On MUSM 4422, a medium-sized loph (i.e., neoformation) runs lingually from the parastyle, slightly directed distolingually, but with its lingual extremity faintly linked to the anteroloph via a very short and thin crestule. On MUSM 4428 (Fig 10E’), this loph is present but reduced to a short spur, whose base is connected to a peculiar anteroloph, short and labially concave. Hence, the parastyle, its anterior arm, its spur and the anteroloph delimit a small and round fossette. On the labial margin, the paracone and mesostyle are equally sized, and the metacone is smaller and usually still visible. The mesostyle is separate mesially from the paracone by a large furrow, that closes with wear. This style can be connected (MUSM 4422, 4431, and 4438) or disconnected (MUSM 4424, 4425, 4427, 4428, 4430, 4439, and 4445; Fig 10U, 10Z and 10E’) PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 53 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru to the metacone (or its anterior arm). Posteriorly, the metacone is strongly connected to the well-curved posteroloph. On dP4s at early stages of wear, the lingual protoloph can be absent (i.e., taeniodont pattern; e.g., MUSM 4422, 4427, and 4428; Fig 10E’) or present, either thin and low (i.e., pseudo-taeniodont pattern; MUSM 4437, 4438, and 4445; Fig 10Z) or high (i.e., non-taeniodont pattern; MUSM 4425, 4431, and 4446; Fig 10U). The labial protoloph is thick and usually straight, but it can also be slightly oblique. On IGM 250320, MUSM 4420, 4427, 4428, 4444, and 4445 (Fig 10Z, 10E’ and 10J’), it is particularly strongly oblique. On MUSM 4425, 4429, 4431, 4432, 4439, 4440, 4442 and 4446, the labial and lingual protolophs are aligned and form a transverse crest linking the paracone to the protocone (Fig 10U). The mure is complete and high, except on MUSM 4429 in which it is low, involving a partial confluence of the mesial mesoflexus with the hypoflexus. It is usually longitudinal (i.e., parallel to the mesiodistal axis of the tooth), but can be more oblique, aligned with the anterior arm of the hypocone (IGM 250320, MUSM 4420, 4421, 4422, 4427, and 4443; Fig 10J’), and sometimes with an oblique protoloph (MUSM 4420 and 4427). The third transverse crest is parallel with the labial protoloph. It is composed of two crests: a short and thick mesoloph stemming from the mesostyle and a mesolophular spur stemming from the anterior arm of the hypocone. On some dP4s (MUSM 4425, 4438, and 4446; Fig 10U), the third crest is complete, with the mesoloph linked to a short mesolophule, whereas they can be unconnected or faintly connected together on other teeth (MUSM 4420, 4427, 4428 and 4443–4445; Fig 10Z and 10E’). On IGM 250320, MUSM 4428 and 4444 (Fig 10E’ and 10J’), the mesoloph seems to be absent or strongly reduced. IGM 250320 appears to not have any mesolophule (Fig 10J’). Most of dP4s have a short metaloph backwardly connected to the posteroloph via a longer posteroloph spur or not, delimiting a small and oval metafossette. However, it can be lingually free (MUSM 4427) or absent (MUSM 4425, 4428, and 4431; Fig 10U and 10E’). On MUSM 4442, there is a short posteroloph spur and a cusp-like structure on the distalmost flexus, which is probably a relic of the metaloph. The latter structures are not linked. On dP4s at the earliest stages of wear, the posteroloph displays a shallow (MUSM 4425, 4430, 4431, 4442; Fig 10U) or deep (MUSM 4427, 4428, 4446; Fig 10E’) furrow, which separates it from the hypocone. P4s are high-crowned, especially at the level of their lingual crown, their labial crown being lower (Hling = 0.81–0.96; Hlab = 0.45–0.79; Fig 10V, 10A’, 10F’ and 10K’). In occlusal view, their crown is oval, labiolingually wider than longer. Mesiolingually, the anteroloph, protocone, its anterior arm and its posterior outgrowth form a long crest, which is separate from the large and well-cuspate paracone by a thin furrow. Distally, the paracone remains free and separate from a long distal crest including the mesostyle, posteroloph, hypocone and its long anterior outgrowth. Therefore, a usually wider and persistant furrow occurs distal to the paracone. The mesial extremity of the anterior outgrowth of the hypocone is connected to the posterior outgrowth of the protocone, and to the whole of the aforementioned structures, thereby forming most of the peripheral margin of the tooth. There is no lingual protoloph on the whole specimens. Accordingly, the most anterior flexus is a fossette, corresponding to the confluence of the paraflexus with the hypoflexus (i.e., taeniodont pattern). In mostcases, the mure and the short anterior arm of the hypocone are thin, linked, and aligned, forming a longitudinal or oblique crest. On MUSM 4450 and 4455, these two structures are not aligned, the mure being more longitudinal than the anterior arm of the hypocone. On MUSM 4456, the mure is not linked to the anterior arm of the hypocone or to the posteroloph. The mure is connected to the lingual extremity of the labial protoloph, which is almost transverse (IGM 251040, MUSM 4449, 4455, 4457, 4459, and 4466; Fig 10A’ and 10K’) or oblique (MUSM 4450–4454, 4456, 4461–4464, 4467, and 4468). The mure is absent on MUSM 4463, in which the labial protoloph is not connected to the anterior arm of the hypocone. On MUSM 4449, a longitudinal crest connects the anteroloph to the labial protoloph. Hence, these strutures and the paracone PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 54 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru delimits a very small rounded fossette. The mesostyle is small and crestiform, poorly distinct from the posteroloph. On MUSM 4452 and 4466 (Fig 10A’ and 10F’), it displays a very short mesoloph lingually free, longer on MUSM 4466 (Fig 10A’) than on MUSM 4452 (Fig 10F’). On P4s in a more advanced stage of wear (MUSM 4451, 4456, and 4468), the mesoloph is longer. On MUSM 4451, its lingual extremity is slightly connected to the mure-anterior arm of the hypocone and to the posteroloph. One P4, MUSM 4453 (Fig 10V), do not show any mesoloph. MUSM 4466 has a very short metalophular spur labially free, as well as a tiny crestule from the posteroloph, which could be a posteroloph spur or a metaloph (Fig 10A’). With wear, the posteroloph often exhibits a thickening in its labial part, which may correspond to its fusion with the mesostyle/mesoloph, metacone/metaloph and/or posteroloph spur (IGM 251040, MUSM 4450, 4455, 4458, 4459, 4463, and 4467; Fig 10K’). This plateform can be free, joined lingually to a tiny mesolophular spur (MUSM 4450) or directly to the mure (MUSM 4455). The different loci documenting upper molars (i.e., M1 vs M2 vs M3) are not easily recognisable (Fig 10W–10Y, 10B’–10D’, 10G’–10I’ and 10L’–10N’). Some upper molars are small, with a rounded occlusal outline and a hypocone clearly labially positioned with respect to the protocone, and thus they could be M3s (determined as M3?s; Fig 10Y, 10D’, 10I’ and 10N’). Other upper molars are square, mesiodistally elongated or transverse. On these upper molars, the hypocone is either more labially situated than the protocone or situated at the level of this cusp (i.e., distal). As for P4s, upper molars are labiolingually asymmetrical with a higher lingual crown (Hling = 0.71–1.07; Hlab = 0.42–0.65). In general, the cusps/styles are crestiform, and not well defined. In most cases (MUSM 4487, 4493, 4502, 4508, 4511, 4525, and 4527; Fig 10C’, 10G’, 10I’ and 10L’), the protocone shows a long and curved anterior arm, which is slightly connected to a shorter and straight anteroloph (+ ?parastyle). These two crests can be also strongly connected (MUSM 4489 and 4520; Fig 10B’) or separated by a narrow (MUSM 4491; Fig 10H’) or wide (MUSM 4505; Fig 10W) furrow. The anteroloph is connected to the mesial aspect of the paracone on some M1s or M2s (MUSM 4483, 4487, 4489, 4495, and 4502; Fig 10B’ and 10G’) and all M3?s (MUSM 4520, 4526, and 4527), whereas it remains separate from the paracone by a narrow furrow such as on MUSM 4482, 4486, 4491, 4494, 4499, 4505, 4513, and 4514 (Fig 10W, 10X, 10H’ and 10M’). On the MUSM 4525 M3?, the paracone is fully fused with the anteroloph (Fig 10I’). On MUSM 4511, the anteroloph bears a short but well-marked crestule lingually free on the paraflexus (Fig 10C’). The lingual protoloph is absent on all upper molars, thereby the paraflexus and hypoflexus are confluent (i.e., taeniodont pattern). The protocone displays a long and strong posterior outgrowth that can end close to the hypocone (MUSM 4495, 4498, 4517, 4519, 4520, 4524, and 4527–4530; Fig 10D’ and 10N’), or can connect to this cusp with wear (MUSM 4521; Fig 10Y). On all upper molars, the hypocone is usually smaller than the other main cusps. It displays a strong and oblique anterior arm, which connects to the mure and tends to be aligned with it. A labial protoloph, strong, short and slightly (e.g., MUSM 4486 and 4529) or strongly (e.g., MUSM 4483, 4489, and 4520; Fig 10B’) oblique, links the paracone to the mure. The anterior arm of the hypocone and the mure are in continuity with the labial protoloph, forming a long and diagonal central crest with a strong obliquity on some upper teeth (e.g., MUSM 4481, 4485, 4489, 4493, 4520; Fig 10B’). The labial protoloph can bear a short crestule mesially free on the paraflexus (MUSM 4498 and 4511; Fig 10C’). Labially, a strong mesostyle is present. It is clearly distinct on some upper molars (MUSM 4482, 4505, 4514, 4516, 4527, and 4530; Fig 10W, 10X and 10D’). It is separated from the paracone by a thin and shallow notch (e.g., MUSM 4483, 4487, 4505, and 4529; Fig 10W) or connected to this cusp (or to the posterior arm of paracone; MUSM 4489, 4516, and 4520; Fig 10B’), thereby forming a labial wall that closes the mesial mesoflexus. Distolabially, a metacone is either well separate from the posterior arm of the PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 55 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru mesostyle by a large but shallow notch (MUSM 4505 and 4514; Fig 10W and 10X), faintly connected to the posterior arm of the mesostyle (MUSM 4482, 4487, 4508, and 4522; Fig 10L’) or more strongly linked to it (MUSM 4516 and 4521; Fig 10Y). The upper molars show an important variation in the development, configuration and orientation of the third crest and metaloph. From the mesostyle, a strong but short mesoloph runs either (i) lingually: it is free (MUSM 4510, 4512, 4514, 4516, 4522, and 4525; Fig 10X and 10I’) or connects to a short mesolophule (MUSM 4471, 4487, 4494, 4513, 4515, and 4520; Fig 10M’); or (ii) backwardly: it is free (MUSM 4483 and 4499), or connects to the metaloph (MUSM 4508 and 4521; Fig 10Y and 10L’), to the posteroloph (via a spur or not; MUSM 4481, 4489, 4491, 4501, 4504, 4511, and 4528; Fig 10B’, 10C’ and 10H’), or seemingly to both crests (MUSM 4482). On MUSM 4521, the mesolophule is not connected to the mesoloph (Fig 10Y). On MUSM 4505, the mesoloph is slightly joined to a tiny mesolophule as well as to the metaloph (Fig 10W). Some upper molars show a distinct metacone, but without metaloph (or almost absent; MUSM 4491; Fig 10H’). If the metaloph is present and not linked to the mesoloph and/or to the posteroloph (via a spur or not; MUSM 4482, 4505, 4508, and 4521; Fig 10W, 10Y and 10L’), it is lingually free (MUSM 4487, 4514, 4516, and 4522; Fig 10X). On MUSM 4502, 4529 and 4530 (Fig 10D’ and 10G’), there is neither the third crest nor the metaloph (i.e., trilophodont pattern). Most of molars at more advanced stages of wear are trilophodont (MUSM 4470, 4476, 4481, 4484, 4492, 4495, 4496, 4498, 4518, 4519, and 4528), but have a enlarged posteroloph, which may include a mesoloph and/or a metaloph and their associated cusps/styles (entirely subsumed within the posteroloph). The flexi are furrow-shaped, especially the anterior flexus (i.e., paraflexus + hypoflexus). Remark The specimens attributed to Microscleromys cribriphilus show a great variation of size. For example, the size range of the lower molars extends from 1.08 mm to 1.78 mm for the length, and from 1.01 mm to 1.57 mm for the width. Likewise, the size range of upper molars is 1.17– 1.71 mm for the length and 1.07–1.66 mm for the width. Due to the sample size that is distributed quite uniformly for the minimum range and without shape variation, we consider that all the material reported here documents a single taxon. Accordingly, we consider the smaller specimens to document smaller individuals. Moreover, it is worth noting that the smallest specimens are often teeth determined as m3?s and M3?s. We attribute the observed size variation between specimens partly resulting of size variation between loci. Common comparisons Basically, among caviomorph rodents, (i) the presence of high-crowned teeth with a taeniodont pattern and oblique loph(-id)s, (ii) the absence or reduction of second transverse cristid on lower molars, and (iii) the absence of metaloph and third transverse crest, or the reduction of the latter (mesoloph) on upper molars, are dental features characterising Chinchilloidea. The specimens described here do not show any heterogeneous thickness of the enamel layer, usually found in most advanced chinchilloid forms (including Incamys) [114, 127, 128]. There is also no heterogeneous thickness of the enamel layer in the alleged chinchilloid Branisamys (Salla, Bolivia, late early Oligocene–late Oligocene; [129–131], but see [4]), Saremmys [132], and in taxa recognised as stem-Chinchilloidea (Eoincamys and Chambiramys) [4]. Furthermore, this character is only insinuated in Scleromys/“Scleromys” [127]. By having highcrowned teeth with a tendency to show oblique loph(-id)s, not forming lamina, and with flexi (-ds) not filled with cement; p4s with a complete tetralophodont pattern or with a more trilophodont pattern; lower molars with a groove on the metalophulid I and without second PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 56 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru transverse cristid (or the latter if present, being strongly reduced); upper teeth with a metaloph reduced or absent, a third transverse crest absent or limited usually to a short mesoloph lingually free or backwardly connected to the posteroloph, the concerned dental specimens from TAR-31 have a morphology matching that of Microscleromys nom. nud. from La Venta (Colombia; late middle Miocene; Fig 24.2G-24.2O, p. 395 [18]; Figs 11–13 [37]). In particular, some specimens from TAR-31 are larger and attribuable to M. paradoxalis, whereas other, smaller, are determinable as M. cribriphilus (S1 File) [18, 37]. In addition, M. cribriphilus differs from M. paradoxalis in having some dP4s showing a non-taeniodont pattern characterised by a complete and high lingual protoloph in continuity to the labial protoloph (this study). Several other chinchilloid genera (Eoincamys, Chambiramys, Incamys, Saremmys, and Scleromys/“Scleromys”) [28, 70, 71, 73, 81, 114, 118, 130–134] have a similar pattern (i.e., highcrowned teeth, homogeneous thickness of the enamel layer; taeniodonty, obliquity of loph(-id) s, no lamina and no cement). Like Microscleromys, Eoincamys, Chambiramys, Incamys and Scleromys/“Scleromys” also show a great variation of the morphology of the second transverse cristid on lower molars, and third transverse crest and metaloph on upper molars in terms of presence/absence, length, orientation and connections. No lower molar from TAR-31 and La Venta has a neomesolophid as long as or longer than on the MUSM 2875 m2 of Chambiramys sylvaticus (Fig 5G, p. 80 [71]). Microscleromys differs from Chambiramys in being slightly higher-crowned, and with more quadrangular upper molars; from Eoincamys, Incamys and Scleromys/“Scleromys” in having upper molars more square-shaped and a reduction more important of the third transverse crest on upper molars (i.e., more frequent trilophodont pattern than in these genera); from Eoincamys ameghinoi, Eoincamys pascuali and Incamys in having a second transverse cristid usually more reduced on lower molars; from Eoincamys pascuali in having a thinner furrow (when it is present) on the metalophulid I, which can be situated in the middle of this lophid; from Saremmys in having lower-crowned teeth, thinner loph (-id)s, a less oblique hypolophid on p4s and lower molars, and some upper molars with a distinct metacone, and long metaloph and mesoloph; from Saremmys and Scleromys/“Scleromys” in having an anterior arm of the hypoconid, which can be present on p4s and lower molars, and with a more curved posterolophid on lower molars; and from Incamys, Saremmys, and Scleromys/“Scleromys” in having a smaller size. Saremmys is documented by two dp4s at early stages of wear (MPEF-PV 11348b and 11349a in Fig 6A and 6B [132]). However, in our opinion, MPEF-PV 11348b would be a dP4 by the proportions between its mesial and distal parts, shape of its outline margin and a pentalophodont pattern closer to an occlusal morphology of a dP4 than dp4. Comparatively to MPEF-PV 11349a, dp4s from La Venta and TAR-31 show a metaconid postionned more distally and strongly connected to the protoconid by a thick second cristid, which corresponds to a metaconid cristid and/or a posterior arm of the protoconid. Unlike dP4s of Microscleromys, MPEF-PV 11348b has a hypocone compressed distolabially-mesiolingually. Moreover, MPEF-PV 11348b displays a more crestiform protocone. Microscleromys differs from Garridomys and Drytomomys in having a smaller size, an anterior arm of the hypoconid that can be present on p4s and lower molars, and a second transverse cristid usually more reduced, and a more curved posterolophid on lower molars; from Garridomys in having a homogeneous thickness of the enamel layer, thinner loph(-id)s, and strong and high protoconid and ectolophid on p4s; from “Scleromys” colombianus and Drytomomys aequatorialis in having a separation between the hypocone and the posteroloph, which is narrower and less persistent at early wear stages on upper molars [28, 29, 127, 135]; and from Drytomomys in having a less oblique hypolophid on lower molars. To sum up, the material from TAR-31 is closer to Microscleromys. After revison, we validate Microscleromys paradoxalis and M. cribriphilus described by Walton [37] from La Venta, and identify both species at TAR-31. PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 57 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Microscleromys sp. 1 Fig 8D and 8E and S1 Table Nom. nud. Microscleromys ?paradoxalis Walton, 1990, Fig 13E, 13D, p. 49. Referred material UNC unnumbered specimen, left P4 (Fig 8D); IGM-DU 85–411, left P4 (Fig 8E). Localities Locality unknown (for UNC unnumbered specimen) and CVP locality of the Fish Bed (for IGM-DU 85–411), La Venta Badlands, upper Magdalena Valley, Huila Department, Colombia. Formation and age Baraya Member of the Villavieja Fm.; late middle Miocene (i.e., Laventan SALMA). Description and comparison UNC unnumbered specimen (Fig 8D) and IGM-DU 85–411 (Fig 8E) are two P4s at advanced states of wear. They have a morphology very similar to those of Microscleromys paradoxalis and M. cribriphilus characterised by a circular/oval outline, a taeniodont pattern, the reduction/absence of third crest and metaloph, and a hypoflexus lingually close due to a connection between the posterior outgrowth of the protocone and the hypocone. The latter structures, as well as the paracone, anteroloph, protocone, posteroloph and mesostyle (and metacone if present) are fused, and form a circular peripheral wall. The paracone is distally separate from this circular wall by a very thin furrow on UNC unnumbered specimen (Fig 8D), whereas both structures are slightly linked together on IGM-DU 85–411 (Fig 8E). The labial protoloph and the mure are mesiolabially oriented on IGM-DU 85–411 (Fig 8E). On UNC unnumbered specimen, the labial protoloph is transverse and the mure is longitudinal (i.e., parallel to the mesiodistal plan of the tooth; Fig 8D). On that tooth, the anterior arm of the hypocone is tiny, oblique, and links the mure to the hypocone. From the mesostyle area runs a short crest, corresponding to a mesoloph, which remains labially free on UNC unnumbered specimen (Fig 8D). On the two P4s, there is no apparent metaloph. In addition, IGM-DU 85–411 does not have any distinct mesoloph (Fig 8E). However, this tooth shows a more advanced stage of wear and a thick labial part of the posteroloph, which could encorporate a mesostyle, the metacone, and/ or the rest of a mesoloph and/or metaloph. These two teeth differ from their counterparts in M. paradoxalis and M. cribriphilus in being clearly larger (S1 File). By their taeniodonty, complete and elevated mure, and the connection between the posterior outgrowth of the procone and hypocone, the pattern of these P4s recalls the morphology of P4s of certain stem-caviomorphs (sensu Boivin et al. [4]; such as Cachiyacuy contamanensis, Cachiyacuy kummeli, Pozomys, Eoespina, and Tarapotomys), the chinchilloid Eoincamys parvus, and some cavioids (Australopracta, Luantus, and Neoreomys) [6, 11, 16, 70, 81, 86, 101, 136, 137]. The material from La Venta differs from Cachiyacuy, Pozomys, E. parvus and Tarapotomys in having a larger size; from C. contamanensis and some P4s of Tarapotomys subandinus in having a more reduced third crest; from C. kummeli in having an anterior arm of the hypocone and third crest (when present) reduced to a mesoloph instead of a mesolophule; from Pozomys in having a complete anteroloph; from Australoprocta, Luantus initialis and L. propheticus in having a slightly larger size; from Australoprocta in having no (or more reduced) metaloph; from L. PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 58 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru initialis and L. propheticus in having a curved lingual edge of the hypocone; from L. propheticus in having a lower crown and a less quick closure of the lingual mesoflexus; from Neoreomys in having a lower crown, thinner crests, more expanded flexi, and a mesoloph not connected to the posteroloph. In summary, these two P4s from La Venta are clearly closer to those of Microscleromys. Their large size is not compatible with that of two species of the genus. Thus, we attribute them to Microscleromys sp. 1. Microscleromys sp. 2 Fig 11 and Table 1 Referred material MUSM 4658, left astragalus. Fig 11. Microscleromys sp. 2 from TAR-31, Peru, MUSM 4658, left astragalus. (A-C) Dorsal, (D, E) lateral, (F, G) plantar, (H, I) medial, (J, K) distal, and (L, M) proximal views. (B, C) Stereomicroscopic views. The illustrations of the fossil specimen are the result of the fusion of numerical multi-focus images obtained with an optical stereomicroscope (A, D, F, H, J, and L) and 3D digital models obtained by X-ray μCT surface reconstruction (B-C, E, G, I, K, and M). https://doi.org/10.1371/journal.pone.0258455.g011 PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 59 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Locality TAR-31, Tarapoto/Juan Guerra, Mayo River, Shapaja road, San Martı́n Department, Western Amazonia, Peru. Formation and age Lower member, Ipururo Fm., late middle Miocene (i.e., Laventan SALMA). Description MUSM 4658 from TAR-31 is a well preserved left astragalus (Fig 11). It is slightly damaged at the level of the lateral ridge of the trochlea (= lateral tibial and lateral fibular facets) and the ectal facet. In dorsal view, the astragalar body is slightly longer (proximodistally) than wide (lateromedially; Fig 11A–11C). The combined length of the neck and head is shorter than that of the trochlea. The latter is characterised by steep-sided lateral and medial margins (fibular facet and medial tibial facet, respectively). It is moderately grooved with slightly asymmetric internal facets. The internal lateral facet of the trochlea (= lateral tibial facet) is extensive and slopes gently, whereas the medial one is more inclined, taller, narrower, and barely longer (Fig 11A–11C and 11J–11M). No astragalar foramen (= superior astragalar foramen sensu Cifelli [138]) is evident. The neck is thick, short and very deflected medially (36.2˚; Fig 11A–11C). The head is rounded and bears a navicular facet wider than long. In medial view (Fig 11H and 11I), the navicular and medial tarsal facets are slightly proximally distinct, but distally continuous. The medial tarsal facet is particularly extensive proximally on the medial aspect of the neck (Fig 11H and 11I). In lateral and medial views, the rims of the trochlea are circular, with a moderate radius of curvature (Fig 11D, 11E, 11H and 11I). In medial view, seven large foramina are recognisable (Fig 11H and 11I). Three foramina are present distally to the area along which the tendons of m. peroneus longus and brevis slide and ventroproximally situated to the most distalmost rim of the medial ridge of the trochlea. They are ventroproximally aligned, the smallest foramen being the most proximal, whereas the largest is in the middle. There are three other foramina on the dorsodistal part of the area along which the tendons of m. peroneus longus and brevis slide, which are dorsoproximally aligned. The last foramen is located proximally to the area along which the tendons of m. peroneus longus and brevis slide. In plantar view (Fig 11F and 11G), the ectal (= calcaneo-astragalar) facet is oval and faintly curved (very slightly concave; i.e., high radius of curvature). Its distal part is not markedly projected laterally (i.e., absence of a protruding lateral process). It is clearly separated from the sustentacular facet by a deep and wide sulcus calcaneus, which is oblique (proximomediallydistolaterally) and divergent distally. As the proximolateral side of the sustentacular facet is gently sloping, the sulcus appears particularly wide. Three main foramina are situated on the sulcus, the largest being the most proximal, close to the medial plantar tuberosity. It might correspond to the plantar astragalar foramen (= inferior astragalar foramen sensu Cifelli [138]). Another foramen is present at the base of the proximolateral slope of the sustentacular facet. This tuberosity is well marked. The sustentacular facet is triangular-shaped and with a major axis proximodistally oriented. It is well extended distally toward the head, and reaches the navicular facet. The distal extremity of the sustentacular facet is wide and concave, whereas its proximal one is narrower and convex. The sustentacular and ectal facets have similar dimensions (i.e., length and width). The navicular and medial tarsal facets are poorly distinct over their entire contact. In distal view (Fig 11J and 11K), two large foramina are present on the distal extremity of the lateral ridge of the trochlea. PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 60 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Comparisons The trochlea of MUSM 4658 is shorter than in Euryzygomatomys, narrower than in Steiromys, Neoepiblema and Coendou, and less grooved than in Cavia, Dolichotis and Proechimys. It has a lateral ridge narrower and less distally positioned than Chinchilla and Dactylomys, and wider (but somewhat sloping) than the medial one, a feature observed on astragali of several other taxa (Platypittamys, Drytomomys, Phoberomys, Abalosia, Actenomys, Dolicavia, Eucelophorus, Eumysops, Pithanotomys, Praectenomys, Dactylomys, Euryzygomatomys, Lagostomus, Proechimys, and Octodon) and particularly accentuated on astragali of Steiromys, Neoepiblema and Coendou. The neck of MUSM 4658 is well differentiated from the body, and is strongly deflected medially as on astragali of Steiromys, Drytomomys, Neoepiblema, Phoberomys, Abalosia, Eumysops, Coendou, Dactylomys, Euryzygomatomys, Myocastor and Proechimys. Particularly, the deflection angle of MUSM 4658 is similar to that of astragali of Abalosia, Eumysops, Myocastor and Proechimys (35–37˚). Conversely, astragali of Platypittamys, Neoreomys, “Scleromys”, Actenomys, Dolicavia, Eucelophorus, Pithanotomys, Praectenomys, Cavia, Chinchilla, Clidomys, Ctenomys, Dinomys, Dolichotis, Galea, Lagostomus and Octodon have their neck being clearly less or not deflected medially. On MUSM 4658, the head is rounded with the medial tarsal facet strongly developed on the medial aspect of the neck in dorsal view as on astragali of Steiromys, Drytomomys, Actenomys, Eucelophorus, Eumysops, Pithanotomys, Coendou, Dactylomys, Octodon, and Proechimys. MUSM 4658 displays an ectal facet narrower than that of astragali of Neoepiblema, Actenomys and Eumysops; shorter than in Pithanotomys; narrower and longer than in Coendou. Contrary to the astragali of Neoreomys, “Scleromys” colombianus, Drytomomys, Neoepiblema, Eumysops, Pithanotomys, Cavia, Chinchilla, Dinomys, Dolichotis, Galea, Myocastor, Octodon and Proechimys cuvieri, there is no well marked lateral process of the ectal facet on MUSM 4658. The ectal facet is less concave than in Steiromys, Neoepiblema, Pithanotomys, Cavia porcellus, Dolichotis and Galea. Several taxa (Steiromys, “Scleromys”, Drytomomys, Neoepiblema, Phoberomys, Abalosia, Actenomys, Dolicavia, Eucelophorus, Eumysops, Pithanotomys, Cavia, Chinchilla, Dactylomys, Dinomys, Dolichotis, Euryzygomatomys, Galea, Lagostomus, Myocastor, Octodon, and Proechimys) have a wide sulcus calcaneus distally divergent like on MUSM 4658, but none seem to have a similar configuration of this sulcus and the proximolateral side of the sustentacular facet, which is gently sloping. In addition, MUSM 4658 differs from astragali of all aforementioned taxa in having a particular triangular-shaped sustentacular facet; from Steiromys, Neoepiblema, Coendou, Lagostomus, and Myocastor in having a sustentacular facet oriented proximodistally; from Dolicavia, Cavia, Chinchilla, Dolichotis, Euryzygomatomys, Galea and Myocastor in having a sustentacular facet not strongly fused with the navicular facet and/or the medial tarsal facet of the neck; and from Steiromys, Abalosia, Eumysops, Pithanotomys and Dactylomys in having no clear separation between the sustentacular facet and the navicular and medial tarsal facets. At first order, the MUSM 4658 astragalus matches very small body-sized extant and fossil rodents. Regression of the body mass in function of trochlear width in living mammals [139] shows that MUSM 4658 belonged to a small rodent having a body mass of about 43 g (Table 2). The smallest body-sized species from TAR-31, Ricardomys longidens, Microscleromys paradoxalis and M. cribriphilus, could be the best candidates. Considering the least-squares regression equation of [140] based on the m1 area in mammals, the body mass of R. longidens is estimated to be ~40 g, that of M. paradoxalis to be ~51 g and that of M. cribriphilus to be ~21 g (Table 2). The values estimated with equation proposed by the same author for rodents are clearly higher (~132, ~161, and ~76 g; Table 2), which makes it difficult to conclude. Given that the dental material from TAR-31 referred to M. cribriphilus is more abundant than that of R. longidens and M. paradoxalis, referral of MUSM 4658 to M. cribriphilus could be a plausible taxonomic PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 61 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Table 2. Metric feature of the MUSM 4658 astragalus (mm) and m1s (mm2) from TAR-31 and La Venta and body mass estimates (g). Material Trochlear Width (mm) MUSM 4658 1.832 Mean area m1 (mm2) Dental locus used N Body mass estimates (g) Regression equation for: Reference 1 43.00 mammals Tsubamoto, 2014 243.26 mammals Legendre, 1986 614.50 rodents 89.98 mammals 262.28 rodents 40.49 mammals 132.40 rodents Microsteiromys jacobsi 7.51 m1 1 Nuyuyomys chinqaska 4.36 m1 6 Ricardomys longidens 2.82 m1? 2 Microscleromys cribriphilus Microscleromys paradoxalis “Scleromys” sp. � 1.97 3.19 14.67 m1? m1� m 34 21.15 2 2 mammals 75.94 rodents 50.90 mammals 161.06 rodents 826.65 mammals 1751.02 rodents based on MUSM 4643 from TAR-31 and IGM unnumbered specimen from La Venta. https://doi.org/10.1371/journal.pone.0258455.t002 option. Nevertheless, as the size ranges of two co-occuring species of Microscleromys are overlapping, we prefer to keep it in open nomenclature as Microscleromys sp. 2. “Scleromys” Ameghino, 1887 “Scleromys” sp. Fig 12 and S1 Table Referred material MUSM 4277, left dp4; MUSM 4278, 4279, right fragmentary dp4s; MUSM 4280, left p4; MUSM 4281, 4282, right lower molars; MUSM 4270–4272, left fragmentary upper molars; MUSM 4273, 4274, left upper molars; MUSM 4275, 4276 right upper molars. Locality TAR-31, Tarapoto/Juan Guerra, Mayo River, Shapaja road, San Martı́n Department, Western Amazonia, Peru. Formation and age Lower member, Ipururo Fm., late middle Miocene (i.e., Laventan SALMA). Description MUSM 4278 is a distal fragment of dp4 preserving only the posterolophid, the hypoconid, its anterior outgrowth, and the distal margin of the ectolophid, hypolophid, and of the entoconid. MUSM 4279 is a mesial fragment of dp4, broken at the level of the ectolophids, the hypolophid and the entoconid (Fig 12C). This tooth also shows several damaged areas, especially at the level of the protoconid and the distal part of the metalophulid I. MUSM 4277 (Fig 12A) is a PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 62 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Fig 12. “Scleromys” sp. indet. from TAR-31, Peru. (A) MUSM 4277, left dp4. (B) MUSM 4281, right lower molar (reversed). (C) MUSM 4279, fragment of right dp4 (reversed). (D) MUSM 4282, right lower molar (reversed). (E–G) MUSM 4280, left p4. (H) MUSM 4273, left upper molar. (I) MUSM 4275, right upper molar (reversed). (J) MUSM 4272, left upper molar. (K–O) MUSM 4276, right upper molar (reversed). (A-E, H-K) Occlusal, (F, N) labial, (G, L) lingual, (M) anterior, and (O) posterior views. The illustrations of the fossil specimens are 3D digital models obtained by X-ray μCT surface reconstruction. https://doi.org/10.1371/journal.pone.0258455.g012 PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 63 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru complete dp4 at a more advanced wear stage than MUSM 4278 and 4279. These three brachydont dp4s are characterised by thick lophids. MUSM 4277 is pentalophodont, probably like MUSM 4279. Despite its degree of wear, MUSM 4279 seemingly displays bulbous lingual cuspids. On this tooth, the metalophulid I is mesially convex and shows a mesiodistal thickening at its centre. The metaconid and protoconid are distally linked by a second cristid, mesially concave, corresponding to a posterior arm of the protoconid and/or a metaconid cristid. The metalophulid I, the second cristid, the metaconid and protoconid delimit a C-shaped anterofossettid. The mesostylid is large and displays a short and strong neomesolophid mesiolabially directed and lingually free. The mesostylid is well separated from the metaconid and entoconid. This cuspid is higher than the mesostylid and metaconid. The hypolophid, distal ectolophid and mesial ectolophid are fused, and form a long cristid stemming from the entoconid and directed toward the protoconid, but without connection to it. This cristid is mesiolabially oriented, but its labial part is more longitudinal than its lingual part. The labial extremity of the neomesolophid is close to the mesial margin of the cristid combining the hypolophid and ectolophids. MUSM 4277 (Fig 12A) shows a similar pattern to that of MUSM 4279 (Fig 12C), but with features deriving from a more advanced stage of wear: loss of relief, lingual closure of the mesial mesoflexid and metaflexid, lingual and labial closure of the distal mesoflexid, and reduction of the anterofossettid and of the mesial mesofossettid to two small and circular fossettids. MUSM 4279 displays a heterogeneous thickness of the enamel layer, which is thicker on the trailing edges and thinner on the leading edges. On MUSM 4277 and 4278, the distalmost cristid (posterolophid + hypoconid + its anterior outgrowth) is curved and there is no anterior arm of the hypoconid, thereby involving the confluence of the metaflexid with the hypoflexid (i.e., taeniodont pattern). The MUSM 4280 p4 (Fig 12E) is slightly damaged at the level of the protoconid, the hypoconid and its anterior outgrowth. MUSM 4280 is hypsodont (Hling = 1.13; Hlab = 1.52; Fig 12F and 12G). Compared to dp4s, MUSM 4280 is tetralophodont, more square-shaped, and with a trigonid clearly narrower than the talonid. The cuspids are crestiform and faintly differentiated from the lophids. On the lingual margin, the metaconid and mesostylid are equally sized and separated by the anterostriid. The metaconid is connected to a metalophulid I, which is very slightly concave and labially free. This crest corresponds to an anterior arm of the metaconid, the anterior arm of the protoconid being absent. Its labial extremity is thin and separated from the protoconid by a narrow and deep furrow, which forms a striid. The second transverse cristid is reduced to a short neomesolophid stemming from the mesostylid. The neomesolophid is mesiolabially directed and labially free. The protoconid is fused with the ectolophid, forming a cristid distolingually oriented. The hypolophid is parallel to the neomesolophid and in continuity of the cristid composed of the protoconid + ectolophid (but the latter being more longitudinal). The hypoconid has no anterior arm (i.e., taeniodont pattern) and it is not distinct from its anterior outgrowth and the posterolophid. The main direction of the posterolophid is the same that those of the neomesolophid and hypolophid. The flexids are furrow-shaped and form striids. On the labial margin, the hypostriid is deep, but ends far from the neck of the crow. On the lingual margin, the anterostriid and mesostriid have a similar depth, which represents more than the double that of the short metastriid. As for p4, the lower molars, MUSM 4281 and 4282, are high crowned (but retaining roots), tetralophodont and taeniodont, with deep furrow-shaped flexids, forming striids, dental structures that fused with wear, forming thick and oblique cristids, and a second transverse cristid reduced to a neomesolophid (Fig 12B and 12D). Contrary to the MUSM 4280 p4, their trigonid and talonid are equally wide. They present a more advanced stage of wear with respect to MUSM 4280: their crown is lower (Hling = 0.82–0.83; Hlab = 0.82–1.19) and their lingual flexids are closed except the metaflexid on MUSM 4281. The metalophulid I is complete on both PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 64 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru molars. On MUSM 4281 (Fig 12B), the neomesolophid is very short and labially free. Consequently, the Y-shaped anterior fossettid corresponds to the confluence between the anteroflexid with the mesoflexid. Conversely, on MUSM 4282 (Fig 12D), the neomesolophid is longer and labially connected to the metalophulid I, thereby delimiting a small and circular anterofossettid. Like on MUSM 4280, the hypolophid, ectolophid and protoconid are in continuity, forming a central cristid. The latter is straight and oblique (i.e., mesiolabially directed) on MUSM 4281. On MUSM 4282, its labial part is mesiolabially directed, while the hypolophid is more transverse. Due to the closure of the anteroflexid and mesoflexus on MUSM 4281 and 4282, the trigonid is delimited by a triangular-shaped structure formed by the metalophulid I, metaconid, mesostylid, entoconid, hypolophid, ectolophid and protoconid. This triangularshaped structure is separate from the distalmost cristid by a very thin furrow-shaped flexid (i.e., confluence of the metaflexid with the hypoflexid). The distalmost cristid, formed by the fusion of the posterolophid, hypoconid and its anterior outgrouwth, is mesially concave. The thickness of the enamel layer is homogenous on MUSM 4282 (Fig 12D). MUSM 4281 displays a homogenous thickness of the enamel layer at the level of the metaflexid but a heterogeneous one (thicker on the trailing edges and thinner on the leading edges) at the level of its mesial and distal margins and those of the hypoflexid (Fig 12B). The hypostriid has a height similar to that of the MUSM 4280 p4. Upper molars have an occlusal pattern similar to that characterising p4 and lower molars (i.e., tetralophodonty, taeniodonty, deep furrow-shaped flexi, forming stria, and dental structures merging with wear, forming thick and oblique crests), which can contribute to confuse them. They show a unilateral hypsodonty characterised by a lingual crown higher that the labial one (Hling = 1.00–1.69; Hlab = 0.51–0.92), whereas the distolabial crown is the highest part of crown on p4 and lower molars. Upper molars are labiolingually wider than lower molars. Upper molars have three roots: a large lingual one and two small labial ones. The mesial margin of the crown is formed by a curved crest, which corresponds to the fusion of the anteroloph, protocone and its posterior outgrowth. On all complete upper molars, this crest is labially connected to the paracone. The labial protoloph is roughly transverse or slightly oblique and in continuity with the mure and anterior arm of the hypocone, which are more distolingually directed. The mesostyle is faintly isolated from the metacone on MUSM 4273 (Fig 12H). On the other upper molars, it is slightly (MUSM 4270 and 4272; Fig 12J) or strongly (MUSM 4274–4276; Fig 12I and 12K) connected to the this cusp and to the paracone. As for the second transverse cristid on p4 and lower molars, the third transverse crest is reduced to a mesoloph stemming from the mesostyle, either lingually free (MUSM 4270 and 4272) or connected to the posteroloph (MUSM 4273 and 4275; Fig 12H and 12I). On MUSM 4272 (Fig 12J), the posteroloph presents, on its mesial margin, a very short crestule (i.e., posteroloph spur), mesially free. On MUSM 4273 (Fig 12H) and 4275 (Fig 12I), the mesoloph and posteroloph (+ posteroloph spur?) delimit a small and circular fossette. On upper molars at a more advanced stage of wear (MUSM 4274 and 4276; Fig 12K), the small posterior fossette disappears due to the fusion between the mesoloph, mesostyle and posteroloph (+ posteroloph spur?). On MUSM 4270 and 4272 (Fig 12J), a tiny notch separtes the hypocone from the posteroloph. On the other upper molars, which show a more advanced stage of wear, the hypocone is strongly linked to the posteroloph. On the most worn upper molar, MUSM 4274, the two remaining flexi (the mesialmost) are fully closed and divided into two parts. Most upper molars (MUSM 4270, 4272 and 4274–4276; Fig 12J, 12I and 12K) display a homogenous thickness of the enamel layer, while MUSM 4273 (Fig 12H) has a slight heterogeneous thickness of the enamel layer (thicker on the trailing edges and thinner on the leading edges) at the level of its mesial and distal margins, and the mesial and distal margins of the anterior flexus. PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 65 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Comparisons These large dental specimens from TAR-31 described here show typical chinchilloid affinities, notably characters documenting Scleromys/“Scleromys”: (i) medium-sized and protohypsodont teeth, (ii) taeniodonty, (iii) dental structures merging with wear, forming thick and oblique crests/cristids, (iv) deep furrow-shaped flexi(-ds), forming striia/striids, (v) upper molars tetralophodont at early stages of wear, becoming trilophodont at more advanced stages of wear, (vi) a metaflexid as being the most persistent lingual flexid on lower teeth, and (vii) an enamel layer with a thickness homogeneous or slightly heterogeneous (i.e., insinuated heterogeneous thickness sensu Kramarz, Vucetich & Arnal [127]; see also [128]). The dental remains from TAR-31 are similar in size to those from La Venta (Colombia; late middle Miocene) attributed to either “Scleromys” schurmanni Stehlin, 1940 [18, 27, 28] or “Scleromys” cf. schurmanni [18]. By its short and labially-free metalophulid I and neomesolophid, the pattern of the MUSM 4280 p4 is particularly close to that of the p4 borne on the IGM 184233 specimen from La Venta attributed to “Scleromys” cf. schurmanni (Fig 24.3E, p. 397 [18]). However, it differs from the latter in having no mesiolingual protuberance of crown, and in showing separated metaconid and mesostylid. As for the material from TAR-31, the lower molars of “Scleromys” cf. schurmanni from La Venta can have a neomesolophid labially free or connected to the metalophulid I. The figured p4 of “Scleromys” schurmanni, also from La Venta (UCMP 38967 in Fig 24.3D, p. 397 [18]), has a complete second transverse cristid composed of a neomesolophid and a shorter posterior arm of the protoconid, contrary to MUSM 4280. The p4s of “Scleromys” schurmanni described by Field [28] show a complete second transverse cristid as well (UCMP 38987 in Figs 4A and 5D, p. 285 [28]) or a second cristid reduced to a neomesolophid linked to the metalophulid I (UCMP 37931 in Figs 4D and 5C, p. 285 [28]). The dp4s of “S”. schurmanni (UCMP 39902 and 38974 in Fig 5A and 5B, p. 285 [28]) seem to be hexalophodont, having more anterior flexids than on dp4s from TAR-31. The TAR-31 teeth are smaller than those of S. quadrangulatus (Pinturas Fm., Argentina; late early Miocene) [133], S. angustus (Santa Cruz Fm., Argentina; late early Miocene) [73] and S. osbornianus (Pinturas and Santa Cruz Fm.; late early Miocene) [118, 133]. Comparatively to S. quadrangulatus, teeth from TAR-31 have a slender closure of the flexi(-ds), a longer metalophulid I on p4, and a paracone area smaller on upper molars at early stages of wear. The MUSM 4280 p4 has a lingual margin more parallel to the mesiodistal axis of the tooth than p4s of S. quadrangulatus and S. angustus. In addition, MUSM 4280 has a neomesolophid, contrary to those species of Scleromys. At early stages of wear, molars of S. angustus and S. osbornianus display a trilophodont pattern [73, 133], contrary to molars from TAR-31, which remain tetralophodont. The dp4s from TAR-31 share a similar pattern in terms of number, types and morphology of the main cristids with the two dp4s from Madre de Dios, Peru: dp4 referred to S. quadrangulatus (MD-61, early Miocene) [141] and dp4 referred to “Scleromys” sp., gr. quadrangulatus-schurmanni-colombianus (MD-67, early middle Miocene) [31]. Nevertheless, they have a metalophulid I connected to the metaconid and protoconid, contrary to dp4s from MD-61 and -67. Unlike dp4s from TAR-31, dp4s from MD-61 and -67 have supernumerary structures on the anteroflexid: a cuspid-shaped structure on the dp4 from MD-67, and a low cristulid connected to the lingual and labial extremity of the metalophulid I on the dp4 from MD-61. The dp4s from TAR-31 display more opened lingual flexids than the dp4 from MD-61. They show other distinctive characters with respect to the dp4 from MD-67: a metaconid isolated from the mesostylid, and a second cristid and a hypolophid-ectolophid cristid, which are more curved. “Scleromys” colombianus and Drytomomys aequatorialis, both from La Venta (and from Nabón region, Ecuador, for D. aequatorialis; late middle Miocene) [28, 29, 135] are recognisable in having larger-sized teeth and a separation between the hypocone and the posteroloph, PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 66 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru wider and more persistent at early stages of wear on upper molars, than on the TAR-31 material. The material from TAR-31 differs from “S.” colombianus [18, 28] and “Scleromys” cf. colombianus [18] in having p4s with a transverse mesial margin and a shorter neomesolophid labially free, and upper molars with a more persistent posterior fossette. It differs from Drytomomys in having a quicker reduction of the anterofossettid on lower molars; from D. aequatorialis in having shorter neomesolophid on p4s; from D. typicus (Ituzaingo Fm., Argentina; late Miocene) [29, 106] in having different morphology of dp4s with a third cristid reduced to a short neomesolophid and without neolophid on the anterofossettid. Comparatively to MUSM 4280, “Drytomomys” sp. (small) and “Drytomomys” sp. (large) from La Venta (“Olenopsis” sp. sensu Walton [18], but see [29]) show a longer neomesolophid labially connected to the metalophulid I on p4s. Moreover, p4s of “Drytomomys” sp. (large) are pentalophodont with a short metaconid cristid, contrary to MUSM 4280. The moderate size, marked hypsodonty and thickness of the crests of these teeth from TAR-31 exclude affinities with Eoincamys (Brazil and Peru; ?late Eocene–early Oligocene) [8, 19, 70, 81], Microscleromys (La Venta, TAR-31; this study) [18] and with the octodontoid Protadelphomys (Sarmiento Fm., Argentina; early Miocene) [142–144]. They are also too high-crowned and slightly larger for Maquiamys (CTA-61, Peru; late Oligocene) [4, 71]. To sum up, the material from TAR-31 has the closest affinities with the specimens of “Scleromys” found in northern South America (La Venta, Colombia and MD-61 and MD-67, Peru). As it would be necessary to deeply revise Scleromys and notably the northern forms (recognized as “Scleromys” [18, 71]), we prefer keeping the material from TAR-31 in open nomenclature as “Scleromys” sp. Discussion The caviomorph fauna from La Venta: Revision and validation of Walton’s nomina nuda Microsteiromys jacobsi, Ricardomys longidens, Microscleromys paradoxalis and Microscleromys cribriphilus, originally described in La Venta in the framework of the Anne H. Walton’s Ph.D [37], have remained nomina nuda given that they were not formally described so far. According to the International Code of Zoological Nomenclature (p. 246 [38]), a nomen nudum is not available but the same name can be made available subsequently for the same concept or for a different concept, in a way meeting the criteria of availability (chapter 4 [38]). Our revision of the original material of these species, combined with the study of the new specimens from TAR-31, allowed us to validate all the concerned taxa. It is worth noting that we have usually not followed the unpublished diagnoses proposed by Walton [37]. In the case of Microscleromys, a classical descriptive and comparative approach combined with statistical analyses on dental dimensions (length and width; S1 File) allowed us to test the taxonomic groups proposed by Walton [18, 37]. Our results (i.e., statistical differences) suggest an inappropriate taxonomic delimitation of these potential groups (S1 File). Thus, we propose a new delimitation of the two Microscleromys species implying changes in the assignation of some specimens (i.e., from M. cribriphilus to M. paradoxalis, and vice versa). In addition, we revised the suprageneric status of this taxon, which was previously assigned to the cavioid family Dasyproctidae [18, 37]. Like Microscleromys, other fossil taxa such as Eoincamys, Incamys and Scleromys, were previously attributed to the dasyproctids [81, 130, 131]. However, Eoincamys, Incamys and Scleromys have then been related to the superfamily Chinchilloidea [70, 114, 133]. As noted by Walton [18], Microscleromys shows an occlusal pattern similar to Scleromys, as well as to chinchilloids in general (i.e., high-crowned teeth with a taeniodont pattern and oblique loph [-id]s, the absence of the second transverse cristid or its reduction on lower molars, and the absence of the metaloph and third transverse crest, or the reduction of the latter to notably a PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 67 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru mesoloph on upper molars). According to the recent phylogenetic analyses of Boivin et al. [4], the phylogenetic position of Microscleromys, but also those of Eoincamys, Incamys and Scleromys were retrieved as stem representatives of Chinchilloidea (but see [127, 145, 146]). Finally, as noted by Walton [18, 37], Ricardomys shows an occlusal morphology similar to Adelphomys and other adelphomyine octodontoids. Here, we assign it to the family Adelphomyidae, following the results of Boivin et al. [4]. The caviomorph assemblage from TAR-31 In being restricted to a single 5–20 cm-thick channelised lens in a very small area (~5 square metres in one piece), TAR-31 records a single attritional accumulation [36], which strongly contrasts with La Venta and Fitzcarrald local faunas (several levels from distinct outcrops spanning a long or short interval, respectively) [26, 33]. Nevertheless, TAR-31 yields a total of nine rodent taxa, with the co-occurrence of the four extant superfamilies: Erethizontoidea (three taxa), Cavioidea (one taxon), Octodontoidea (two taxa) and Chinchilloidea (three taxa). Compared with other Laventan caviomorph assemblages from low latitudes of South America, TAR-31 ranks second just in front to the fauna from Fitzcarrald Arch (eight taxa) [32, 33], but far from La Venta, which includes 28 taxa (21 excluding the indeterminate genera and specie; La Victoria and Villavieja formations, Colombia; see [18, 28] and this work). Like TAR-31, the faunas from La Venta and Fitzcarrald Arch, as well as that from MD-67 (Madre de Dios; similar outcrop conditions as in TAR-31) [31], show representatives of the four caviomorph superfamilies. Without considering the validation of the nomina nuda from La Venta, TAR-31 records a new erethizontoid (Nuyuyomys chinqaska gen. et sp. nov.) and perhaps a new stemcaviid, here described as Caviidae gen. et sp. indet. (see Systematic Palaeontology section). The single specimen assigned to this taxon does not allow for its formal description as a new species. TAR-31 shares two taxa or closely related taxa with MD-67 (“Scleromys” sp. and Nuyuyomys chinqaska) and one with the assemblages from Fitzcarrald local fauna and La Victoria Fm. of La Venta (“Scleromys”). TAR-31 shares with the Villavieja Fm. of La Venta a representative of “Scleromys”, as well as three species only recorded in La Venta so far: Microscleromys paradoxalis, M. cribriphilus and Ricardomys longidens. In striking contrast, there is no taxon in common between TAR-31 and most middle Miocene assemblages at mid- and high latitudes in South America (Argentina, Bolivia; Chile) [10, 30, 51, 87, 105, 123, 126, 147–152]. The only assemblage showing similarities with TAR-31 is that from the Marino Fm. (Divisadero Largo area, Mendoza, Argentina) [153], in yielding Scleromys sp. Nevertheless, Scleromys (and “Scleromys”) had a wide geographic (Argentina, Brazil, Chile, Colombia, and Peru) and a long stratigraphical range (late Oligocene–late middle Miocene) [15–19, 27, 28, 31, 33, 34, 73, 133, 141, 154]. Moreover, some doubts were expressed about the cogeneric afinities between Scleromys spp. from northern versus southern South America [18, 71]. Astragalus of Microscleromys from TAR-31: Morpho-functional implications The trochlea of MUSM 4658 does not have a mediolateral widening nor a high degree of asymmetry between its medial and lateral rims, thereby suggesting that parasagittal movements would be more favoured during plantar- or dorsi- flexion of the foot [57, 61, 155]. The trochlea is however moderately grooved with shallower and less sharp crests than those of astragali of cursorial-jumping taxa in which lateral movements are strongly restricted (cavioids: Cavia, Dasyprocta, Dolichotis, Galea) [61]. The ectal facet is long with a high radius of curvature and not projected laterally, which would give proximodistal and mediolateral mobility with the calcaneus (astragalo-calcaneal joint = sub-astragalar joint) [57]. The narrowing and the PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 68 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru proximodistal orientation of the sustentacular facet (here long reaching the navicular facet) would indicate parasagittal movements at the astragalo-calcaneal joint, limiting the inversion of the foot [57, 61]. In addition, the sulcus calcaneus is particularly wide, thereby suggesting the presence of a strong ligament linked to the dorsal side of the calcaneus [57]. This character is found in numerous caviomorphs, but associated with a proximodistal sustentacular facet, it would confer more resistance of the ankle by limiting the general mobility of the foot with respect to the leg [156, 157]. The neck is long and medially deflected with a medial tarsal facet on its dorsal and medial aspects, and a round head, characters typically found in arboreal taxa such as Coendou or Sciurus, but also in some terrestrial echimyids (Proechimys) [57, 61]. In addition to the increase of the articular surface of the sustentacular facet with the calcaneus [57], these featurs would therefore be facilitated lateral and rotator movements of the foot at the astragalo-navicular joint and at the contact between the astragalus and the medial tarsal bone (= first sesamoid) [61, 155]. Moreover, the extension of the medial tarsal facet on the neck would permit to stabilize the astragalus during inversion of the foot [57, 155, 158]. In summary, MUSM 4658 shows an intermediary morphology between morphotypes of the cursorial-jumping and climbing locomotory groups recognised by Ginot et al. [57]. Some of its characters (i.e., ectal facet slightly concave and not projected laterally, neck medially deflected, round head, medial tarsal facet extended on the dorsal and medial aspects of the neck) would enhance mediolateral movements particularly at the level of the distal joints with the navicular and medial tarsal bone, whereas others (i.e., moderately groveed and slightly asymmetric trochlea with no mediolateral widening, narrowing and the proximodistal orientation of the sustentacular facet, wide sulcus calcaneus) would tend to favor parasagittal movements especially of the astragalo-tibial joint (= cruro-astragalar joint). Such an astragalar morphology matches that of terrestrial generalists with a possible higher trend toward arboreal locomotion. Age of TAR-31 and biostratigraphical implications The caviomorph fauna from TAR-31 shares four taxa with the assemblage characterising the lower part of the Villavieja Fm. at La Venta (see above). Similar affinities are observed on their non-rodent mammalian content (e.g., Miocochilius anomopodus, Neosaimiri fieldsi and Megadolodus molariformis; Fig 29.5, p. 508 [26]; see also [36] and the section Material and methods of this paper). An age ranging between 13.18–12.47 Ma can therefore be hypothesised for TAR-31, corresponding with the timespan of the magnetostratigraphically-constrained lower part of the Villavieja Fm. (C5AAn–C5AR.1R chrons) [26, 49]. The MUSM 4283 p4 of Caviidae gen. et sp. indet. from TAR-31 is particular in having more primitive characters (i.e., protohypsodonty, presence of flexids/fossettids, and a hypoflexid not reaching the lingual margin of the tooth) than the caviids found in other caviomorph faunas at low latitudes of South America from Laventan deposits such as Prodolichotis pridiana (La Venta and Fitzcarrald Arch), Dolichotinae gen. 2 large and Dolichotinae gen. 2 small (La Venta), and Guiomys sp. (MD-67) [18, 31, 33] or from sligthly younger deposits (Mayoan SALMA; CTA-44 Top, CTA-43) [34]. Similarly, it differs from middle Miocene caviids from southern South America (Cardiomys? andinus, Cardiomys? buemulensis, Eocardia robusta, Eocardia robertoi, Guiomys unica, Microcardiodon huemulensis and Microcardiodon williensis) [51, 87, 105]; in having of flexids/fossettids, and a hypoflexid, which does not reach the lingual margin of the tooth, characters otherwise found only in early Miocene stem-caviids such as Chubutomys or Luantus [52, 101, 103]. Thus, the discovery of a tooth with such characters suggests that this kind of morphotype may have subsisted until the late middle Miocene, at least in some representatives of the superfamily at low latitudes of the continent. Occurring in both MD-67 and TAR-31 localities, Nuyuyomys chinqaska likely spans the entire middle Miocene interval. PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 69 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru Palaeobiogeographical implications The Miocene epoch is marked by important environmental changes, particularly in low-latitude regions of South America [159]. During the latest Oligocene–early Miocene interval, Andean deformations are generalised, in particular by the Eastern Cordillera of the Northern and Central Andes (beginning of the “Quechua phase” as defined by Steinmann [160]; [161– 165]). However, the Eastern Cordillera of Northern Andes had not reached its full elevation at that time, with a major uplift episode during late Miocene–Pliocene times [166–168]. In relation with the uplift of this region, the Solimões Basin in Brazil is occupied by swamps and shallow lakes [169–173]. These bodies of water belonged to a vast wet system (mega-wetland), which also covered the Andean foreland retroarc basins in Peru and Colombia, and had a north connection with the Caribbean Sea [174]. This system reaches its maximal extension from ~16.0 to 11.3 Ma and forms the Pebas System, which corresponds to a dynamic mosaic of swampy, lake, fluvial and fluvio-marine environments [174, 175]. With our results, the geographical ranges of Ricardomys longidens, Microscleromys paradoxalis and M. cribriphilus are now extended southerly to Peruvian Amazonia for the Laventan interval. More generally, strong taxonomic affinities between the vertebrate assemblages of La Venta (Colombia) and Fitzcarrald, Madre de Dios (MD-67), Iquitos, Contamana, and TAR-31 in Peruvian Amazonia support the existence of a single “Pebasian” biome throughout the middle–earliest late Miocene, and more specifically during the Laventan SALMA, as shown by their terrestrial and aquatic components (crocodylomorphs, chelonians, marsupials, rodents, primates, notoungulates, astrapotheres, litopterns, sirenians, and/or cetaceans; [21, 26, 31–34, 36, 176–178] and see this work). These moderate or strong faunal similarities suggest that the Eastern Cordillera of the North Andes and Pebas System would have not constituted barriers for mammalian dispersals during this interval at least for a region close to the Andes (Western Corridor) [36, 44]. Conclusions The TAR-31 locality in Tarapoto area, Peru, documents nine caviomorph taxa encompassing all four living superfamilies. This assemblage encompasses: Microsteiromys jacobsi gen. et sp. nov., Nuyuyomys chinqaska gen. et sp. nov., Erethizontoidea gen. et sp. indet. 1 and 2 (Erethizontoidea), Caviidae gen. et sp. indet. (Cavioidea), Ricardomys longidens gen. et sp. nov. and Octodontoidea gen. et sp. indet. (Octodontoidea), plus Microscleromys paradoxalis gen. et sp. nov., M. cribriphilus gen. et sp. nov., and “Scleromys” sp. (Chinchilloidea). The study of an astragalus suggests that at least a representative of Microscleromys was a terrestrial generalist rodent. Although very restricted in terms of area (5 square meters) and volume investigated (~550 kg wet-screened) with respect to other assemblages, this locality yields the most diverse middle Miocene caviomorph fauna recorded in Western Amazonia. In addition, it is the second most diverse for this time window in low-latitude regions of South America, well after La Venta. The geographical range of Ricardomys longidens, Microscleromys paradoxalis and M. cribriphilus is now expanded 1,100 km to the south, during the Laventan SALMA, as a further testimony of the extent of the Pebasian biome at those times. Supporting information S1 File. Exploring the potential species number of Microscleromys from TAR-31 and La Venta. (DOCX) PLOS ONE | https://doi.org/10.1371/journal.pone.0258455 November 3, 2021 70 / 80 PLOS ONE Miocene caviomorphs from Tarapoto, Peru S2 File. Data and corresponding script(s) for each dental locus used for statistical analyses. (ZIP) S1 Table. Dental measurements (in millimeters) of rodent material from TAR-31 (Tarapoto, Peru; late middle Miocene) and La Venta (Colombia; late middle Miocene). The previous assignation of the material from La Venta are specified (Walton, 1990, 1997). � indicates the specimens selected by Walton (1990) as holotypes, ��� indicates the specimens selected in this study as holotypes. (XLSX) S2 Table. Extinct and extant caviomorph taxa used for dental comparisons in this study. For the references, see the manuscript. Fm., Formation; INGEMMET, Instituto Geológico Minero y Metalúrgico, Lima; MACN, Museo Argentino de Ciencias Naturales, Buenos Aires; MLP, Museo de Ciencias Naturales de La Plata; MMP, Museo Municipal De Ciencias Naturales Lorenzo Scaglia, Mar del Plata; MNHN-Bol, Museo Nacional de Historia Natural, La Paz, Bolivia; MNHN, Musée National d’Histoire Naturelle, Paris; MUSM, Museo de Historia Natural de la Universidad Nacional Mayor San Marcos, Lima; UM, Université de Montpellier. (XLSX) Acknowledgments Many thanks to M. Roddaz (GET, Toulouse, France), M.A. Custódio and R. Ventura (GEOCRON/IG, Brası́lia, Brazil) and whoever helped us in the field and in the lab. We are particularly grateful to our drivers Giancarlo and Manuel, for their long standing help during the yearly field seasons. We warmly thank M.R. Ruiz-Monachesi (IBIGEO, Salta, Argentina) for discussing on statistics, his relevant suggestions and aid with respect to the Bayesian models and the osteological preparation of the Galea. We are much indebted to M.C. Madozzo-Jaén (Lillo, San Miguel de Tucumán, Argentina) and to M.E. Pérez (MEF, Trelew, Argentina) for discussing on phylogeny and homology of dental structures in cavioids. Many thanks to A. Álvarez and M.D. Ercoli (INECOA, San Salvador de Jujuy, Argentina) for providing us photos of collection material for dental comparisons. Christine Argot (MNHN, Paris, France), M. Reguero (MLP, La Plata, Argentina), L. Chornogubsky and A.G. Kramarz (MACN, Buenos Aires, Argentina), and M. Taglioretti (MMP, Mar del Plata, Argentina) kindly granted access to the collections under their care. We are much indebted to M. Gómez Pérez, the team of Museo Geológico Nacional—Servicio Geológico Colombiano and J.D. Carrillo (MNHN) for the correspondance between the field numbers and the collection numbers for the material from La Venta. We warmly thank B. Rondeau and L. Lena (LPG, Nantes, France) for access to a Keyence Digital Microscope facility; C. Cazeveille (Institut des Neurosciences de Montpellier [INM], France) for access to a electron microscope scanning facility; and A.-L. Charruault and R. Lebrun (ISEM, Montpellier, France) for μCT scan acquisitions and treatments. We thank the MRI and the LabEx CeMEB for access to the electron microscope and μCT scanning facilities (ISEM, Montpellier). We are indebted to A.-L. Charruault (ISEM, Montpellier, France) for the cast preparation of the rodent material from TAR-31. We gratefully thank the E. Beucler and O. Bourgeois (LPG, Nantes, France) in order to have allowed MB realizing a journey in Montpellier. This is ISEM publication 2021-225-Sud. Author Contributions Conceptualization: Myriam Boivin, Laurent Marivaux, Pierre-Olivier Antoine. 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Análise de Canais do YouTube como Objetos de Aprendizagem de Suporte ao Estudo de Cálculo I em Cursos da Área de Ciências Exatas Analyzing YouTube Channels as Objects of Learning to Support the Study of Calculus I in Exact Sciences Courses O Isabela Aparecida DIVINO1* Mariele Rodrigues MOREIRA1 EL Daniel Alfonso SIERRA1 Rodrigo Naoto SHIRAISHI1 Victoria Alejandra Salazar HERRERA2 1 Universidade Estadual de Campinas, Campinas - SP, BRASIL CECS -Universidade Federal do ABC, Santo André – SP, BRASIL * i265342@dac.unicamp.br PR 2 Resumo. A videoaula deixou de ser apenas uma coadjuvante da aprendizagem, e tem sido cada vez mais agregada à rotina de estudos de alunos em diferentes níveis educacionais. O uso de videoaulas do YouTube como ferramenta complementar é cada vez mais crescente, independente da área de estudo. Entretanto, é escassa a literatura em relação à eficácia dos modelos de ensino utilizados nestas videoaulas. Tendo em vista a dificuldade por parte dos alunos na aprendizagem em matemática, o objetivo deste trabalho foi realizar o levantamento e análise dos principais canais de Matemática do YouTube assistidos por alunos dos cursos da área de Ciências Exatas da Universidade Virtual do Estado de São Paulo como complementaridade aos estudos. Para isso, aplicaram-se os doze princípios da aprendizagem multimídia formulada por Richard Mayer: Coerência, Sinalização, Redundância, Contiguidade espacial, Contiguidade temporal, Segmentação, Pré-treinamento, Modalidade, Multimídia, Personalização, Voz e Imagem. Foram coletadas informações dos canais acessados pelos estudantes por meio de um formulário e selecionados os cinco canais com maior número de visualizações na plataforma YouTube. Após a análise constatou-se que boa parte dos princípios está presente na didática dos professores que dirigem os canais, sendo os que mais apareceram: redundância, contiguidade temporal, modalidade, personalização, voz e imagem. Ainda assim, detalhes relacionados à questões visuais e de acessibilidade na reprodução dos vídeos podem ser melhorados. Palavras-chave: Objetos de aprendizagem. Tecnologias da informação e comunicação. Ensino superior. YouTube. EL O Abstract. Video lessons are no longer just an adjunct to learning, they have been increasingly added to the study routine of students at different educational levels. The use of YouTube video lessons as a complementary tool is increasingly growing, regardless of the area of study. However, there is little literature regarding the effectiveness of the teaching models used in these videos. Considering the difficulties of students in learning mathematics, the objective of this work is to carry out a survey and analysis of the main Mathematics channels on YouTube watched by students, as complementarity to studies, from courses in the STEAM area of the Virtual University of the State of São Paulo. To achieve such goal the twelve principles of multimedia learning formulated by Richard Mayer were applied: Coherence, Signaling, Redundancy, Spatial Contiguity, Temporal Contiguity, Segmentation, Pre-training, Modality, Multimedia, Personalization, Voice, and Image. Information was collected from the channels accessed by students using forms and the five channels with the highest number of views on the YouTube platform were selected. After the analysis, it was found that a most of the principles are present in teaching strategies of the teachers who manage the channels, highlighting redundancy, temporal contiguity, modality, personalization, voice and image. Still, details related to visual issues and accessibility when playing videos can be improved. PR Keywords: Learning objects. Information and communication technologies. University. teaching, YouTube. 1. Introdução A internet tem aumentado de forma exponencial sua influência no dia a dia das pessoas em todos os âmbitos, no Brasil o número de usuários chegou a representar 81% da população em 2020 (FOLHA, 2021) se constituindo numa ferramenta de democratização do conhecimento e da informação. Em todos os planos de ensino, formais ou não formais, se usa este canal de comunicação, seja para organizar as plataformas de estudo ou para pesquisas aprofundadas em tópicos específicos. A metodologia tradicional de estudos foi eficiente numa era onde o acesso à informação era restrito. Atualmente, com uma sociedade conectada o tempo todo, as pessoas buscam aprendizado online em sites e aplicativos sociais. Essa mudança progressiva faz com que as escolas tenham que se adaptar, buscando estratégias de ensino baseadas em materiais de aprendizagem digitais (SOUZA e MORALES, 2015). EaD em Foco,2024, 14(1): e2090 |2 O Algumas ferramentas de aprendizagem virtual vêm sendo utilizadas para enriquecer o ensino. Entre elas podem ser citados os objetos de aprendizagem. A IEEE Standard for Learning Object Metadata (2020) define Objetos de Aprendizagem (OAs) como: “qualquer entidade digital ou não digital que possa ser usada, reutilizada ou referenciada durante o uso de tecnologias que suportam o ensino”. Como mencionado por Coradini (2009), os objetos de aprendizagem fazem referência a conteúdo multimídia, conteúdos instrucionais, software em geral entre outros. Do mesmo modo, o site Younder (2021) aponta os objetos de aprendizagem como recursos educacionais utilizados para potencializar a transmissão dos conteúdos, citando como exemplos podcasts, vídeos, fóruns online, infográficos e e-books. Neste universo de OAs, o vídeo foi escolhido pelo fato de juntar o meio visual, texto, animação e áudio e ser uma ferramenta que as pessoas utilizam com frequência. Dessa forma é importante citar a plataforma YouTube, que é o maior site de compartilhamento de vídeos do mundo, e entre outros usos, é utilizado para difundir informação educativa. Além de livre acesso mundial, as pessoas conseguem interagir, dar feedbacks e curtidas (CURRAN et al., 2020). PR EL Existem diversos canais de YouTube, podendo ser caracterizados como repositórios de OAs, que oferecem um conteúdo atraente, rigoroso, embasado cientificamente e que auxilia nos planos de estudo. Exemplos conhecidos como o canal alemão “Kurzgesagt – In a Nutshell” com tradução para o inglês e o português, oferecem vídeos com os mais diversos temas como Astrologia, História ou Matemática. No Brasil podemos ressaltar os canais associados a ScienceBlogs Brasil (2021) como BlaBlalogia, Minuto da Física e Marcelo Gleiser, os quais são revisados por profissionais especialistas que verificam a rigorosidade científica do seu conteúdo e dão o selo de aprovação. Levando estas definições em consideração, é possível concluir que vídeos de YouTube, usados como suporte nos processos de ensino-aprendizado, são Objetos de Aprendizagem digital e, portanto, é possível analisar sua qualidade usando a teoria da aprendizagem multimídia. A aprendizagem multimídia resume-se em uma representação mental construída pelo aluno, a partir da junção de falas e imagens. Alguns exemplos dessa abordagem incluem gráficos, vídeos, mapas e animações. Um recurso multimídia não trabalha apenas conteúdos determinados como por exemplo livros e computadores, mas sim abrange diversas características como palavras dentro de textos escritos ou falados, além de informações gráficas como figuras, fotos, animações, gráficos e mapas. Nesse sentido, Mayer (1999) acredita que o meio não tem relação direta com a aprendizagem, então a questão a ser resolvida passa a ser como desenvolver recursos multimídia para aperfeiçoar os conteúdos e mensagens abordados. Para isso, o professor divide dois tipos de abordagem para produção dos recursos multimídia: a centrada na tecnologia e a centrada nos alunos/aprendizes. A primeira abordagem possibilita o acesso a novas tecnologias, assim é necessário que os alunos/aprendizes se adaptem a elas, a segunda abordagem é o contrário, são as novas tecnologias que devem se adaptar às necessidades das pessoas facilitando o entendimento do conteúdo (MIRANDA, 2009). EaD em Foco,2024, 14(1): e2090 |3 Para que a aprendizagem multimídia aconteça de fato é necessário que alguns processos aconteçam: seleção de palavras para o trabalho verbal sobre o assunto; seleção de imagens relevantes para o trabalho visual; organizar as palavras de forma que mantenham coerência dentro do modelo mental verbal; organizar as imagens de forma mantenham coerência dentro do modelo mental visual; e juntar todas as informações de forma que tenha harmonia entre as representações verbais visuais e conhecimento prévio (MAYER et al., 2004). O Para Mayer (2009) a cognição humana pode ser organizada em: Pressuposto do canal duplo: existem dois sistemas não-equivalentes de processamento de informação verbal/auditivo e visual/pictórico; pressuposto da capacidade cognitiva: a quantidade de informação processada simultaneamente em cada canal é limitada: pessoas aprendem melhor com a utilização de palavras e imagens do que somente com palavras (MAYER,2001). Esta teoria se aplica em recursos de aprendizagem multimídia como vídeos, animações e jogos. EL Existem diversos trabalhos de pesquisa que avaliam objetos de aprendizagem aplicando os princípios da Teoria de Aprendizagem Multimídia formulada por Mayer (2009). Silva e Montané (2017) avaliam e aprimoram dois Objetos de Aprendizagem da temática Modelos Atômicos consultando a professores de ciências exatas e aplicando os princípios de Mayer (2009). Por outra parte, Almeida et al. (2014) analisam Objetos de Aprendizagem da temática Sistema Digestivo incluindo vídeos de YouTube e materiais audiovisuais do Banco Internacional de Objetivos Educacionais (objetoseducacionais2.mec.gov.br) entre outras fontes. PR O Youtube não é uma ferramenta desenvolvida para fins educacionais e pedagógicos, apesar de ser atualmente usado também para divulgar aulas. E caso o educador escolha este repositório para suas práticas educacionais, é importante que acompanhe e aprove o conteúdo a ser estudado por seus alunos na plataforma, para que de fato, a aprendizagem seja relevante. Segundo Cortez (2010), a recomendação é que o professor assista os vídeos com antecedência e proponha a partir dele algum exercício, para que o vídeo não substitua o ensino em aula, mas sim, complemente. Mesmo com essa recomendação, a literatura atual ainda é escassa com relação à avaliação dos canais de Youtube voltados à aprendizagem, bem como as metodologias necessárias para se realizar essa avaliação. É necessário que se hajam estudos nesse sentido, a fim de que se padronize meios para dizer sobre a qualidade do vídeo, e por fim a recomendação de utilizá-lo ou não em complementaridade ao conteúdo lecionado em sala de aula. Estrategicamente, o professor pode recomendar o Youtube para fins de aprendizagem, sobretudo em conteúdos em que os alunos apresentam mais dificuldade. Segundo o Programa Internacional de avaliação de estudantes (PISA), matemática e ciências são as disciplinas com pior desempenho entre os estudantes. No Brasil, apenas 27% dos estudantes alcançaram em 2022 o nível 2 de proficiência, que é o patamar mínimo considerado, enquanto em outros países, a média é de 69% dos estudantes atingindo esse nível. (Pimentel, 2023). São inúmeros os canais EaD em Foco,2024, 14(1): e2090 |4 no Youtube que trazem a proposta de ensino em matemática, mas ainda não se sabe sobre a preferência dos alunos e a qualidade do conteúdo disponível. EL O Diante disso este trabalho tem como objetivo principal a análise de vídeos em YouTube da temática Cálculo I (Cálculo Diferencial e Cálculo Integral) segundo a ementa do plano de Estudos dos cursos de exatas da Universidade Virtual do Estado de São Paulo (UNIVESP), aplicando os princípios da teoria de Aprendizagem Multimídia formulada por Mayer (2009). Para atingir este objetivo foi realizado um levantamento dos canais utilizados pelos alunos em complementaridade a aprendizagem de Cálculo, e posteriormente, a análise desses canais. O levantamento foi feito usando um formulário on-line para consultar tutores e estudantes sobre seus canais preferidos e mais usados e após esta contribuição foram escolhidos os canais com maior quantidade de subscrições. A análise feita seguiu a teoria de Aprendizagem Multimídia de Mayer (2009), observando vídeos da plataforma YouTube sobre os conteúdos da disciplina Cálculo I, com o intuito de revisar sua qualidade e capacidade de complementar o aprendizado de Cálculo por estudantes de ensino superior. Este estudo é relevante vista a ausência de estudos que validem desde o ponto de vista pedagógico a qualidade de OAs desse tipo sendo que são os mais comuns entre os estudantes. 1.1. Princípios de Aprendizagem Multimídia de Mayer Nesta seção serão descritos os princípios de aprendizagem multimídia (MAYER, 1999 e MAYER, 2014) utilizados neste trabalho para analisar os canais de YouTube que contém vídeos relacionados com Cálculo I. Eles estão separados de acordo com objetivos específicos. PR Inicialmente apresentam-se os princípios que visam reduzir o processamento desnecessário (evitar sobrecarga cognitiva), eles são: Coerência, Sinalização, Redundância, Contiguidade espacial e Contiguidade temporal. Coerência: se aprende melhor quando informações desnecessárias (palavras, figuras, símbolos, sons etc.) são excluídas, é importante que a apresentação seja o mais simples possível. Sinalização: se aprende melhor quando a organização do conteúdo é explicada, para que o aprendiz possa se focar no que é essencial, favorecendo a organização mental. Um exemplo desse princípio são vídeos que apresentem um menu de conteúdos e minutos na legenda. Este princípio também é válido para destacar elementos durante a apresentação do material como setas, cores destaque, grifos, letras maiores etc. Redundância: se aprende melhor com imagens e narração, mas não com imagens, narração e legenda (do que está sendo narrado), pois isto ocasiona sobrecarga do canal visual Contiguidade espacial: se aprende melhor quando as palavras (informação verbal) e imagens (informação visual) correspondentes estão espacialmente próximas, gastando menos recursos EaD em Foco,2024, 14(1): e2090 |5 cognitivos para estabelecer estas conexões sem necessidade de buscá-las na tela. Aqui é importante esclarecer que que as imagens não devem incluir palavras descritivas (MAYER, 2014) Contiguidade temporal: se aprende melhor quando palavras (informação verbal) e imagens (informação visual) correspondentes aparecem ao mesmo tempo, gastando menos recursos cognitivos estabelecendo estas conexões Existem também, princípios que visam favorecer a administração do processamento essencial (a representação do material na memória) em que a sobrecarga diminui os recursos cognitivos para realizar o processamento gerador (organização e integração das representações mentais produzidas), eles são: Segmentação, Pré-treinamento e Modalidade. O Segmentação: se aprende melhor quando o recurso é apresentado em partes, nas quais o usuário pode definir o ritmo (cada um tem um tempo diferente de processamento). Por exemplo, dividir o conteúdo que será apresentado em partes dentro do próprio vídeo, ou em vários vídeos. Desta maneira o conteúdo é processado em um ritmo adequado, diminuindo a sobrecarga cognitiva e permitindo maior controle sobre o conteúdo (MAYER, 2014). EL Pré-treinamento: se aprende melhor quando já se sabe os nomes e as características dos principais conceitos antes de entrar em mais detalhes. Aqui seria interessante observar se a videoaula parte do básico para o avançado, ou se já parte do avançado, mas deixa alguma sugestão como: “clique no card no topo do vídeo (ou no link da legenda) e assista um vídeo sobre X antes de assistir a esta aula.” PR Modalidade: se aprende melhor com imagens e textos falados do que com figuras e textos escritos (competem no canal visual). Outros princípios visam promover o princípio gerador (organização e integração das representações mentais produzidas), eles são: Multimídia, Personalização, Voz e Imagem. Multimídia: se aprende melhor com palavras e imagens do que só com palavras. Personalização: o estilo formal de conversação não favorece a aprendizagem, portanto, se aprende melhor quando as palavras estão em estilo informal, sendo recomendado o uso de comentários em primeira e segunda pessoa (MAYER, 2014). Voz: se aprende melhor quando a voz da narração é humana do que quando a voz é de máquina. A imagem de quem fala pode trazer um sentido de presença social, mas não necessariamente se aprende melhor quando a imagem de quem está falando está na tela, pois dependendo da quantidade de informações fornecidas com a imagem pode favorecer sobrecarga mental. EaD em Foco,2024, 14(1): e2090 |6 2. Metodologia Este trabalho é uma pesquisa exploratória, pois utilizou um questionário para levantamento de canais utilizados por estudantes e após esse levantamento foi aplicada uma análise de canais com abordagem qualitativa e mediante o método indutivo baseadas nos princípios de Mayer. O público-alvo escolhido foram os estudantes de cálculo por ser uma disciplina considerada como complexa pelos estudantes de vários cursos, e sendo que o contexto é numa instituição que trabalha só na modalidade EaD as videoaulas estão todas disponíveis no YouTube. EL O O questionário contou com 14 questões e foi disponibilizado por 21 dias. A escolha dos canais de Cálculo Matemático I (Cálculo Diferencial e Cálculo Integral) da plataforma YouTube foi feita a partir da indicação dos alunos dos cursos da área de Ciências Exatas que estão cursando ou já cursaram a disciplina de Cálculo Matemático I na UNIVESP através de um formulário de pesquisa que foi dividido em três partes: 1- Termo de Consentimento Livre e Esclarecido; 2- Questões de rastreamento do perfil socioeconômico dos participantes para que possamos entender um pouco sobre os motivos que os levam a acessar estes conteúdos; 3- Questões que irão mostrar quais canais eles utilizam e por qual motivo fazem uso deste objeto de aprendizagem. Caso o participante tenha interesse ele poderá deixar o contato de e-mail ao final do questionário para que possamos enviar o artigo ao final da pesquisa. PR Da coleta de dados foram selecionados cinco canais de Cálculo Matemático da Plataforma YouTube dentre os citados pelos alunos no formulário mostrado no. O critério de escolha foi a quantidade de visualizações de cada canal. Foram assistidas as cinco últimas videoaulas postadas. Os canais foram analisados pela apresentação de seus conteúdos, se estavam de forma a facilitar a aprendizagem ou gerar sobrecarga mental - dificultando a aprendizagem - a partir dos 12 princípios da aprendizagem multimídia de MAYER (2009). A qualidade do conteúdo apresentado não foi levada em consideração, mas sim como ele foi transmitido e em como foi feita a produção midiática do material. 3. Resultados e Discussão Os últimos dados levantados pela UNIVESP (2021), mostram que há um total de 48.3131 alunos matriculados nas diferentes graduações ofertadas, e que 50% desses alunos são mulheres. Têm faixa etária entre 17 - 80 anos e a grande maioria (72%) vieram de escola pública. Em complemento, em (UNIVESP, 2020) verifica-se que 79% dos alunos são responsáveis ou contribuem com a renda familiar e 85% recebem entre 2 a 4 salários mínimos, a “grande maioria dos alunos já atua no mercado de trabalho e compõe a renda da família, que optam pelo EaD por ter um tempo reduzido para dedicação aos estudos” (UNIVESP, 2020). Nos cursos atingidos por nosso questionário (ciência de dados, Licenciatura em Matemática e Engenharia da computação), há um total de 15.656 estudantes, e, apesar de nossa amostra corresponder a uma pequena parte desses estudantes, conseguimos atingir pessoas de 20 a 67 anos de idade, das quais a maioria trabalha e não tem mais de 4 horas diárias para estudar. EaD em Foco,2024, 14(1): e2090 |7 Como um dos objetivos deste trabalho era coletar um conjunto de canais que fossem acessados pelos alunos para poder analisá-los mediante os princípios de Mayer, a coleta de informações via formulário não focou na quantidade de respondentes, e sim na quantidade de canais sugeridos. Obteve-se um total de 27 respostas ao formulário, sendo todos alunos da UNIVESP. Apesar de ser uma amostra pequena, serão detalhadas algumas características sociocomportamentais, lembrando que o critério da escolha dos canais foi a maior quantidade de subscrições e visualizações dos canais mencionados nas respostas ao questionário, isto é o questionário foi aplicado no intuito de saber quais canais são comumente acessados pelos alunos, mas a escolha dos canais foi um critério externo às respostas dos respondentes do questionário. O Dos respondentes, 18 foram do sexo masculino e 9 do sexo feminino, com a faixa etária de 20 a 67 anos, pertencentes aos cursos de: Ciências de dados, Licenciatura em Matemática e Engenharia da Computação, apesar de ter sido disponibilizado para todos os cursos da área de exatas da universidade. Todos os cursos descritos possuem a disciplina de Cálculo. EL Segundo análises do perfil socioeconômico dos entrevistados, 23 dos 27 estudantes possuem ocupação remunerada, enquanto 4 dedicam-se exclusivamente à UNIVESP. O que corrobora com estudos recentes onde 48% dos universitários possuem jornada dupla, sendo 50% destes com mais de 8 horas de dedicação no trabalho, precisando enfrentar desgaste físico, mental e acúmulo excessivo de tarefas diariamente. A renda mensal encontra-se acima de 1,5 salário-mínimo per capita familiar para 85% dos estudantes, o que representa cerca de R$1818,00 reais, enquanto 15% possuem menos de 1,5 salário mínimo de renda per capita familiar por mês. Esse último dado é semelhante à amostra geral apresentada em (UNIVESP, 2020) onde 85% dos alunos têm uma renda entre 2 e 4 salários mínimos. PR Com relação ao tempo de dedicação aos estudos, 11 relataram que estudam até 2 horas por dia, 11 relataram que estudam mais de 2 horas por dia, enquanto 5 relataram que não conseguem estudar diariamente. O tempo diário utilizando internet para atividades acadêmicas foi relatado em 11% dos estudantes sendo acima de 5 horas por dia, os outros 89% usam até 4 horas diárias. Sobre a utilização de aulas do YouTube como apoio às aulas institucionais, 15 estudantes relataram utilizar destes recursos frequentemente, representando mais da metade dos entrevistados. Ao serem questionados sobre os motivos que os levaram a buscar outros recursos educacionais para aprenderem a disciplina de Cálculo I, 29,6% responderam que as aulas no ambiente virtual não eram didáticas, 44,5% responderam que as aulas eram didáticas, porém não eram suficientes para compreensão, já 18,5% justificaram o uso para reforçar o aprendizado. Houve ainda justificativa com relação à duração das aulas institucionais, mais longas, segundo um entrevistado, enquanto as aulas encontradas no YouTube eram sucintas e de fácil compreensão. Além do YouTube, recursos como livros, artigos e programas interativos também são utilizados para estudo. EaD em Foco,2024, 14(1): e2090 |8 Os canais utilizados pelos alunos como recurso de apoio aos estudos em Cálculo I encontram-se listados no Quadro 1, sendo 17 no total. Os canais com maior número de inscritos e de acessos, bem como seus vídeos mais recentes, foram selecionados para análise, são eles: Ferreto matemática, Me salva!, Matemática Rio, Equaciona e Marcos Aba. A partir da escolha dos canais mais visualizados, foi realizada uma análise baseada nos 12 princípios de Mayer (2009) nos quatro últimos vídeos postados nos canais. No Quadro 2, apresentam-se as informações recolhidas, as quais se dispõem da seguinte maneira: quando o canal cumpre o tópico, é colocado “sim” e quando não cumpre, coloca-se “não”. Quadro 1: Canais do YouTube utilizados pelos estudantes como apoio nos estudos para a disciplina de Cálculo I. Professor Douglas Maioli Acesse aqui O Canal do YouTube professordouglasmaioli Professor Murakami – Matemática rapidola rapidola Ferreto Matemática professorferretto equacionamatematica EL Equaciona com Paulo Pereira respondeaí Me salva! mesalva Canal Univesp univesptv Matemática para todos marciorafaelbarros Omatematico.com mmatematicogrings Matemática - Ester Velasquez matemateca Help engenharia com Denis Rodrigues helpengenharia Matemática Rio com Rafael Procopio matematicario Marcos Aba marcosaba Matemática da Tamires matematicadatamires Universo narrado universonarrado Cláudio Possani claudiopossani PR Responde aí Dicasdemat Sandro Curió dicasdemat Fonte: Elaborado pelos autores (2022) Quadro 2: Canais do YouTube escolhidos e analisados segundo os tópicos da Teoria de aprendizagem e multimídia de Mayer (2009). Ferreto Matemática Me salva! Matemática com Rafael Procopio Equaciona com Paulo Pereira Marcos Aba Coerência Sim Não Sim Sim Sim Sinalização Sim Sim Não Sim Não Redundância Sim Sim Sim Sim Sim Critérios EaD em Foco,2024, 14(1): e2090 |9 Não Não Não Sim Sim Contiguidade temporal Sim Sim Sim Sim Sim Segmentação Sim Sim Não Sim Não Pré treinamento Sim Não Sim Não Sim Modalidade Sim Sim Sim Sim Sim Multimídia Não Sim Não Não Sim Personalização Sim Sim Sim Sim Sim Voz Sim Sim Sim Sim Sim Imagem Sim Sim O Contiguidade espacial Sim Sim Sim Fonte: Elaborado pelos autores (2022) Uma descrição mais detalhada de cada canal é apresentada nas seguintes subseções. EL 3.1. Ferreto Matemática O canal Ferreto matemática possui quase 3 milhões de inscritos e milhares de visualizações em vídeos. Algo interessante no canal, é a presença de aulas ao vivo. Observamos que existem inúmeras aulas temáticas fracionadas, de maneira que um conteúdo todo, por exemplo de razão, fica disposto em diversos vídeos, o que ganha muitos pontos no tópico segmentação. Voz e personalização também são pontos fortes, bem como coerência e redundância. PR Outro aspecto muito atrativo são vídeos de revisão para ENEM, com resolução de problemas. São, no geral, os vídeos mais assistidos. O canal cumpre com a maioria dos princípios, porém poderia melhorar em quesitos de apresentação de figuras, o que faz com que não seja cumprida a contiguidade espacial juntamente com a multimídia: o professor sempre mostra lousas e cartazes na tela, mas há baixa presença de figuras propriamente ditas, por exemplo, ao explicar fração, poder-se-ia utilizar uma figura e fracioná-la, ao invés de somente apresentar uma imagem com texto escrito. 3.2. Me salva! O canal “Me salva” é constituído por vários professores e aborda inúmeras disciplinas, mas para elaboração dessa tabela, foram vistos somente os conteúdos de matemática. As aulas são sempre de revisão para ENEM, resumos ou resolução de problemas. Utilizam métodos de “escrita à mão”, algo parecido com o caderno, onde o vídeo se baseia no professor filmando a própria mão num quadro branco resolvendo os problemas utilizando-se muitas setas, rabiscos, esquemas, coisas que ensinam bem sem poluir a imagem. A fala é sempre informal, mas, muito compreensível, humana e o conteúdo é interessante. O que podemos observar de ponto EaD em Foco,2024, 14(1): e2090 | 10 negativo, é principalmente a extensão dos vídeos: mais de uma hora, outras quase 2 horas. Isso porque os professores fazem coletâneas de aulas, abordando muitos conteúdos num vídeo só. E mesmo vídeos que enfatizam apenas um material, acabam sendo bem extensos, e não há legendas de minutos, o que dificulta e torna ainda mais maçante a visualização. Segundo Mayer (2014) “quanto mais simples e objetiva for a apresentação do conteúdo, mais livre ficará a memória operacional para processar um número maior de conhecimento”. EL 3.3. Matemática com Rafael Procópio O A contiguidade espacial também acaba sendo insuficiente neste canal. Recursos de sinalização são bem utilizados, porém, figuras e imagens não estão presentes. Obviamente, em disciplinas de cálculo, a numeração acaba por ser mais presente, mas a utilização de imagens e figuras, vinculada a palavras, é mais eficiente, o que se pode ver no item multimídia. Além disso, como as aulas são filmagens de resolução de problemas num papel, há um certo momento em que ocorre uma “poluição visual”, pois na folha utilizada, espalham-se números e setas para todos os lados, às vezes rabiscos e rasuras, o que acaba deixando confuso. PR O canal do Rafael Procópio apresenta os conceitos de uma forma limpa e organizada. Levando em conta o princípio da coerência, ele desenvolve exercícios mostrando o passo a passo numa lousa tirando as informações que não aportam na solução do problema e sendo simples e direto. Pode-se classificar os vídeos em dois grupos: 1) vídeos curtos (de 2 a 5 minutos) apresentando dicas de cálculos simples e curiosidades e 2) vídeos maiores (10 a 40 minutos) com conteúdo formal e completo. Nota-se que não há inclusão de divisão dos temas em barra de reprodução ou tempo sendo insuficiente em sinalização, em compensação mostra no começo do vídeo o desenvolvimento da aula e a duração. As palavras e as imagens estão espacialmente próximas, quando se fala de um mesmo conceito. Quando apresenta uma imagem fala dela e não deixa um texto explicativo na tela (Contiguidade Espacial e Temporal). A narração é muito clara com um tom de voz calmo, mas não monótono, além de ser informal, mas ao mesmo tempo, sem perder rigorosidade. As aulas sempre vão do básico ao avançado, seguindo o critério de Pré-treinamento. Baseados nos critérios de Meyer, podemos sugerir algumas melhorias: Indicar na barra de tempo à divisão dos temas do vídeo (sinalização e segmentação), usar software gráfico que aprimore os traços a mão alçada, linhas retas (Contiguidade Espacial) e melhorar a qualidade das imagens, levando em conta que imagens já feitas em programas de design ficam mais claras que feitas a mão durante a aula. (Multimídia). 3.4. Equaciona com Paulo Pereira Canal de um professor e mestre em Matemática, oferece videoaulas curtas e descontraídas. Dentre os princípios da aprendizagem multimídia que o canal apresenta estão: coerência - a EaD em Foco,2024, 14(1): e2090 | 11 apresentação dos conteúdos é bastante simples, somente a equação sendo resolvida na tela; sinalização - como o canal se dedica a resolução de exercícios em vídeos curtos, não tem menu de minutos, mas o professor utiliza bastante a mão e caneta de cor vermelha para destacar informações em azul; redundância - os vídeos consistem em uma equação na tela (imagem) e a fala do professor (narração); contiguidade temporal - a resolução do exercício se dá de maneira simultânea; segmentação - antes de iniciar a resolução dos exercícios, ele apresenta aos alunos qual a estratégia para resolução da atividade, ou seja, quais etapas serão cumpridas para resolução do exercício; personalização - utiliza linguagem informal e comentários em primeira pessoa, aproximando seu público-alvo usando termos “engraçados e chamativos”. Outros princípios que também são cumpridos são o da contiguidade espacial e modalidade. O Idealmente as ferramentas multimídia devem utilizar ambos os canais (visual e auditivo), para favorecer a aprendizagem evitando sobrecarga cognitiva (BRAGA et al., 2019) o que pode ser apreciado em vídeo aulas oferecidas no canal, onde não há uso simultâneo de texto escrito e imagens, ou de informações excessivas na tela. PR 3.5. Canal Marcos ABA EL Dentre os princípios que não apresenta estão: pré-treinamento - o canal parte do avançado, porém explica conceitos básicos de matemática para esclarecer melhor o porquê de cada passo do exercício e o princípio multimídia - utiliza somente equações. Como sugestões para o canal adequar-se aos princípios que ainda não apresenta, sugerimos o uso de links de conteúdos relacionados aos temas na descrição dos vídeos para auxiliar e complementar o entendimento dos alunos e utilizar, quando possível, imagens ou desenhos para ilustrar o raciocínio desenvolvido nas equações. Fundado pelo professor Marcos com mais de 3,48 mil inscritos e 3,1 mil vídeos, o canal aborda diversos temas dentro da matemática e da física, desde assuntos mais básicos até matérias avançadas de nível universitário (para este estudo avaliamos apenas aulas de matemática). Uma característica forte do canal é que cada tema principal é dividido em várias aulas explicativas respeitando o tempo de raciocínio dos diferentes alunos. É notável que o professor gosta do método tradicional usando caneta e papel ou caneta e lousa para resolução e explicação dos exercícios, facilitando a compreensão e visualização das contas matemáticas de forma simples sem excesso de informações. O canal vem crescendo exponencialmente e novos professores têm se juntado à equipe, atendendo assim diversos pedidos específicos de seguidores resolvendo suas dúvidas em vídeos explicativos rápidos em média de 20 minutos. Para melhorar a sinalização seria ideal utilizar canetas de cores diferentes durante a resolução dos exercícios. O professor, na maioria dos vídeos, utiliza apenas uma cor de caneta. E para melhorar a segmentação de alguns vídeos que é feito uma explicação prévia sobre o tema abordado antes do exercício seria o ideal dividir em 2 blocos dentro do mesmo vídeo separando melhor as etapas introdução e exercício. EaD em Foco,2024, 14(1): e2090 | 12 3.6. Aulas Cálculo - Univesp 3.7. Considerações gerais sobre os canais O A fim de compreender melhor sobre a necessidade dos alunos de buscarem conteúdo complementar àqueles que lhes são oferecidos pela UNIVESP, decidimos analisar as aulas de Cálculo disponíveis no canal da UNIVESP no YouTube, Observando as 4 últimas aulas postadas, notamos que alguns dos tópicos de Mayer (2009) deixam a desejar naquilo que é proposto: se usam poucos acessórios de sinalização, bem como não há nenhum menu de conteúdos que facilite o aluno a se localizar. O pré-treinamento não é aplicado, uma vez que os vídeos tratam de conteúdos diferentes, mas não há um direcionamento sobre conteúdos anteriores, nem introdução na fala da professora. A modalidade pode ser melhorada também, pois usa-se muitos textos e poucas figuras na narração. A imagem da educadora o tempo todo na tela, acaba em uma sobrecarga visual desnecessária também. Nota-se que há muita propriedade e um excelente conteúdo nas aulas, mas esses tópicos, se melhorados, poderiam influenciar na maior afinidade dos alunos para com o Canal UNIVESP. PR EL Após o levantamento detalhado para cada canal, considera-se importante ressaltar que a apresentação de passo a passo à mão faz com que se desenhem figuras com baixa qualidade. Em todos os canais faz falta o uso de imagens pré-desenhadas ou prontas, que facilitem a visualização apoiadas pelas explicações feitas pelo narrador. Também é preciso um uso maior da segmentação: dividir a barra de tempo (ou reprodução) no YouTube indicando as partes dos vídeos e os temas e indicar ao começo do vídeo como vai ser organizado (simular a um planejamento de aula), atualmente existem ferramentas de inteligência artificial que ajudam com a segmentação de vídeos. Em alguns canais faltou uma melhor organização indicando o avanço dos temas e os links com os conteúdos prévios. Pensando em aproveitar as vantagens observadas dos canais do YouTube, seria de grande valia que instituições de ensino, como é o caso da UNIVESP, adotassem técnicas de engajamento, modulando o tom de voz e fazendo vídeos curtos ou técnicas como o uso de nomes “engraçados” para as equações ou o uso de desenhos que provoquem emoções e sentimento de pertinência também podem ajudar na melhora da qualidade das aulas, (MAYER, 2011). É importante ressaltar que é possível que os materiais da UNIVESP contenham resolução de exercícios em outros médios no AVA utilizado pela instituição, mas aqui foi analisado apenas o elemento videoaula. 4. Conclusão Segundo Schwartz (2013), os vídeos publicados no YouTube se caracterizam pela facilidade em assistir, manobrar e absorver o conteúdo. Porém, o conteúdo assistido é apenas superficial, uma vez que é possível compreender conhecimentos contextuais ou mais profundos em situações verídicas do cotidiano. Esse pensamento reforça o fato de que é necessária uma avaliação do material disponível no YouTube e uma reflexão por parte dos professores que se propõem a construção desses canais, no sentido de sempre evoluir e simplificar seus ensinamentos. EaD em Foco,2024, 14(1): e2090 | 13 Também pode ser um dos motivos pelos quais os estudantes procuram este tipo de ferramenta para o seu aprendizado, visto que podem usar menor quantidade de tempo. É importante considerar que tais canais têm como objetivo realizar o suporte à aprendizagem informalmente, e não estão associados a uma instituição de ensino. O que ocorre de maneira diferente da UNIVESP, a qual oferece graduação em ensino superior e por este motivo deve seguir uma ementa, de acordo com os requisitos do Ministério da Educação, situação que traz maior responsabilidade na elaboração dos conteúdos apresentados. EL O Através do levantamento dos canais observou-se, adicionalmente, a busca dos alunos por um conteúdo mais dinâmico e ilustrativo, os levando à procura por conteúdos além dos disponibilizados pela UNIVESP. O objetivo inicial foi fazer um levantamento real dos canais mais assistidos pelos alunos que passam pela disciplina de Cálculo, e a partir desse levantamento caminhar para o objetivo principal, isto é a realização de uma análise pedagógica dos canais listados, o que permitiu trazer apontamentos importantes sobre o perfil de ensino que os alunos têm preferência. Em todos os canais analisados, o aprendizado é focado na resolução de exercícios, com uso de caneta ou lousa, deixando ver os cálculos mais ilustrativos e práticos. Também ressalta o bom uso da voz, usando uma narrativa fluida e descontraída sem perder rigorosidade, criando uma conexão com os alunos e ajudando a perder o medo das matemáticas. Estes dois fatos (solução passo a passo e voz informal) normalmente não são aplicados nas aulas da UNIVESP, vista a formalidade por ser uma IES, e poderiam se implementar considerando que podem ajudar a quebrar barreiras de comunicação na EaD e fazer as aulas mais atraentes para os estudantes. PR Diante dos dados apresentados podemos concluir que os doze princípios da teoria de aprendizagem de Mayer (2009) são eficientes num processo de ensino virtual, e que a adesão dos alunos a materiais que seguem esse pressuposto (ou boa parte dele) é alta. Ainda assim, há muito o que ser implementado nos canais gratuitos disponíveis no YouTube, para que possam atingir todos os princípios. Além disso, a crítica construtiva às videoaulas apresentadas pela UNIVESP na disciplina de cálculo foi feita de forma isolada, sem considerar o material completo que a instituição disponibiliza para os estudantes. EaD em Foco,2024, 14(1): e2090 | 14 Biodados e contatos dos autores Isabela DIVINO é nutricionista pelo Centro Universitário de Jaguariúna (2017) e atuou na prática clínica durante 5 anos. Durante o mestrado foi bolsista no programa de especialização em Processos didáticos-pedagógicos para cursos de modalidade a distância. Doutoranda e Mestra em Ciências da Nutrição e do esporte e metabolismo pela da Faculdade de Ciências Aplicadas da UNICAMP. Trabalha no Laboratório de Biologia Celular e Tecidual, desenvolve pesquisas com ênfase no Músculo esquelético e regeneração. Atualmente bolsista CAPES. ORCID: https://orcid.org/0000-0002-7007-7842 O Contato: +55 (19) 99494-0084 EL Rodrigo Naoto SHIRAISHI é doutorando na Faculdade de Ciências Médicas da UNICAMP pelo programa de Fisiopatologia Médica e técnico em laboratório no Hemocentro da mesma universidade. Graduado em Ciências Biológicas pela UNICAMP, destacou-se em pesquisas sobre leucemia promielocítica aguda, com foco nos efeitos do extrato de Chá Verde em modelos animais. Possui experiência como facilitador EAD na UNIVESP, estagiou na empresa de biotecnologia Helixxa e no Instituto Nacional de Pesquisa e Conservação de Peixes Continentais. Seus conhecimentos incluem análise química e bioquímica, obtidos durante seu curso técnico na ETECAP e estágio no Instituto Agronômico de Campinas. PR ORCID: https://orcid.org/0000-0001-6778-6886 Contato: +55 (19) 99825-2442 Mariele Rodrigues MOREIRA é doutoranda e Mestra em Nutrição e Metabolismo pelo Programa de Ciências da Nutrição e do Esporte e Metabolismo da Faculdade de Ciências Aplicadas da UNICAMP em que trabalhou com desenvolvimento e análise de um alimento clean label. Durante o mestrado foi bolsista no programa de especialização em Processos didáticos-pedagógicos para cursos de modalidade a distância da UNIVESP onde além da formação teórica teve a prática atuando como tutora nos cursos de graduação. Graduada em Ciências com habilitação plena em Química que permitiu atar como professora por dez anos em escolas públicas, particulares e cursinho pré-vestibular. Seus interesses de pesquisa atuais incluem educação alimentar e nutricional com ênfase em medicina culinária. ORCID: https://orcid.org/0000-0001-8056-8269 Daniel Alfonso SIERRA é doutorando na Faculdade de Engenharia Civil, Arquitetura e Urbanismo da UNICAMP. Completou o seu mestrado na mesma faculdade, na EaD em Foco,2024, 14(1): e2090 | 15 área de Recursos Hídricos, Energéticos e Ambientais. Seus interesses de pesquisa incluem redes de abastecimento de água, adutoras, sistema de proteção contra transitórios hidráulicos, com destaque para a modelação de transitórios hidráulicos e na otimização do dimensionamento de dispositivos de proteção. ORCID: https://orcid.org/0000-0003-0699-4512 Contato: +55 19992196674 O Victoria HERRERA é professora do Centro de Engenharia Modelagem e Ciências sociais Aplicadas da Universidade Federal do ABC (UFABC). Formada em engenharia eletrônica pela Universidad Mayor de San Andrés, mestre e doutora em Energia pela UFABC. Atua em elaboração de objetos de aprendizagem para engenharias, modelagem de sistemas dinâmicos, diagnóstico de falhas e educação a distância. EL ORCID: https://orcid.org/0000-0001-8056-8269 Referências Bibliográficas PR ALMEIDA, R. et al. Avaliação de objetos de aprendizagem sobre o sistema digestório com base nos princípios da Teoria Cognitiva de Aprendizagem Multimídia. Ciência & Educação (Bauru), 20, p. 1003-1017, 2014. BRAGA, A., et al. A teoria cognitiva da aprendizagem multimídia no desenvolvimento de atividades de alfabetização matemática. Scientia Plena, 15(7), 2019. https://doi.org/10.14808/sci.plena.2019.074803 CORADINI, F.R. Objetos de Aprendizagem: uma proposta de recurso pedagógico nos anos iniciais do ensino fundamental. 2009. CORTEZ, L.C. OS VÍDEOS DO YOUTUBE COMO RECURSO DIDÁTICO. 2010. 52 f. Monografia (Especialização) - Curso de Pós-graduação Latu Sensu em Mídias Integradas na Educação, Universidade Federal do Paraná, Curitiba, 2010. CURRAN, V., et al. YouTube como um recurso educacional na educação médica: uma revisão de escopo. Educador de ciências médicas, 30 (4), p.1775-1782, 2020. Disponível em: https://doi.org/10.1007/s40670-020-01016-w FOLHA DE SÃO PAULO. Acesso à internet cresce em 2020 com 64% das casas da classe D/E conectadas.18 ago, 2021. Disponível em: https://rb.gy/bduzev - Acesso em: 23 mar, 2022. EaD em Foco,2024, 14(1): e2090 | 16 IEEE STANDARD FOR LEARNING OBJECT METADATA. Us-Canada: Ieee LTSC, 16 nov. 2020. Disponível em: 10.1109/IEEESTD.2020.9262118 Acesso em: 13 mar. 2024. MAYER, R. Research-based principles for the design of instructional messages. Document Design, [S.L.], v. 1, n. 1, p. 7-19, 5 nov. 1999. John Benjamin’s Publishing Company. http://dx.doi.org/10.1075/dd.1.1.02may MAYER, R. Multimedia Learning. 1. ed. New York/USA: Cambridge University Press, 2001. MAYER, R. et al. A personalization effect in multimedia learning: Students learn better when words are in conversational style rather than formal style. Journal of educational psychology, v. 96, n. 2, p. 389, 2004. O MAYER, R. Applying the science of learning to multimedia instruction. In: Psychology of learning and motivation. Academic Press, 2011. p. 77-108. EL MAYER, R. Cognitive Theory of Multimedia Learning. Chapter. In The Cambridge Handbook of Multimedia Learning, edited by Richard E. Mayer, 43–71. Cambridge Handbooks in Psychology. Cambridge: Cambridge University Press, 2014. MIRANDA, G. Ensino online e aprendizagem multimídia. Relógio d'Água Editores, 2009. SOUZA, C.A. e MORALES, O. Coleção Mídias Contemporâneas. Convergências Midiáticas, Educação e Cidadania: aproximações jovens. Vol. II, Ponta Grossa: UEPG/PROEX, 2015 PR PIMENTEL, C. Repórter da Agência Brasil. Pisa: menos de 50% dos alunos sabem o básico em matemática e ciências. 2023. Disponível em: https://rb.gy/iegouv Acesso em: 13 mar. 2024. SILVA, A.M.; MONTANÉ, F. Objetos de Aprendizagem baseados na teoria da Aprendizagem Multimídia. Redin-Revista Educacional Interdisciplinar, 2017, vol. 6, no 1. SCIENCE BLOG BRASIL. Disponível em: https://www.blogs.unicamp.br/sbbr/ Acesso em: 23 mar, 2022. SCHWARTZ, M. Khan academy: The illusion funder standing. Online Learning Journal, v. 17, n. 4, 2013. UNIVESP. UNIVESP em números 2021: TCESP. São Paulo: Governo do Estado de São Paulo, 2021. 19 p. Disponível em: https://rb.gy/d0wb63 - Acesso em: 09 ago, 2022 UNIVESP. GUIA GERAL Produção de Disciplinas. São Paulo: Governo do Estado de São Paulo, 2020. 19 p. Disponível em: https://apps.univesp.br/academico/guia-disciplina/ - Acesso em: 09 ago, 2022 YOUNDER. Objetos de aprendizagem: aprimorando em: https://rb.gy/ro9hbm - Acesso em: 23 mar, 2022. EaD em Foco,2024, 14(1): e2090 os treinamentos. Disponível | 17 COMO CITAR ESTE TRABALHO PR EL O ABNT: DIVINO, I. A. et al. Análise de Canais do YouTube como Objetos de Aprendizagem de Suporte ao Estudo de Cálculo I em Cursos da Área de Ciências Exatas. EaD em Foco, v. 14, n. 1, e2090, 2024. doi: https://doi.org/10.18264/eadf.v14i1.2090 EaD em Foco,2024, 14(1): e2090 | 18 

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Borrelia lusitaniae and Green Lizards (Lacerta viridis), Karst Region, Slovakia Viktória Majláthová,* Igor Majláth,† Marketa Derdáková,*‡ Bronislava Víchová,* and Branislav Pet’ko* In Europe, spirochetes within the Borrelia burgdorferi sensu lato complex are transmitted by Ixodes ricinus ticks. Specific associations are described between reservoir hosts and individual genospecies. We focused on green lizard (Lacerta viridis) as a host for ticks and potential host for borreliae. In 2004 and 2005, a total of 146 green lizards infested by ticks were captured, and 469 I. ricinus ticks were removed. Borrelial infection was detected in 16.6% of ticks from lizards. Of 102 skin biopsy specimens collected from lizards, 18.6% tested positive. The most frequently detected genospecies was B. lusitaniae (77.9%–94.7%). More than 19% of questing I. ricinus collected in areas where lizards were sampled tested positive for borreliae. B. garinii was the dominant species, and B. lusitaniae represented 11.1%. The presence of B. lusitaniae in skin biopsy specimens and in ticks that had fed on green lizards implicates this species in the transmission cycle of B. lusitaniae. he causative agents of Lyme borreliosis, spirochetes of the Borrelia burgdorferi sensu lato complex, are maintained in natural foci by circulation between the vector ticks in the Ixodes ricinus complex and reservoir hosts of various vertebrate taxa. The B. burgdorferi s.l. complex encompasses 12 species (1–3); 4 species have been clearly established as pathogenic to humans: B. afzelii, B. garinii, B. burgdorferi s. s., and B. spielmanii (4–6). B. valaisiana and B. lusitaniae, which were previously considered nonpathogenic, may cause disease as well (7,8). Different species are associated with distinct ecologic features, levels of pathogenicity, and clinical symptoms in patients. In Europe, I. ricinus ticks infest a wide variety of vertebrate hosts, such as mammals, birds, and lizards. The verte- T *Parasitological Institute of Slovak Academy of Sciences, Košice, Slovakia; †University of P.J. Šafárik in Košice, Košice, Slovakia; and ‡Institute of Zoology of Slovak Academy of Sciences, Košice, Slovakia brate hosts are necessary to maintain the tick population and may also serve as reservoirs for the pathogen. Therefore, the identification of reservoir host species is essential to clarify the transmission patterns of B. burgdorferi s.l. in natural foci. The importance of rodents for maintaining B. afzelii (9), and of birds for B. garinii and B. valaisiana (10), in endemic regions of Slovakia is now indisputable. The National Park Slovak Karst is within the region in which B. burgdorferi s.l. in questing ticks and birds has been reported (V. Taragel'ová, unpub. data). In this area, 2 lizard species occur sympatrically, the common wall lizard (Podarcis muralis) and the green lizard (Lacerta viridis). The green lizard, the dominant species, is frequently infested by immature stages of I. ricinus ticks (11). The importance of lizards in the maintenance cycles of B. burgdorferi s.l. spirochetes is still controversial. In Italy, B. lusitaniae was detected in blood and tissue samples of P. muralis (12). Furthermore, Psammodromus algirus, the most abundant lizard species in North Tunisia, was found to be the primary host for immature stages of I. ricinus. Thus, it could play a role in the circulation of borreliae (13). B. burgdorferi s.s., B. andersonii, and B. bisettii were detected in the blood of 9 lizard species in the southeastern United States (14). On the other hand, several other studies in the United States have shown that the lizards Sceloporus occidentalis and Elgaria multicarinata are reservoir-incompetent for borreliae because they possess borreliacidal factor in their blood (15,16). However, 2 lizard species, Eumeces inexpectatus and Anolis carolinensis, can sustain B. burgdorferi s. s. infection (17). In the Slovak Karst (southeastern part of Slovakia), the green lizard is the major host for immature stages of I. ricinus ticks (11). Therefore, the main aim of this study was to find out whether green lizards can participate in the maintenance cycles of B. burgdorferi s.l. in natural foci and Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 12, December 2006 1895 RESEARCH whether an association with specific borrelial genospecies exists. Materials and Methods Study Area The study was conducted in the National Park Slovak Karst. This area represents a part of the Inner Carpathians in southeastern Slovakia (48°36′ N, 20°52′ E). The climate is warm with low humidity and average temperatures of − 4°C in January and 18°C in July. The average rainfall is 700 mm/year. merase (5 U/µL), 0.5 µL deoxynucleoside triphosphate (dNTP) mix (10 mmol/L) (Fermentas, Vilnius, Lithuania), 1.25 µL of each primer (10 pmol/µL) (Invitrogen, Paisley, Scotland), and 2.5 µL DNA template. To verify that DNA had been successfully isolated from each tick, primers for the fragment of the tick’s mitochondrial cytochrome b gene (620 bp) were used (19). Negative samples were excluded from the further analysis. Positive samples were examined for the presence of B. burgdorferi s. l. by amplifying a portion of the 5S (rrfA)-23S (rrlB) rDNA intergenic spacer (20). PCR products were subjected to electrophoresis on a 1% agarose gel, stained with ethidium bromide, and visualized with a UV transilluminator. Tick and Lizard Collection This survey was conducted in 2004–2005, from May to September, when lizards and ticks are active. Questing I. ricinus nymphs and adults were collected by flagging the vegetation in areas where lizards were sampled. Ticks were immediately stored in 70% ethanol. Green lizards were captured along hiking paths by hand or by noosing, in which a loop made from fishing nylon was attached to the end of a wooden stick and dangled in front of a lizard, which would be captured as it walked through the loop. Animals were characterized by sex and age (adult, subadult, juvenile) and examined for ticks. Ticks were removed with forceps immediately after capture and stored in 70% ethanol. Biopsy specimens (a 2-cm distal part of the tail and a 1-mm × 1.5-mm piece of skin from collar scales) were taken from each lizard with sterile scissors and put in separate vials with 70% ethanol. Ticks were identified to the species and sex. Only I. ricinus ticks were further examined for B. burgdorferi sensu lato. DNA Isolation Immediately before extraction, ticks and tissues were dried for 30 min to evaporate the ethanol. Each sample was cut with a disposable sterile scalpel. Tissue DNA from lizards’ tails and scales was extracted by using DNeasy tissue kit (Qiagen, Hilden, Germany). Extraction steps were conducted according to the manufacturer’s protocol. Genomic DNA from ticks was isolated by alkaline hydrolysis (18). Incubation time was extended from 5 to 30 min. Isolated DNA was stored at −20°C. PCR PCR amplification was performed in a 25-µL reaction mixture from the MasterTaq DNA polymerase kit (Eppendorf AG, Hamburg, Germany) containing 10.4 µL deionized water, 5 µL 5× TaqMaster PCR Enhancer, 2.5 µL 10× Taq buffer (with 15 mmol/L Mg2+), 1.5 µL 25mmol/L solution of Mg (OAc)2, 0.1 µL Taq DNA poly- 1896 RFLP Analysis The positive PCR products of the 5S-23S rDNA intergenic spacer regions were further analyzed by restriction fragment length polymorphism (RFLP). Previously extracted DNA of B. afzelii, B. garinii, B. valaisiana, and B. burgdorferi s.s. were used as positive controls. For each positive sample, 13 µL amplified DNA was digested at 65°C overnight in a solution containing 5 U of Tru1I (300 U/mL) and 1× Buffer R (Fermentas). Electrophoresis was conducted in 16% polyacrylamide gel at 150 V for 3 h. The gels were stained with SYBR gold nucleic acid gel stain (Molecular Probes, Leiden, the Netherlands) for 20 min, and bands were visualized with a UV transilluminator. RFLP profiles that differed from the known profiles of positive controls were further analyzed by sequence analysis. DNA Sequencing of PCR Products Sequencing was performed at the Department of Molecular Biology (Faculty of Natural Sciences Commenius University, Bratislava, Slovak Republic). PCR of the 5S-23S rDNA intergenic spacer was conducted according to the protocol described above. For the fla gene amplicons, DNA strands were sequenced as described previously (21). PCR products were purified by using a QIAquick PCR purification kit (Qiagen). The complementary strands of each sequenced product were manually assembled. Sequences were compared with GenBank entries by Blast N2.2.13 (22). Homologous sequences were aligned by using the CLUSTAL W Multiple Sequence Alignment Program (version 1.81) (23). Sequence similarity among the sequences were calculated by EMBOSS Align, a pairwise alignment algorithm (http://www.ebi.ac.uk/emboss/align). The accession numbers of 5S-23S rDNA intergenic spacer sequences obtained in this study are DQ539339 and DQ539340. Accession numbers of flagellin sequences obtained in this study are DQ788618, DQ788619, and DQ788620. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 12, December 2006 Borrelia lusitaniae and Green Lizards, Slovakia Data Analysis and Statistics To estimate the probability of a tick’s becoming infected after engorging on a green lizard and to measure the degree of infectiousness of infected animals, specific infectivity Is (24) and transmission coeficient βH-T (9) were calculated. Individual infectivity (i) is defined as the proportion of larvae derived from an individual lizard that are infected (i = li/lh, li is the number of larvae that become infected, and lh is the total number of larvae derived from that host). The specific infectivity (Is) of a reservoir host species is defined as the sum of individual infectivities and number of individual lizards sampled (Is = ΣiS/nS, n is the number of individual ticks captured). The host-to-tick transmission coefficient (βH–T) is defined as the portion of the sum of individual infectivities and the number of lizards that infected >1 larva (βH–T = ΣiS/niS (ni is the number of individual hosts that gave rise to at least 1 infected tick). Differences in the prevalence of B. burgdorferi s.l. in I. ricinus were evaluated statistically with the 2-tailed χ2 test (degrees of freedom [df] = 1). A value of p<0.05 was considered statistically significant. Results Lizards and Infestation with Ticks One hundred forty-six (84 male, 52 female, and 10 subadult) of 165 (89 male, 61 female, and 15 subadult) captured green lizards were infested by ticks during the study period. In total, 469 (199 larvae and 270 nymphs) ticks were removed and further identified as I. ricinus. Male lizards were infested with 363 ticks (131 larvae and 232 nymphs), which represented 77.4% of all collected ticks. Moreover, 53 tails and 102 skin biopsy specimens were taken from the captured lizards. B. burgdorferi Prevalence in Ticks Collected from Lizards DNA isolation was successful in 464 ticks (197 larvae and 267 nymphs), from which the fragment of cytochrome b gene was amplified. These ticks were further analyzed for the presence of B. burgdorferi s.l. In total, 77 (16.6%) ticks carried borreliae. The infection prevalence between nymphs (15.2%) and larvae (17.6%) did not differ significantly (p = 0.49669, df = 1) (Table 1). Twenty-nine percent of tick-infested lizards carried >1 infected tick. Infected lizards yielded ≈2 infected larvae per host. Genotyping with PCR-RFLP identified the following species: B. lusitaniae, B. afzelii, B. garinii,B. burgdorferi s.s., and B. valaisiana. Of the B. burgdorferi–positive ticks, most (77.9%) were infected with B. lusitaniae. The presence of this species was significantly higher than that of other species (p<0.001). B. lusitaniae was detected in 26 (86.7%) larvae. B. afzelii, B. garinii, and B. burgdorferi s. s. each were found in 1 larva. Of the 47 B. burgdorferi–infected nymphs, 34 (72.3%) were infected with B. lusitaniae, 5 (10.6%) with B. afzelii, 2 (4.3%) with B. burgdorferi s. s., and 1 (2.1%) with B. garinii. A mixed infection of B. lusitaniae and B. burgdorferi sensu stricto was found in 5 (10.6%) nymphs (Table 1). Nymphs and larvae did not differ significantly in the prevalence of B. lusitaniae. Male lizards were parasitized by 61 (79.2%) of 77 infected ticks. Variability of detected genospecies was higher in ticks collected from male than from female lizards. Larvae that fed on female lizards were only infected with B. lusitaniae. Out of 7 infected nymphs collected from females, B. lusitaniae was present in 5 and B. afzelii in 1 tick; B. lusitaniae and B. burgdorferi s.s. were detected as mixed infection in 1 nymph. The specific infectivity from lizards to larval ticks was highest for B. lusitaniae. The specific infectivity of female lizards was slightly higher than that of males (Table 2). B. burgdorferi Prevalence in Lizards Isolated genomic DNA from tails and skin biopsy specimens from collar scales was tested for the presence of B. burgdorferi sensu lato. None of 53 tested tail samples was positive. Of 102 skin biopsy specimens collected from green lizards, 19 (18.6%) tested positive. Differences in infection prevalence between sexes (18.2% in males vs. 23.7% in females) were not significant. Of 9 skin biopsy specimens from subadult individual lizards, 2 (22.2%) were borrelia positive. The most frequently detected genospecies was B. lusitaniae (94.7%), which was present in 18 samples. One lizard was infected with B. afzelii. B. burgdorferi Prevalence in Questing Ticks Cytochrome b was amplified in 325 of 331 (71 female, 73 male, and 187 nymph) questing ticks. Therefore, only these 325 ticks (71 female, 71 male, and 183 nymph) were analyzed further for the presence of B. burgdorferi s.l. Sixty-three (19.3%) ticks tested positive. B. burgdorferi Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 12, December 2006 1897 RESEARCH prevalence in female ticks was the same as in male ticks (22.5%), and it was lower in nymphs (16.9%). RFLP analysis of the amplified products resulted in 5 distinct profiles. Of the 63 positive ticks, 21 (33.3%) were infected with B. garinii, 19 (30.2%) were infected with B. afzelii, 8 (12.7%) were infected with B. burgdorferi s. s., 7 (11.1%) were infected with B. lusitaniae, and 7 (11.1%) were infected with B. valaisiana. One nymph was infected simultaneously with B. garinii and B. valaisiana (Table 3). Sequence Analysis Representative samples of RFLP profiles that were different from the known profiles of positive controls were sequenced. The fragment of the 5S-23S rDNA intergenic spacer obtained from the B. burgdorferi s.l.–positive nymph (538N) from lizard belonged to B. lusitaniae. It was 100% identical to a Borrelia-positive skin biopsy specimen (277S) sampled from a lizard. Both obtained sequences were 100% identical with the Turkish B. lusitaniae strain Tr213 (AB 091802) and 98.9%, 98.4%, and 94.5% similar to PotiBL37 (AY 463167), PotiB2 (L30131), and PotiB3 (L30132) strains from Portugal, respectively. To better characterize B. lusitaniae circulating in ticks and lizards from Slovak Karst, the fla gene from a B. burgdorferi s. l.–positive questing adult tick and skin biopsy specimen from collar scale was amplified and sequenced. The flagellin sequence of B. lusitaniae detected in a skin biopsy specimen (277 S) was 100% identical and 99.6% similar to B. lusitaniae detected in questing adult ticks (43 ZLIF, 47 ZMLIM), respectively. Genotypes 277S and 43ZLIF were 100% identical with the Turkish B. lusitaniae strain Tr213 (AB091812) as well as with the Polish strain D23–04 (DQ 016623). Genotype 47ZLIM was 99.6%, 99.6%, and 99.4% similar to Tr213, D23–04, and PotiB2 (DQ111036), respectively. 1898 Discussion The role of lizard species in maintaining B. burgdorferi s.l. has not been clearly elucidated yet. In United States, some lizard species have sustained borrelial infection (14,17); however, other species are incompetent reservoir hosts (15,16). The reservoir competence of lizards seems to be species specific. Therefore the aim of our study was to establish whether a relationship exists between green lizards, the dominant lizard species in the Slovak Karst, and B. burgdorferi s.l., which circulates in this area. Seventeen percent of ticks that fed on lizards were infected with B. burgdorferi s.l. Seventy-eight percent of all infected ticks were infected with B. lusitaniae. Moreover, 18.6% of skin biopsy specimens from lizards were positive for B. burgdorferi s.l., and almost all (94.7%) were infected with B. lusitaniae. Similarly, B. lusitaniae have been detected in blood and tissue samples of Podarcis muralis in Tuscany in Italy, where borreliae were detected in 2 of 14 tested whole tails from lizards (12). At the beginning of our study, we also collected the distal tip of a lizard’s tail because this method is minimally invasive and convenient for obtaining a tissue sample. The tissue at the tail, however, is squamous and keratinized, and none of the collected samples was borreliae positive. Therefore, we also obtained skin biopsy specimens from collar scales. These are elongated and extend from the skin on the ventral side, so collecting them is minimally invasive and perhaps more likely to detect infection with B. burgdorferi s.l. because most of the immature I. ricinus ticks parasitize at the dorsal area (pers. observation). Furthermore, collar scales were chosen to avoid detecting the borreliae that persist in the skin after feeding of the infected ticks, which may enable infection of ticks by “extended co-feeding” (25). In this manner, incompetent host species may contribute to the circulation of B. burgdorferi s.l. in nature. For example, in England, Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 12, December 2006 Borrelia lusitaniae and Green Lizards, Slovakia I. ricinus ticks cofeeding on sheep become infected with B. burgdorferi, although sheep themselves are refractory to infection (26). In Europe the principal importance of cofeeding to Lyme disease ecology has been suggested to be the extent of the range of vertebrate host species that contribute significantly to the maintenance of B, burgdorferi s.l. spirochetes in nature (27). Therefore, cofeeding transmission could also be responsible for B. afzelii, B. garinii, and B. burgdorferi s.s. infection in larvae that fed on lizards collected in our study, even though skin biopsy results yielded mostly B. lusitaniae. Another possible explanation for the presence of non–B. lusitaniae spirochetes is that these larvae may have been infected transovarially (28). Cofeeding transmission might explain why individual lizards with borreliae negative skin biopsy specimens carry borreliae-positive larvae. Because the quantity of borreliae is low in the vertebrate host and may lodge in deeper organs, detecting them in skin biopsy specimens may not always be possible (29). Thus, a negative skin biopsy result does not prove conclusively that the lizard is not infected. Despite the fact that male lizards hosted >75% of all host-feeding ticks, as well as 79.2% of all infected ticks, the specific infectivity and host-to-tick transmission coefficient were almost the same for male and female lizards. The seasonal activity of green lizards and different patterns in male and female behavior were monitored in the Slovak Karst (I. Majlath, unpub. data). Larger numbers of ticks feeding on male lizards are associated with higher male activity in spring months, when tick activity peaks as well. Male lizards end hibernation first and are active when the air temperature reaches 10°C–12°C. They need to restock the energy that was depleted during winter and to gain energy for fighting other male lizards to compete for territory and females, for seeking female lizards, and for mating. Female activity increases in summer months when they are incubating eggs. As determined by PCR, the overall prevalence of infection in our sample of questing ticks (19.3%) is consistent with 20.5% found in southern Czech Republic (20) but lower than that reported for a geographically close area in western Slovakia (40%–49%) (30). The total prevalence was higher in adults (22.5%) than in nymphs (19.6%), which is in agreement with the general pattern of increasing Borrelia prevalence through the life stages of ticks as their adults feed on a multiple hosts (31). The total prevalence of borreliae in male and female ticks was identical, but the distribution of genospecies was different. B. garinii was the predominant genospecies in this locality. B. garinii and B. valaisiana are the most commonly reported species in central Europe (32). The high prevalence of B. lusitaniae in borrelia-positive larvae and nymphs as well as skin biopsy specimens from lizards suggests that green lizards are susceptible and transmission competent for B. lusitaniae. On the other hand, a lack or low prevalence of other genospecies in ticks that had fed on lizards may suggest that these genospecies could be negatively selected against by green lizards. A similar suppressive effect of Madeiran wall lizard (Podarcis dugesii) on the transmission of spirochetes was observed (33). Borreliacidal activity against B. burgdorferi s.s. was observed in the lizards S. occidentalis and E. multicarinata in North America (15,16). These findings add to the growing support for the hypothesis that there are Borrelia species-specific associations with specific reservoir host species that result from Borrelia species–specific interactions with host serum complement (29). Significant differences were found in B. lusitaniae prevalence in fed larvae compared with questing nymphs (p<0.001, df = 1); none of 183 examined nymphs was infected by this genospecies. This finding raises the questions of whether borreliae are eliminated during molting and thus do not contribute to the transmission cycle or whether we were just unable to detect it. Significant differences were found in B. lusitaniae prevalence also in fed nymphs compared with questing adults (p<0.01, df = 1). The infection prevalence decreased from 74.5% in fed nymphs to 5% in questing adults. Reduction of infection prevalence has been observed in B. afzelii from 47% in nymphs engorged on the rodents to 7% in questing nymphs (9). The occurrence of B. lusitaniae in ticks is frequent in some areas of the Iberian Peninsula and North Africa, where the organism often represents the only species of B. burgdorferi s.l. complex (13,34). In the rest of the Europe, it has been isolated or detected less frequently, with low prevalence in ticks (30,35,36). The prevalence of B. lusitaniae is the highest in southern Europe and can be exported to other areas by hosts such as birds (37). The 5S-23S rDNA and flagellin sequences of B. lusitaniae–positive ticks and skin biopsy specimens in our study were 100% identical to the B. lusitaniae strain Tr213 from a tick in Turkey (38). The distribution of this borrelial species may be associated with the distribution range of reservoir hosts, including lizards, that inhabit drier and warmer areas. These ecosystems are less abundant in central Europe than in the Mediterranean. Thus, lizards may influence the transmission cycle of borreliae in some localities in which they are the predominant host for ticks. In our study, we found B. lusitaniae in skin biopsy specimens and ticks that fed on green lizards. These findings implicate this species of lizard in the transmission cycle of B. lusitaniae. The competence of other lizard species that feed ticks should be also investigated. The low prevalence of B. lusitaniae in questing ticks, however, indicates that the ecology of Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 12, December 2006 1899 RESEARCH B. lusitaniae in endemic foci of central Europe is more complex. Further studies that analyze the circulation of B. burgdorferi s.l. among a broader spectrum of host species should be undertaken. Acknowledgments We thank Jean Tsao and Slavka Barlakova for their critical reading of the manuscript and helpful comments and Marcela Mirekova and Renata Ivanova for technical assistance. This work was partly supported by the Slovak Agency of Research and Development, APVV-51-009205 project (M.D.), VEGA 2/6163/26 to (B.P.), and VEGA 1/1284/04 (I.M.). Mrs Majláthová is a doctoral student at the Parasitological Institute of the Slovak Academy of Sciences. Her research interests lie in ecology and epidemiology of tickborne pathogens. References 1. Wang G, van Dam AP, Schwartz I, Dankert J. Molecular typing of Borrelia burgdorferi sensu lato: taxonomic, epidemiological, and clinical implications. Clin Microbiol Rev. 1999;12:633–53. 2. Masuzawa T, Takada N, Kudeken M, Fukui T, Yano Y, Ishiguro F, et al. Borrelia sinica sp. nov., a Lyme disease–related Borrelia species isolated in China. Int J Syst Evol Microbiol. 2001;51:1817–24. 3. Richter D, Schlee DB, Allgower R, Matuschka FR. Relationships of a novel Lyme disease spirochete, Borrelia spielmani sp. nov., with its hosts in Central Europe. Appl Environ Microbiol. 2004;70:6414–9. 4. van Dam AP, Kuiper H, Vos K, Widjojokusumo A, de Jongh BM, Spanjaard L, et al. Different genospecies of Borrelia burgdorferi are associated with distinct clinical manifestations of Lyme borreliosis. Clin Infect Dis. 1993;17:708–17. 5. Wang G, van Dam AP, Dankert J. Phenotypic and genetic characterization of a novel Borrelia burgdorferi sensu lato isolate from a patient with Lyme borreliosis. J Clin Microbiol. 1999;37:3025–8. 6. Foldvari G, Farkas R, Lakos A. Borrelia spielmanii erythema migrans, Hungary. Emerg Infect Dis. 2005;11:1794–5. 7. Collares-Pereira M, Couceiro S, Franca I, Kurtenbach K, Schafer SM, Vitorino L, et al. First isolation of Borrelia lusitaniae from a human patient. J Clin Microbiol. 2004;42:1316–8. 8. Diza E, Papa A, Vezyri E, Tsounis S, Milonas I, Antoniadis A. Borrelia valaisiana in cerebrospinal fluid. Emerg Infect Dis. 2004;10:1692–3. 9. Hanincova K, Schafer SM, Etti S, Sewell HS, Taragelova V, Ziak D, et al. Association of Borrelia afzelii with rodents in Europe. Parasitology. 2003;126:11–20. 10. Hanincova K, Taragelova V, Koci J, Schafer SM, Hails R, Ullmann AJ, et al. Association of Borrelia garinii and B. valaisiana with songbirds in Slovakia. Appl Environ Microbiol. 2003;69:2825–30. 11. Majlath I, Majlathova V. Green lizard (Lacerta viridis) as host for ectoparasites. Proceedings of abstracts and papers of the 5th International Conference on Ticks and Tick-Borne Pathogens. 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Lyme disease in California: interrelationship of Ixodes pacificus (Acari: Ixodidae), the western fence lizard (Sceloporus occidentalis), and Borrelia burgdorferi. J Med Entomol. 1989;26:272–8. 17. Levin M, Levine JF, Yang S, Howard P, Apperson CS. Reservoir competence of the southeastern five-lined skink (Eumeces inexpectatus) and the green anole (Anolis carolinensis) for Borrelia burgdorferi. Am J Trop Med Hyg. 1996;54:92–7. 18. Guy EC, Stanek G. Detection of Borrelia burgdorferi in patients with Lyme disease by the polymerase chain reaction. J Clin Pathol. 1991;44:610–1. 19. Black WC, Roehrdanz RL. Mitochondrial gene order is not conserved in arthropods: prostriate and metastriate tick mitochondrial genomes. Mol Biol Evol. 1998;15:1772–85. 20. Derdakova M, Beati L, Pet’ko B, Stanko M, Fish D. 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Comparing the relative potential of rodents as reservoirs of the Lyme disease spirochete (Borrelia burgdorferi). Am J Epidemiol. 1989;130:143–50. 25. Gern L, Rais O. Efficient transmission of Borrelia burgdorferi between cofeeding Ixodes ricinus ticks (Acari: Ixodidae). J Med Entomol. 1996;33:189–92. 26. Ogden NH, Nuttall PA, Randolph SE. Natural Lyme disease cycles maintained via sheep by co-feeding ticks. Parasitology. 1997;115: 591–9 27. Randolph SE, Gern L, Nuttall PA. Co-feeding ticks: epidemiological significance for tick-borne pathogen transmission. Parasitol Today. 1996;12:472–9. 28. Piesman J, Donahue JG, Mather TN, Spielman A. Transovarially acquired Lyme disease spirochetes (Borrelia burgdorferi) in fieldcollected larval Ixodes dammini (Acari: Ixodidae). J Med Entomol. 1986;23:219. 29. Kurtenbach K, De Michelis S, Etti S, Schafer SM, Sewell HS, Brade V, et al. Host association of Borrelia burgdorferi sensu lato-the key role of host complement. Trends Microbiol. 2002;10:74–9. 30. Gern L, Hu CM, Kocianova E, Vyrostekova V, Rehacek J. Genetic diversity of Borrelia burgdorferi sensu lato isolates obtained from Ixodes ricinus ticks collected in Slovakia. Eur J Epidemiol. 1999;15:665–9. 31. Hubalek Z, Halouzka J. Prevalence rates of Borrelia burgdorferi sensu lato in host-seeking Ixodes ricinus ticks in Europe. Parasitol Res. 1998;84:167–72. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 12, December 2006 Borrelia lusitaniae and Green Lizards, Slovakia 32. Hubalek Z, Halouzka J. Distribution of Borrelia burgdorferi sensu lato genomic groups in Europe, a review. Eur J Epidemiol. 1997;13:951–7. 33. Matuschka FR, Eiffert H, Ohlenbusch A, Richter D, Schein E, Spielman A. Transmission of the agent of Lyme disease on a subtropical island. Trop Med Parasitol. 1994;45:39–44. 34. De Michelis S, Sewell HS, Collares-Pereira M, Santos-Reis M, Schouls LM, Benes V, et al. Genetic diversity of Borrelia burgdorferi sensu lato in ticks from mainland Portugal. J Clin Microbiol. 2000;38:2128–33. 35. Le Fleche A, Postic D, Girardet K, Peter O, Baranton G. Characterization of Borrelia lusitaniae sp. nov. by 16S ribosomal DNA sequence analysis. Int J Syst Bacteriol. 1997;47:921–5. 36. Wodecka B, Skotarczak B. First isolation of Borrelia lusitaniae DNA from Ixodes ricinus ticks in Poland. Scand J Infect Dis. 2005;37:27–34. 37. Poupon M-A, Lommano E, Humair PF, Douet V, Rais O, Schaad M, et al. Prevalence of Borrelia burgdorferi sensu lato in ticks collected from migratory birds in Switzerland. Appl Environ Microbiol. 2006;72:976–9. 38. Guner ES, Hashimoto N, Takada N, Kaneda K, Imai Y, Masuzawa T. First isolation and characterization of Borrelia burgdorferi sensu lato strains from Ixodes ricinus ticks in Turkey. J Med Microbiol. 2003;52:807–13. Address for correspondence: Viktória Majláthová, Parasitological Institute, Slovak Academy of Sciences, Hlinkova 3, 040 01 Košice, Slovakia; email: majlat@saske.sk Search past issues Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 12, December 2006 1901 

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Manuscript to be reviewed Short-chain soluble polyphosphate fertilizers increased soil P availability and mobility by reducing P fixation in two contrasting calcareous soils Jawad Ali Shah 1 , Guixin Chu Corresp. 1 1 Department of Envirnmental Science and Engineering, College of Life Science, Shaoxing University, Shaoxing, Zhejiang, P. R. China Corresponding Author: Guixin Chu Email address: chuguixin@usx.edu.cn Short-chain polyphosphate fertilizers have been increasingly applied in agriculture, but little is known about the chemical behaviors of polyphosphate in soils. Herein, a cylinder experiment was carried out to investigate the influences of different P types (i.e. mono- ammonium phosphate (MAP), phosphoric acid (PA) and ammonium polyphosphate (poly-P) and their application methods (single vs split) on the mobility and availability of P in soil through a column millimeter-scale slice cutting method; meanwhile a soil microcosm experiment (560-day) was conducted to investigate the effects of different P types on phosphorus dynamic transformation. Polyphosphate addition significantly increased P mobility. The average distance of P downward movement (81.5 mm) in soil profile in the poly-P application treatment increased by 33.6% and 81.1%, respectively, compared to the MAP and PA treatments. Different P application methods also markedly influenced phosphorus mobility. For instance, the average distance of P vertical movement in the split P application treatment was 21.2% higher than in the single application treatment, indicating that split P addition significantly increased P downward movement. Moreover, polyphosphate application decreased soil P fixation by blocking the transformation of the applied-P from labile to recalcitrant forms (HCl-P and residual-P). Overall, our findings provide meaningful information to current phosphorus fertilization practice in increasing soil P mobility and bioavailability. We suggest that polyphosphate could be regarded as an alternative P source used in agriculture, and split polyphosphate application is recommended as an effective P fertilization strategy. PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed 1 Type of contribution: Research paper 2 Number of text pages: 20 3 Number of tables: 05 4 Number of figures: 04 5 6 Title: 7 Short-chain soluble polyphosphate fertilizers increased soil P availability and mobility by 8 reducing P fixation in two contrasting calcareous soils 9 Authors: Jawad Ali SHAH1 and Gui-xin CHU 1* 10 Affiliations: 11 1. 12 China. 13 * Corresponding author: Prof. CHU Gui-xin 14 Address：Department of Environmental Science and Engineering, School of Life Science, 15 Shaoxing University, Huancheng West Road 508, Shaoxing 312000, P. R. China. 16 Tel: +86 17857505869; Fax: +86 575 8834 1905 17 Email: chuguixin@usx.edu.cn, ali_jawad009@yahoo.com College of Life Science, Shaoxing University, Shaoxing City, 312000, Zhejiang Province, 18 19 20 21 PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed 22 23 24 ABSTRACT 25 Short-chain polyphosphate fertilizers have been increasingly applied in agriculture, but little is 26 known about the chemical behaviors of polyphosphate in soils. Herein, a cylinder experiment 27 was carried out to investigate the influences of different P types (i.e. mono- ammonium 28 phosphate (MAP), phosphoric acid (PA) and ammonium polyphosphate (poly-P) and their 29 application methods (single vs split) on the mobility and availability of P in soil through a 30 column millimeter-scale slice cutting method; meanwhile a soil microcosm experiment (560-day) 31 was conducted to investigate the effects of different P types on phosphorus dynamic 32 transformation. Polyphosphate addition significantly increased P mobility. The average distance 33 of P downward movement (81.5 mm) in soil profile in the poly-P application treatment increased 34 by 33.6% and 81.1%, respectively, compared to the MAP and PA treatments. Different P 35 application methods also markedly influenced phosphorus mobility. For instance, the average 36 distance of P vertical movement in the split P application treatment was 21.2% higher than in the 37 single application treatment, indicating that split P addition significantly increased P downward 38 movement. Moreover, polyphosphate application decreased soil P fixation by blocking the 39 transformation of the applied-P from labile to recalcitrant forms (HCl-P and residual-P). Overall, 40 our findings provide meaningful information to current phosphorus fertilization practice in 41 increasing soil P mobility and bioavailability. We suggest that polyphosphate could be regarded 42 as an alternative P source used in agriculture, and split polyphosphate application is PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed 43 recommended as an effective P fertilization strategy. 44 Keywords: Phosphorus; Ammonium polyphosphate; Mobility; Availability; P transformation; 45 Calcareous soil 46 1. Introduction 47 Phosphorus (P) is an essential and yield-limiting macro-element for higher plants. However, 48 about two-thirds of the total cultivated soil belongs to P-deficient soil (e.g. plant available P <10 49 mg P kg-1) in China (Cao et al., 2012; Zhang et al., 2019), and approximately 5.7 billion ha of 50 area was deficient of soil available P on global scale (Hinsinger, 2001). Chemical synthesized 51 orthophosphate fertilizers (ortho-P) have been excessively applied in agriculture in the past 52 several decades (Chowdhury et al., 2017). MacDonald et al. (2012) addressed that P input was 53 23.8 Tg P·y−1 globally in 2000, resulted in an annual P surplus of 11.5 Tg of P·y−1. However, 54 only a small fraction of the applied P was absorbed by plants (Mueller et al., 2012), and plant- 55 accessible P only accounted for 6 % (range 1.5 to 11%) of total P in soils (Stutter et al., 2012). 56 On the other hand, P is a non-renewable resource, the exploitable P ore will be depleted within 57 60-100 yr, because ca 31 million tons of P was exploited annually (Cordell et al., 2009). 58 Therefore, set up an effective P management strategy is imperative to recycle P more efficient 59 and avoid running out of the finite P resource. 60 The applied P in soils undergoes a sequence of intricate transformation processes. In neutral or 61 calcareous soil, the freshly added P easily transformed from dibasic calcium phosphate (DCP) to 62 brushite dicalcium phosphate dihydrate, (DCPD), then to octacalcium phosphate (OCP), finally PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed 63 to hydroxyapatite (HAP) (Weihrauch & Opp, 2018; Ge et al., 2019). When P is applied through 64 basal application method, a relative high P concentration appears at P application site (Chien et 65 al., 2010; Li et al., 2019). In this way, the added-P progressively converts to octa-calcium 66 phosphate (Ca8H2(PO4)6·5H2O, OCP) within 3 to 5 months, following by constitutes β-tri- 67 calcium phosphate (β-TCP) after 8 to 10 months, finally turns into hydroxyapatite 68 Ca10(PO4)6(OH)2. In contrast, more added P remains in available form when P fertilizer is split 69 repeated applied at low application dose in crop growing season (Khatiwada et al., 2014; 70 González-Jimenez et al. 2019). Consequently, a high crop yield and phosphorus use efficiency 71 (PUE) can be achieved (Saarela et al., 2006). Unfortunately, at present, basal application of P 72 fertilizer remains used as main P fertilization technology in most agriculture areas. 73 Different P sources (ortho-P vs poly-P) significantly influenced phosphorus availability and 74 transformation (McBeath et al., 2005; Wang & Chu, 2015; Gao et al., 2019). Polyphosphates 75 (poly-P) are superior over ortho-P fertilizers (Hamilton et al., 2018). This may largely be 76 ascribed to its slow release characteristic (Kulakovskaya et al., 2012; Hamilton et al., 2016). 77 Following this, polyphosphate application significantly increased soil available P (Gao et al., 78 2019) and improved crop yield (Holloway et al., 2004). However, other studies showed that 79 polyphosphate (ammonium polyphosphate, APP) had no obvious advantage or even less than 80 ortho-P fertilizer (granular monoammonium phosphate, MAP) in increasing soil P availability 81 and crop yield (Ottman et al., 2006). These controversial or paradoxical results implied that the 82 physiochemical behaviors (i.e. mobility, availability, and transformation) of polyphosphate in 83 soils need to be further explored. PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed 84 In this study, a soil cylinder experiment and a soil microcosm experiment were carried out in 85 contrasting calcareous soils. We hypothesize that split repeated polyphosphate application 86 outcompetes conventional orthophosphate in increasing soil P availability and mobility. 87 Therefore, the objectives of our study were to (i) compare the effects of different P types and 88 their application methods on soil P mobility; (ii) clarify the response of soil P availability and 89 transformation to different P addition, (iii) finally put forward an appropriate P fertilization 90 strategy to increase soil P availability and improve PUE in calcareous soil. 91 2. Materials and Methods 92 2.1 Soils sampling and soil description 93 Experiments were conducted using two calcareous soils (loam and clay soils). According to 94 FAO/WRB soil taxonomy, both were classified as Calcisol Fluvisols. Loam soil was taken from 95 experimental station of Shihezi University (44°18′ N, 86°02′ E) and clay soil was collected from 96 147 State Farm (44°37′ N, 86°10′ E) in Shihezi region. Soil samples were air dried, stones and 97 small visible plant residues were manually removed. All samples were ground to pass through a 98 2 mm sieve prior to measurement of soil properties. The selected soil physical and chemical 99 properties are presented in (Table 1). 100 Insert “(Table 1)” PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed 101 2.2 Different P sources Description 102 Three types of P sources including (i) mono-ammonium phosphate (powder MAP, NH4H2PO4) 103 with purity of 99% and density of 1.803 g cm-3 and 26.9% of P (Shengao chemical reagent co., 104 LTD, China); (ii) phosphoric acid (fluid PA, H3PO4) with purity of 85% and density of 1.685 g 105 cm-3 and 27.1% of P (Fuyu special chemicals co., LTD, China), and (iii) ammonium 106 polyphosphate (fluid APP, H6P4O13, n=4) with purity of 85% and density of 2.1g cm-3 and 31.3% 107 of P (Aladdin Industrial Corporation, U.S.A) were employed in this study. 108 2.3 Experiment Ⅰ Soil cylinder experiment 109 Soil cylinder experiment was carried out in a transparency Plexi glass cylinders (350 mm height, 110 64 mm internal diameter). Five holes (2 mm diameter) were drilled at bottom of the cylinder to 111 maintain aerobic condition. A 10-mm-thick layer of fine sand was placed at the bottom of the 112 cylinder and placed two pieces of filter paper on the surface of the sand layer. A totally of 1302 g 113 loam soil and 1331 g clay soil were placed into each cylinder. Another two pieces of filter paper 114 was placed on the top of the cylinder. Soil bulk densities of 1.35 g cm-3 and 1.38 g cm-3 were 115 designed for loam and clay soil, respectively. Three P types (i.e., powder MAP, fluid PA, and 116 fluid APP) were applied to the each cylinder following the methods of: (i) Single application, the 117 total amount of P fertilizer was uniformly mixed into the upper 20 mm of soil layer and watered 118 four times over the four-week-long incubation period; (ii) split repeated application, the fertilizer 119 was initially dissolved with 50 ml milli-Q water in a 250 ml beaker individually except MAP PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed 120 which was applied directly and then fertilizer solution was injected into soil at the center site of 121 the cylinder using a syringe, P fertilizer was four times evenly added at equivalent ratio during 122 entire 28-days period. The irrigation water was poured into a Markov bottle, and hanged the 123 bottle 1.5 m above each cylinder (Fig. 1). The flow rate of the emitter was controlled at 6 drops 124 per second by connecting each emitter to individual Markov bottle. The total water column 125 irrigated were 401.6 mL and 515.8 mL cylinder-1 for loam and clay soils, respectively. P 126 application rates were 58.5 mg P cylinder-1 for loam and 59.8 mg P cylinder-1 for clay soils, 127 respectively. The P fertilizer application rate was two to three times higher than in normal 128 farmland (Du et al., 2013; Wang & Chu, 2015). Each treatment replicated three times giving a 129 total of 42 cylinders (containing 6 CK treatments with no fertilizer either in loam or clay soil, 18 130 cylinders for single application of MAP, PA and APP either in loam or clay soil and another 18 131 cylinders for split application of MAP, PA and APP either in loam or clay soil). All cylinders 132 were covered with a piece of parafilm, and incubated at room temperature during incubation 133 period. 134 After 28-day incubation, all cylinders were placed into a -80℃ freezer for 12 hours to keep 135 soil column hard enough. The frozen cylinders were cut into the given thick soil slices by placing 136 the cylinder to a high-speed spindle of the lathe with a sharp blade at millimeter-scale (Type: 137 CW6163C, made in Dalian, China) (Supplementary Fig). Each cylinder was vertically cut into 138 20 slices with a 5-mm thickness for each slice, then another 10 slices with a 20-mm-thickness 139 were cut. Each slice was immediately placed in a plastic bag and transferred to fridge. 140 All collected soil slices were used to determine soil moisture, soil WE-P (water extracted PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed 141 phosphorus) and Olsen-P. Briefly, to determine WE-P, an aliquot 2 g soil was taken in a 100 mL 142 centrifuge tube with 50 mL triple de-ionized (TDI) water. Shaking the soil sample for 1 hour at 143 25℃, following centrifugation for 15 min at 900 rpm, decant supernatant was separated and WE- 144 P was measured using malachite green colorimetric method at an absorbance of 610nm (Masson 145 et al., 2001). Olsen-P was determined in 0.5 M NaHCO3 extracts (pH 8.5), using the 146 molybdenum blue method (Olsen et al., 1954). 147 148 An exponential decay equation model was employed to describe the P downward P movement in soil cylinder. 149 Y = (Y0 - Plateau) ×exp (-K × X) + Plateau 150 Where X represents the depth of the soil column expressed as mm. Y indicates the content of 151 phosphorus in soil column in mg/kg. Y0 is the Y value when X (depth) is zero. It is expressed in 152 the same units as Y; Plateau is the Y value at infinite depths, expressed in the same units as Y; K 153 is the rate constant, expressed in reciprocal of the X axis depth units (mm-1); Half-depth is in the 154 depth units of the X axis. It is computed as ln(2)/K, which means the movement depth when the 155 concentration of P drop down to the half. 156 (1) Insert “(Figure 1)” 157 2.4 Experiment Ⅱ Soil incubation experiment 158 To explore the influences of different P types on phosphorus transformation, a 560-day 159 microcosm incubation experiment was set up. Herein only loam soil was used as described in the 160 previous section. In brief, a total of 253 g soil was thoroughly mixed with different P fertilizers PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed 161 (MAP, PA and APP) at the addition rate of 40.3 mg P pot-1. The mixed soil was placed in a 162 plastic pot (5.7 cm in height, 6.6 cm in diameter) with soil bulk density of 1.30 g cm-3, all pots 163 were incubated under room condition for 560 days. Tap water was added periodically per week 164 to keep water holding capacity of 60% (WHC). Each treatment was replicated four times giving 165 a total of 16 pots (including 4 CK treatments having no fertilizer). 166 After 65-, 140-, 230-, 320-, 560-day incubation, soil samples were collected from each pot. All 167 soil samples were air dried and grounded for determination of different soil inorganic P fractions 168 according to Guppy et al. (2000) and modified by Aulakh et al. (2003). Briefly, soil P sequential 169 extraction followed: (1) resin-P: an aliquot 0.500g soil sample was mixed with 30 mL deionized 170 water in a 500 mL centrifuge tube, two Cl-saturated anion exchange resin strips were added to 171 remove resin-P, the sample was shaken for 16 h. After shaking the two Cl-saturated anion 172 exchange resin strips were taken in a 100 mL centrifuge tube and 30 mL 0.7 moL/L NaCl was 173 added subsequently, shaking the sample for 1 h. (2) NaHCO3-P: the soil residue in the previous 174 step was treated with 30 mL 0.5 moL/L NaHCO3 to remove NaHCO3-P, shaking the sample for 175 stay overnight, and centrifugation for 30 min. (3) NaOH-P: the soil residue was treated with 30 176 mL 0.1 M NaOH and 1 mL of 4 M NaCl to remove NaOH-P, shaking the sample for 16 h, and 177 subsequently centrifugation for 30 min. (4) HCl-P: the soil residue was treated with 30 mL 178 1moL/L HCl, shaking stay over. (5) Residue-P: aliquot 0.5 g anhydrous MgSO4 and 5 mL of 179 H2SO4: HClO4 (20:1) acid solution to remove residue-P. Inorganic P concentration in each of the 180 extracts was determined using malachite green colorimetric method at an absorbance of 610 nm 181 (Masson et al., 2001). The total P content of soil samples was determined using molybdenum PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed 182 183 blue spectrophotometry method after digestion with HClO4-H2SO4 (Masson et al., 2001). The proportion of applied P fertilizer transformed to different P fractions in soils was figured 184 out referring to Aulakh et al. (2003). Individual parameters were calculated as 185 Amount of fertilizer P recovered as Pi in soil (kg P ha-1) 186 = [amount of Pi in P-fertilized treatment (kg P ha-1)] - [amount of Pi in no-P control (kg P ha-1)] 187 (2) 188 Total P recovered in soil (kg P ha-1) = [sum of P recovered in different fractions in the soils (kg P 189 ha-1) × (2.325×106 kg ha-1) 190 (3) Where 2.325×106 kg P ha-1 is soil mass of 0-150 mm soil layer computed using field bulk 191 density of 1.55 g cm-3. 192 Total fertilizer P recovered in soil (kg P ha-1) 193 = [total P recovered in P-fertilized treatment (kg P ha-1)] – [total P recovered in no-P control (kg 194 P ha-1)] 195 Fertilizer P present in any specific soil fraction (%) 196 = [(Pi in P - fertilized treatment ) - (Pi in no - P control )](kg P ha -1 ) Total fertilizer P recovered in soil (kg P ha -1 ) (4) (5) 197 2.5 Statistical analyses 198 Data were analyzed using the SPSS 11.5 statistical program (SPSS Inc., Chicago, USA) with 199 two-way ANOVA at a significance level of p < 0.05. A Duncan multiple range test was carried 200 out to test the significant differences between different treatments. Microsoft Excel 2003 and 201 Graphpad Prism 5.0 software (Graphpad software, Inc., USA) were used for data processing and PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed 202 images making. All results in figures and tables were presented as mean of three or four 203 replicates with a standard deviation (SD), a statistical significance level of p < 0.05 was used for 204 all analyses. 205 3. Results 206 3.1 Different P application methods on P mobility and availability 207 Soil Olsen-P decreased with soil depth (0-100 mm) increasing across all P sources (Fig. 2). 208 Compared to the single application treatment, the significant higher values of Olsen-P were 209 always appeared in the repeated PA and APP application at 0-40 mm depth (p < 0.05). The 210 vertical movement of Olsen-P reached the same value with CK (baseline) were 80 mm and 95 211 mm, respectively, in the PA and APP repeated application treatment, while those were merely 42 212 mm and 50 mm in the single application treatment in loam soil. Similar tendency was also 213 observed in clay soil, indicating that repeated P application significantly promoted P vertical 214 migration. 215 An exponential decay model was employed to describe Olsen-P movement in soil cylinder 216 (Table 2). Rate constant (K) and half-depth were key parameters to reflect the effects of different 217 application methods on P movement. The steeper the response curve (higher K value) and lower 218 half-depth, the shorter distance the added-P moved, and vice versa. In clay soil, K value (Rate 219 constant) in the repeated P application was lower than that in the single application treatment 220 across all treatments. The values of half-depth increased by 39.7%, 49.8%, 158.6% in the PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed 221 repeated application treatment, respectively, relative to the single MAP, PA and APP application 222 treatments. 223 Insert “(Figure 2, and Table 2)” 224 3.2 Different P types on P mobility and availability 225 Compared to the treatments of MAP and PA application, soil P mobility was significantly 226 increased by APP application (Fig. 3). The distance of WE-P downward movement in the single 227 P application treatment in loam soil followed order of APP (80 mm) > MAP (60 mm) > PA (35 228 mm). Similarly, the distance of WE-P downward movement in clay soil followed the order of 229 APP (83 mm) > MAP (62 mm) > PA (55 mm). Moreover, the exponential decay equation model 230 showed that K value in loam soil followed the order of APP (0.054 mm-1) < MAP (0.065 mm-1) 231 < PA (0.115 mm-1), and the half-depth followed the order of APP (12.70 mm) > MAP (10.66 232 mm) > PA (6.02 mm) when P fertilizer applied as single application method (Table 3). Likewise, 233 K value in the repeated application treatment (loam soil) followed order of APP (0.039 mm-1) < 234 MAP (0.044 mm-1) < PA (0.062 mm-1), the half-depth followed the sequence of APP (17.92 mm) 235 > MAP (15.88 mm) > PA (11.24 mm). The half-depth in the APP treatment were 4.84% and 236 31.5%, respectively, greater than the MAP and PA treated clay soils. These results indicate that 237 APP fertilization significantly improved P mobility and availability. 238 Insert “(Figure 3, and Table 3)” PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed 239 3.3 Inorganic P transformation 240 During whole incubation time, resin-P and NaHCO3-P (labile P) notably decreased, while 241 NaOH-P (moderately labile P) and HCl-P (recalcitrant soil P) showed an increasing tendency, 242 (Fig. 4). Furthermore, soil resin-P, NaHCO3-P and NaOH-P in the APP added treatment were 243 significantly higher than in the PA and MAP treatments. For instance, at the end of incubation 244 (560 d), resin-P (5.33 mg kg-1) and NaHCO3-P (73.7 mg kg-1) in the APP addition treatment 245 increased by 51.6% and 191% respectively, relative to the MAP and PA application treatments 246 (on average). In addition, When P fertilizer was added as polyphosphate, NaOH-P was increased 247 by 24.6% and 39.8%, respectively, relative to the MAP and PA treatments. The influence of the 248 different P types on non-labile P distribution proportion followed order of MAP (87.8%) > PA 249 (73.7%) > APP (54.5%) (Table 4), indicating that polyphosphate addition significantly retarded 250 the transformation of the added-P from labile to non-labile P forms, thus reduced P fixation Insert “(Figure 4, and Table 4)” 251 252 4. Discussion 253 Generally, short-chain soluble polyphosphate fertilizers are made up from different inorganic P 254 components at a certain proportion (i.e. ortho-P, pyro-P, triple-P, tetra-P). Therefore, poly-P 255 can’t be directly absorbed by plants unless it be hydrolyzed into orthophosphate (H2PO4- or 256 HPO42-) (Kulakovskaya et al., 2012). In this study, phosphorus downward movement in soil 257 column was significantly influenced by different types of P fertilizers (Table 2, Fig. 3). PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed 258 Application of slow release fertilizers (poly-P) significantly increased P movement. We found 259 that soil P vertical movement well-fitted the nonlinear exponential regression (0.81 < R2 < 0.99), 260 which provides a reliable evidence to our hypothesis that split repeated polyphosphate 261 application was superior over the single application method in increasing P mobility. Following 262 this, when P fertilizer is applied in single basal method, the freshly added-P is easily fixed due to 263 high P concentration in fertilization placement (Yang et al., 2012). A in situ imaging of liquid- 264 cell atomic force microscopy (AFM) showed that the formation of calcium phosphates (Ca-P) 265 nanoclusters was markedly increased as P presented at high concentration [50 mM (NH4)2HPO4] 266 (Wang and Putnis, 2020). Besides, the direction of P diffusion from P application site to outside 267 is opposite to the direction of water movement caused by low soil water potential (Hedley & 268 McLaughlin, 2005). Therefore, the diffusion of H2PO4-/HPO42- would inevitably be blocked by 269 water movement (Hinsinger, 2001). In contrast, when P fertilizer is applied though split repeated 270 method, the concentration of P in P application site is relative low, resulted in the distribution P 271 in soil was scattering and dispersive (Hettiarachchi et al., 2006; McLaughlin et al., 2011). Our 272 results were consistent with the findings by Lombi et al. (2004) who noted that fluid sources of P 273 enhanced phosphorus mobility and liability compared with granular P fertilizers. The reasons 274 may likely attributed to: (і) poly-P fertilizers belong to slow-release fertilizer, they move in 275 condensed form in soil before it be hydrolyzed (McBeath et al., 2007), and it hardly interacted 276 with soil mineral particles of Fe2O3/Al2O3 (Hamilton et al., 2016; Hamilton et al., 2018); (іі) 277 poly-P may mobilize soil native P via chelating reaction. In this way, Mcbeath et al. (2007) 278 showed that poly-P activated recalcitrant oxidation forms of soil P (Fe2O3 and MnO2). Similarly, PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed 279 Wang et al. (2019) found that poly-P fertilizer treatment significantly increased P availability in 280 calcareous soil. Besides, poly-P fertilizer application decrease soil rhizosphere pH value at 0.1- 281 0.5 unit (Hamilton et al., 2018; Rahmatullah et al., 2006; Mcbeath et al., 2007: Du et al., 2013). 282 Likewise, our data showed that the poly-P fertilizer application treatment decreased solution pH 283 by ~0.34 unit than MAP and PA fertilizer (Table 5), which might be another possible reason for 284 polyphosphate mobilizing native soil P. Although the effects of poly-P applied to agricultural 285 soils on increasing soil P availability and reducing its fixation is promising, nevertheless further 286 study is needed by using edge-cutting technologies to fully elucidate the mechanisms of 287 mobilizing effect by poly-P application and their interaction with soil particles in a wide-range of 288 soil conditions. Insert “(Table 5)” 289 290 5. Conclusion 291 The mobility, effectiveness, and availability of fertilizer P were significantly increased with 292 ammonium-polyphosphate application compared with MAP and PA application. Compared to P 293 fertilizer basal application, split repeated P application markedly promoted soil P movement. 294 Moreover, the added-P transformation from labile (resin-P and NaHCO3-P) to non-labile forms 295 (HCl-P and residue-P) in the poly-P treated soil was significantly retarded, in contrast to MAP 296 and PA application treatments. Therefore, considering the scarcity of P resource and low P 297 fertilizer use efficiency, polyphosphate fertilizers coupled with split repeated application method 298 is recommended as an effective P management strategy in increasing soil available P decreasing PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed 299 P fixation in calcareous soil. 300 301 Funding 302 This work was jointly supported by National Natural Science Foundation of China (No. 303 41161047), and the scientific development and technology innovation project of Xinjiang 304 Production and Construction Group (XPCG), China (No. 2017BA041) and the Shenzhen Batian 305 ecological engineering Co., Ltd. 306 307 Competing Interests 308 The authors declare that they have no competing interests. 309 310 Acknowledgement 311 Thanks are given to Dr. & Prof. Wakelin A Steve from New Zealand for his helpful comments 312 and revision. 313 314 Author Contributions 315 Jawad Ali SHAH: methodology, sampling, measurement, data collection and analysis writing- 316 original draft, interpretation, and approved the final draft. 317 CHU Gui-xin: project administration, conceptualization, experiment-design, supervision, 318 funding acquisition, resources, critical manuscript revision, and approved the final draft. 319 Data Availability PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed 320 The following information was supplied regarding data availability: 321 The raw data is available as a Supplemental File. 322 323 References 324 Aulakh MS, Kabba BS, Baddesha HS, Bahl GS, Gill MPS. 2003. 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Global Change Biology 18:1904-1917 DOI 385 10.1111/j.1365-2486.2012.02653.x. 386 Masson P, Morel C, Martin E, Oberson A, Friesen D. 2001. Comparison of soluble P in soil water extracts 387 determined by ion chromatography, colorimetric, and inductively coupled plasma techniques in PPB 388 range. Communications of Soil Science and Plant Analysis 32:2241–2253 DOI 10.1081/CSS-120000280. 389 McBeath TM, Armstrong RD, Lombi E, McLaughlin MJ, Holloway RE. 2005. Responsiveness of wheat 390 (Triticum aestivum) to liquid and granular phosphorus fertilisers in southern Australian soils. Australian 391 Journal of Soil Research 43:203–212 DOI 10.1071/SR04066. 392 Mcbeath TM, Lombi E, Mclaughlin MJ, Bünemann EK. 2007. Polyphosphate fertilizer solution stability 393 with time, temperature, and pH. Journal of Plant Nutrition and Soil Science 170:387–391 DOI 394 10.1002/jpln.200625166. 395 Mclaughlin MJ, Mcbeath TM, Smernik R, Stacey SP, Ajiboye B, Guppy C. 2011. The chemical nature of 396 p accumulation in agricultural soils; implications for fertilizer management and design: an Australian 397 perspective. Plant and Soil 349:69–87 DOI 10.1007/s11104-011-0907-7. 398 399 Mueller ND, Gerber JS, Johnston M, Ray DK, Ramankutty N, Foley JA. 2012. Closing yield gaps through nutrient and water management. Nature 490:254–257 DOI 10.1038/nature11420. 400 Olsen SR, Cole CV, Watanable FS, Dean L. 1954. Estimation of Available Phosphorus in Soils by 401 Extraction with Sodium Bicarbonate. Washington, DC: United States Department of Agriculture, Circular 402 Nr. 939. 403 Ottman MJ, Thompson TL, Doerge TA. 2006. Alfalfa yield and soil phosphorus increased with topdressed PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed 404 granular compared with 405 10.2134/agronj2005.0257. fluid phosphorus fertilizer. Agronomy Journal 98:899–906 DOI 406 Rahmatullah GMA, Wissemeier AH, Steffens D. 2006. Phosphate availability from phosphate rock as 407 related to nitrogen form and the nitrification inhibitor DMPP. Journal of Plant Nutrition and Soil Science 408 169:675–678 DOI 10.1002/jpln.200520577 409 Saarela I, Salo Y, Vuorinen M. 2006. Effects of repeated phosphorus fertilisation on field crops in Finland I. 410 Yield responses on clay and loam soils in relation to soil test P values. Agricultural and Food Science 411 15:106–123 DOI 10.2137/145960606778644548. 412 413 Shaw K. 1959. Determination of organic carbon in soil and plant material. European Journal of Soil Science 10:316–326 DOI 10.1111/j.1365-2389.1959.tb02353.x. 414 Stutter MI, Shand CA, George TS, Blackwell MSA, Bol R, MacKay RL, Richardson AE, Condron LM, 415 Turner BL, Haygarth PM. 2012. Recovering phosphorus from soil: a root solution? Environmental 416 Science & Technology 46:1977–1978 DOI 10.1021/es2044745. 417 Wang J, Chu G. 2015. Phosphate fertilizer form and application strategy affect phosphorus mobility and 418 transformation in a drip-irrigated calcareous soil. Journal of Plant Nutrition and Soil Science 178:914– 419 922 DOI 10.1002/jpln.201500339. 420 Wang L, Putnis CV. 2020. Dissolution and precipitation dynamics at environmental mineral interfaces 421 imaged by in situ atomic force microscopy. Accounts of Chemical Research 53:1196-1205 DOI 422 10.1021/acs.accounts.0c00128. 423 Wang X, Gao Y, Hu B, Chu G. 2019. Comparison of the hydrolysis characteristics of three polyphosphates 424 and their effects on soil phosphorus and micronutrient availability. Soil Use and Management 35:664-674 PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed 425 426 427 DOI 10.1111/sum.12526. Weihrauch C, Opp C. 2018. Ecologically relevant phosphorus pools in soils and their dynamics: The story so far. Geoderma 325:183–194 DOI 10.1016/j.geoderma.2018.02.047. 428 Yang J, Wang Z, Zhou J, Jiang H, Zhang J, Pan P, Han Z, Lu C, Li L, Ge C. 2012. Inorganic phosphorus 429 fractionation and its translocation dynamics in a low-P soil. Journal of Environmental Radioactivity 430 112:64–69 DOI 10.1016/j.jenvrad.2012.03.011. 431 Zhang W, Zhan X, Zhang S, Ibrahima KHM, Xu M. 2019. Response of soil Olsen-P to P budget under 432 different long-term fertilization treatments in a fluvo-aquic soil. Journal of Integrative Agriculture 433 18:667–676 DOI 10.1016/S2095-3119(18)62070-2. 434 435 PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed Table 1(on next page) Selected physical and chemical properties of the tested soils PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed Table 1. 1 2 3 4 5 6 7 8 9 Soil properties Texture a (<0.01mm %) pH b EC c OM d (g kg-1) Total-N e(g kg-1) Olsen-P f (mg kg-1) Water-P g (mg kg-1) Total-P h (g kg-1) Olsen-K i (mg kg-1) CaCO3 j (%) Loam 37.0±0.02 7.83±0.03 0.53±0.01 21.0±0.23 0.85±0.05 24.2±3.52 3.54±0.39 1.22±0.12 518±2.07 16.1±0.00 Clay 71.4±0.02 8.15±0.05 1.22±0.02 15.6±0.03 0.71±0.02 11.9±2.18 3.58±0.01 0.95±0.01 291±5.53 15.0±0.00 10 the mean ± standard deviation (SD), n=3 at a significance level of p < 0.05. 11 aTexture 12 bpH 13 cSoil 14 dOrganic 15 eSoil 16 fOlsen 17 gWater-P 18 hTotal P was measured by the perchloric acid digestion method (Olsen et al. 1954). 19 iOlsen K was measured by the flame photometry method (Bao 2000). 20 jCaCO 3 Selected physical and chemical properties of the tested soils Data presented was determined by the Bouyoucos hydrometer method and Katschinski classification System (Bouyoucos 1962). was determined at soil to milli-Q water ratio of 1:2.5 w/v using pH meter EC was measured using a 1:2.5 ratio of soil to Milli-Q water. C was measured by the wet-oxidation technique (Shaw 1959). total N was measured by the semimicro-Kjeldahl method; (Bao 2000). P was measured by the Olsen method (Olsen et al. 1954). was measured using a 1:25 ratio of soil to milli-Q water. was measured by neutral titration method (Bao 2000). PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) were as Manuscript to be reviewed 21 PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed Table 2(on next page) Exponential equations simulating the influences of different P fertilization methods on soil P downward movement (Olsen-P, mg kg-1) PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed Table 2. Exponential equations simulating the influences of different P fertilization methods on soil P downward movement (Olsen-P, mg kg-1) 1 P form MAP Application method Single Repeated Single Repeated PA Loam soil Clay soil Equation y = 192.03e-0.059x+19.07 K (mm-1) 0.059 Half-depth (mm) 11.8 R2 0.97 Equation y = 216.05e-0.056x+10.45 K (mm-1) 0.056 Half-depth (mm) 12.49 R2 0.98 y = 144.70e-0.053x+24.70 y = 152.68e-0.105x+17.12 0.053 0.105 13.1 6.57 0.94 0.87 y = 155.72e-0.040x+7.38 y = 153.98e-0.059x+13.12 0.040 0.059 17.45 11.72 0.96 0.98 y = 181.36e-0.063x+20.94 y = 187.05e-0.056x+17.25 0.063 0.056 10.9 12.3 0.90 0.97 y = 151.40e-0.040x+8.10 y = 286.92e-0.060x+10.58 0.040 0.060 17.56 11.51 0.97 0.81 0.023 29.77 0.94 Single Repeated y = 785.95e-0.044x+12.65 0.044 15.9 0.99 y =471.57e-0.023x-40.17 Data were presented as the mean value of three replicates and standard deviation (SD) at a significance level of p < 0.05. APP 2 PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed Table 3(on next page) Exponential equations simulating the influences different P sources on phosphorus downward movement (water-P, mg kg-1) PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed 1 Table 3. Exponential equations simulating the influences different P sources on phosphorus downward movement (water-P, mg kg-1) Application method P form Loam soil Clay soil Equation y = 192.21e-0.065x+12.79 K (mm-1) 0.065 Half-depth (mm) 10.7 R2 0.97 Equation y = 104.94e-0.041x+9.56 K (mm-1) 0.041 Half-depth (mm) 16.72 R2 0.94 y =163.38e-0.115x+12.02 y = 410.54e-0.054 x +12.96 y = 95.8e-0.044x+15.30 0.115 0.054 0.044 6.02 12.7 15.9 0.92 0.95 0.92 y = 89.25e-0.052x+14.25 y = 182.075e-0.040x+8.53 y =98.48e-0.031x+6.02 0.052 0.040 0.031 13.33 17.53 22.43 0.93 0.96 0.93 PA y = 153.71e-0.062x+14.29 0.062 11.2 0.93 y = 90.86e-0.031x+8.94 APP y = 496.43e-0.039x+5.87 0.039 17.9 0.97 y =334.01e-0.014x-80.51 Data were presented as the mean value of three replicates and standard deviation (SD) at a significance level of p < 0.05. 0.031 0.014 22.47 50.93 0.91 0.96 MAP Single PA APP MAP Repeated 2 3 PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed Table 4(on next page) The influences of MAP, PA and APP applications on different inorganic P fractionations in soil (after 560-day incubation) PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed 1 Table 4. The influences of MAP, PA and APP applications on different inorganic P fractionations in soil (after 2 560-day incubation) 3 4 5 Treatment Resin-P (%) NaHCO3-P (%) NaOH-P (%) HCl-P (%) Residue-P (%) MAP PA APP 0.23±0.06 1.07±0.03 1.64±0.15 6.13±0.2 20.0±0.1 36.7±0.4 5.82±0.8 5.19±0.1 7.25±0.9 21.7±0.6 58.2±0.9 45.4±0.5 66.1±1.0 15.5±0.8 9.10±0.1 Abbreviation: MAP, mono-ammonium phosphate; PA, phosphoric acid; APP, ammonium polyphosphate. Data were presented as the mean value of four replicates and standard deviation (SD) at a significance level of p < 0.05. PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed Table 5(on next page) pHs in MAP, PA and APP fertilizer and mixed with soils. pHs in MAP, PA and APP fertilizer and mixed with soils. PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed 1 2 3 4 5 6 7 8 9 Table 5. pHs in MAP, PA and APP fertilizer and mixed with soils. Treatment pH (fertilizer) pH (fertilizer mixed in loam soil) pH (fertilizer mixed in clay soil) MAP PA APP 5.15±0.08 2.01±0.05 1.36±0.02 7.81±0.07 7.99±0.09 7.55±0.06 8.37±0.03 8.38±0.09 8.06±0.05 Abbreviation: MAP, mono-Ammonium phosphate; PA, phosphoric acid; APP, ammonium polyphosphate. 10 PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed Figure 1 Schematic diagram of soil cylinder experiment. PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed Figure 2 The effect of different P application methods on phosphorus mobility and availability. Data are presented as mean ± standard deviation (SD) of three replicates at a significance level of p < 0.05. The three lines represent MAP, PA and APP, respectively. T PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed Figure 3 The influences of different P sources on soil phosphorus mobility and availability. Data are presented as mean ± standard deviation (SD) of three replicates at a significance level of p < 0.05. The upper and lower rows represent single application and rep PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed Figure 4(on next page) The effect of different P sources and their application methods on the changes of soil resin-P, NaHCO3-P, NaOH-P, HCl-P and residue-P. Data are presented as mean ± standard deviation (SD) of four replicates at a significance level of p < 0.05 (n PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed Fig. 4 PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) Manuscript to be reviewed PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021) 

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Journal of Learning Development in Higher Education ISSN: 1759-667X Special Issue 22: Compendium of Innovative Practice October 2021 ________________________________________________________________________ Watch party lectures: synchronous delivery of asynchronous material Carolina E. Kuepper-Tetzel University of Glasgow Emily Nordmann University of Glasgow Keywords: instruction; lectures; synchronous and asynchronous learning; student engagement; Covid-19 The challenge When the Covid-19 pandemic hit in early 2020, the majority of university activities on campus were suspended across the world, and teaching pivoted online (Nordmann et al., 2020). The challenge was to provide students who were enrolled and expecting a face-to-face, oncampus learning experience with a similar university experience that would encourage them to engage with learning and to develop a sense of belonging (Peacock et al., 2020). As year leads for first- and second-year Psychology at our university, our cohorts were particularly large with approximately 600 first-year students and 250 second-year students. Each week students attend three to five lectures and one practical lab session, and previously all teaching was delivered in person on campus. In the process of planning our move online, we realised the need to develop an approach that offered both asynchronous and synchronous elements. Synchronous sessions are vital, not only for community building, but there is also a positive association between scheduled teaching of lectures and practicals and academic achievement (Torenbeek et al., 2012). However, with students studying from home in different countries, under a variety of circumstances, and with differential access to internet, study space and technology, it was important to offer all essential teaching material asynchronously too (Nordmann et al., 2020). Kuepper-Tetzel and Nordmann Watch party lectures: synchronous delivery of asynchronous material In the initial period of emergency remote teaching directly following the first lockdown, we kept in touch with students through online remote social activities such as meeting online for a watch party of psychology-related movies and used the integrated chat box function to write comments or thoughts. This was when the concept of ‘watch party lectures’ was born. The response Watch party lectures combine asynchronous and synchronous teaching approaches. Lecturers record short videos of directly instructed content; for one-hour lectures, lecturers aim to record 2-3 short videos for a maximum of 40-45 minutes. These pre-recorded videos are uploaded onto the virtual learning environment (VLE). However, the crucial element of watch party lectures is the scheduled, synchronous time during which these pre-recordings are watched together via screen sharing. Watch parties purposely differ from a flipped classroom approach in which students are required to complete the pre-recordings in their own time before coming to class. While, in general, the flipped classroom approach has shown positive effects on student performance across educational sectors and disciplines (Strelan et al., 2020), students, particularly younger cohorts, may become overwhelmed by the added requirement of independent learning and, consequently, not complete the learning activities prior to class (Tomas et al., 2019). This may be more pronounced now as students report additional strains on mental health and wellbeing during the pandemic (e.g., Chirikov et al., 2020; Mechili et al., 2020; Wang et al., 2020). Thus, watch party lectures offer a scheduled time during which the core teaching content is delivered. Perhaps more importantly, watch party sessions allowed for meaningful interactions between lecturers and students while watching the videos. Students would post questions or comments in the chat box, and the lecturer could address them immediately, for example, by providing links to additional resources. We observed more engagement during watch party lectures from a more diverse body of students than we have experienced in face-to-face lectures, which reflects students’ preference for using the chat box during instruction (see Vu and Fadde, 2013). Collaborative co-creation of knowledge that combines social and cognitive Journal of Learning Development in Higher Education, Issue 22: Compendium October 2021 2 Kuepper-Tetzel and Nordmann Watch party lectures: synchronous delivery of asynchronous material processes aligns well with the theoretical framework of Community of Inquiry (Garrison et al., 2001; Cleveland-Innes et al., 2019); and is an essential aspect of watch party lectures. Discussion took place not only between lecturers and students, but also between peers who would frequently answer questions or provide follow-up opinions and thoughts to points raised in the chat. Besides the ongoing discussions, lecturers enriched the watch party sessions with short quiz activities, either to review previously taught material or to prompt upcoming content. Quizzing during lectures not only provides students with a short break from watching the videos, but is also a beneficial strategy to increase retention and transfer of knowledge (Thomas et al., 2020). Although multiple platforms would be capable of presenting watch parties, we used Zoom (Zoom Video Communications Inc, 2016). In addition to being adopted as a core technology by our institution, Zoom had no known restrictions in terms of international access and was also available for students on mobile devices, and we were not made aware of any cases in which students who wished to engage with the sessions were not able to do so. Recordings of the watch party sessions including the chat conversations were also made available to students for asynchronous engagement, although the community and active learning benefits of the sessions would have been limited. The feedback from students on the watch party lectures has been overwhelmingly positive, for example: I like that all lectures are watch party style, it makes it feel more interactive and 'normal’. None of my other subjects have done this so every week I look forward to my psychology lectures. (Anonymous, 2020a) Online live lecture watch parties are amazing because it feels a lot more engaging than just having to watch the already recorded lecture alone. (Anonymous, 2020b) In student evaluations, our approach was praised as an effective way to engage with the material, ask questions during the session, and catch-up afterwards. Students appreciated Journal of Learning Development in Higher Education, Issue 22: Compendium October 2021 3 Kuepper-Tetzel and Nordmann Watch party lectures: synchronous delivery of asynchronous material the different activities during the watch party sessions, citing that these kept them focused and encouraged further elaboration on the topic. As expected, students highlighted the chat box conversations as a positive feature of watch party lectures. Student feedback was so positive that the format was adopted by other lecturers and departments at both our own and other institutions. Recommendations Lecturers interested in implementing this approach may experience an initial hesitation about watching themselves during the watch party session, but from our own experiences and from speaking to our colleagues, this was not perceived as an issue, or was one that quickly became normal. Lecturers enjoyed the interactions in the chat box, which allowed them time to construct more elaborate answers to student questions. When moving back to face-to-face lectures, we recommend integrating text-based chats during lectures. Lecturers could encourage students to post comments and questions during the lecture using existing tools (e.g., Teams, forums on the VLE) and then take brief Q&A breaks during the lecture. After the lecture, the lecturer could reply to questions that require more elaborate answers or additional resources. In conclusion, watch party lectures demonstrate the benefits of strategically combining asynchronous and synchronous approaches. When considering what the new normal of lectures will look like, it is crucial to consider their social and community aspects (Nordmann et al., 2021), and we strongly recommend integrating both asynchronous and synchronous elements regardless of the modality or format of teaching delivery. References Chirikov, I., Soria, K.M., Horgos, B, and Jones-White, D. (2020) ‘Undergraduate and graduate students’ mental health during the COVID-19 pandemic’, UC Berkeley: Center for Journal of Learning Development in Higher Education, Issue 22: Compendium October 2021 4 Kuepper-Tetzel and Nordmann Watch party lectures: synchronous delivery of asynchronous material Studies in Higher Education, viewed 3 June 2021. Available at: https://escholarship.org/uc/item/80k5d5hw (Accessed: 30 June 2021). Cleveland-Innes, M., Gauvreau, S., Richardson, G., Mishra, S. and Ostashewski, N. (2019) ‘Technology-enabled learning and the benefits and challenges of using the Community of Inquiry theoretical framework’, International Journal of E-Learning & Distance Education, 34(1), pp.1-18. Garrison, D.R., Anderson, T. and Archer, W. (2001) ‘Critical thinking, cognitive presence, and computer conferencing in distance education’, American Journal of Distance Education,15(1), pp. 7-23. Available at: https://doi.org/10.1080/08923640109527071 (Accessed: 30 June 2021). Mechili, E.A., Saliaj, A., Kamberi, F., Girvalaki, C., Peto, E., Patelarou, A.E., Bucaj, J. and Patelarou, E. (2020) ‘Is the mental health of young students and their family members affected during the quarantine period? Evidence from the COVID‐19 pandemic in Albania’, Journal of Psychiatric and Mental Health Nursing, 28(3), pp.317-325. Available at: https://doi.org/10.1111/jpm.12672 (Accessed: 5 July 2021). Nordmann E., Horlin, C., Hutchison, J., Murray, J.A., Robson, L., Seery, M.K. and MacKay, J.R.D. (2020) ‘Ten simple rules for supporting a temporary online pivot in higher education’, PLoS Computational Biology, 16(10), pp.e1008242. Available at: https://doi.org/10.1371/journal.pcbi.1008242 (Accessed: 5 July 2021). Nordmann, E., Hutchison, J. and MacKay, J.R.D. (2021) ‘Lecture rapture: the place and case for lectures in the new normal’, EdArXiv. Available at: https://doi.org/10.35542/osf.io/h8zt6 (Accessed: 5 July 2021). Peacock, S., Cowan, J., Irvine, L. and Williams, J. (2020) ‘An exploration into the importance of a sense of belonging for online learners’, The International Review of Research in Journal of Learning Development in Higher Education, Issue 22: Compendium October 2021 5 Kuepper-Tetzel and Nordmann Watch party lectures: synchronous delivery of asynchronous material Open and Distributed Learning, 21(2), pp. 18-35. Available at: https://doi.org/10.19173/irrodl.v20i5.4539 (Accessed: 5 July 2021). Torenbeek, M., Jansen, E. and Suhre, C. (2013) ‘Predicting undergraduates' academic achievement: the role of the curriculum, time investment and self-regulated learning’, Studies in Higher Education, 38(9), pp. 1393-1406. Available at: https://doi.org/10.1080/03075079.2011.640996 (Accessed: 5 July 2021). Strelan, P., Osborn, A. and Palmer, E. (2020) ‘The flipped classroom: a meta-analysis of effects on student performance across disciplines and education levels’, Educational Research Review, 30, pp. 100314. Available at: https://doi.org/10.1016/j.edurev.2020.100314 (Accessed: 5 July 2021). Thomas, A.K., Smith, A.M., Kamal, K. and Gordon, L.T. (2020) ‘Should you use frequent quizzing in your college course? Giving up 20 minutes of lecture time may pay off’, Journal of Applied Research in Memory and Cognition, 9(1), pp. 83-95. Available at: https://doi.org/10.1016/j.jarmac.2019.12.005 (Accessed: 5 July 2021). Tomas, L., Doyle, T. and Skamp, K. (2019) ‘Are first year students ready for a flipped classroom? A case for a flipped learning continuum’, International Journal of Educational Technology in Higher Education, 16(1), pp.1-22. Available at: https://doi.org/10.1186/s41239-019-0135-4 (Accessed: 5 July 2021). Wang, X., Hegde, S., Son, C., Keller, B., Smith, A. and Sasangohar, F. (2020) ‘Investigating mental health of US college students during the COVID-19 pandemic: cross-sectional survey study’, Journal of Medical Internet Research, 22(9), pp. e22817. Available at: https://doi.org/10.2196/22817 (Accessed: 5 July 2021). Vu, P. and Fadde, P.J. (2013) ‘When to talk, when to chat: student interactions in live virtual classrooms’, Journal of Interactive Online Learning, 12(2), pp.41-52. Available at: http://www.ncolr.org/jiol/issues/pdf/12.2.1.pdf (Accessed: 5 July 2021). Journal of Learning Development in Higher Education, Issue 22: Compendium October 2021 6 Kuepper-Tetzel and Nordmann Watch party lectures: synchronous delivery of asynchronous material Zoom Video Communications Inc. (2016) Security guide. Available at: https://d24cgw3uvb9a9h.cloudfront.net/static/81625/doc/Zoom-Security-WhitePaper.pdf (Accessed: 30 June 2021). Author details Carolina E. Kuepper-Tetzel is a Senior Lecturer in Psychology at the University of Glasgow and a Senior Fellow of the HEA. She is an expert in applying findings from cognitive science to education and an enthusiastic science communicator. She is a member of The Learning Scientists and the founder of the Teaching Innovation & Learning Enhancement (TILE) network. You can follow her work on Twitter: @pimpmymemory. Emily Nordmann is a Senior Lecturer in Psychology at the University of Glasgow and was awarded the British Psychological Society HE Psychology Teacher of the Year in 2020. She is an expert in lecture capture and a strong proponent of open educational resources and the teaching of reproducible research methods. You can follow her work on Twitter: @emilynordmann. Journal of Learning Development in Higher Education, Issue 22: Compendium October 2021 7 

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materials Article A Parameter Study for 3D-Printing Organized Nanofibrous Collagen Scaffolds Using Direct-Write Electrospinning Frank A. Alexander Jr. 1, *, Lee Johnson 2, *, Krystaufeux Williams 3 and Kyle Packer 4 1 2 3 4 * The Geneva Foundation, Tacoma, WA 98402, USA Meadowave LLC, NE Washington, DC 20012, USA Chemistry Division, Center for Corrosion Science and Engineering (CCSE), US Naval Research Laboratory, SW Washington, DC 20375, USA; krystaufeux.williams@nrl.navy.mil Womack Army Medical Center, Fort Bragg, NC 28310, USA; kyle.t.packer.mil@mail.mil Correspondence: falexander@genevausa.org (F.A.A.J.); ljohnson@meadowave.com (L.J.) Received: 1 November 2019; Accepted: 27 November 2019; Published: 10 December 2019



 Abstract: Collagen-based scaffolds are gaining more prominence in the field of tissue engineering. However, readily available collagen scaffolds either lack the rigid structure (hydrogels) and/or the organization (biopapers) seen in many organ tissues, such as the cornea and meniscus. Direct-write electrospinning is a promising potential additive manufacturing technique for constructing highly ordered fibrous scaffolds for tissue engineering and foundational studies in cellular behavior, but requires specific process parameters (voltage, relative humidity, solvent) in order to produce organized structures depending on the polymer chosen. To date, no work has been done to optimize direct-write electrospinning parameters for use with pure collagen. In this work, a custom electrospinning 3D printer was constructed to derive optimal direct write electrospinning parameters (voltage, relative humidity and acetic acid concentrations) for pure collagen. A LabVIEW program was built to automate control of the print stage. Relative humidity and electrospinning current were monitored in real-time to determine the impact on fiber morphology. Fiber orientation was analyzed via a newly defined parameter (spin quality ratio (SQR)). Finally, tensile tests were performed on electrospun fibrous mats as a proof of concept. Keywords: electrospinning; collagen; direct-write electrospinning; scaffolds 1. Introduction Electrospinning is a well-established technique for creating nanofibers that can be used for a variety of applications. The core technique was first demonstrated more than a century ago [1]; however, recent interest in biomaterials has caused a resurgent interest in it as a method for developing scaffolds for regenerative medicine and wound dressings [2]. In a classic far-field electrospinning (FFES) setup, a syringe pump perfuses a polymer solution through a conductive syringe tip to a grounded collector with a high voltage power supply. When enough voltage is applied between the syringe and collector circuit, charge buildup in the viscoelastic fluid pumped through the syringe deforms the meniscus into a “Taylor cone” and a nanofiber jet erupts from the tip of the cone when the electrical force overcomes surface tension. As the nanofiber jet is drawn closer to the surface, the carrier liquid evaporates and nanofibers form. Dried fibers accumulate onto the grounded collector electrode surface located below the syringe. Typically, in FFES the syringe tip is held a large distance (centimeters) from a target. This results in nanofibers collecting in random “pile of spaghetti”-like orientations. Alongside bioprinted hydrogels [3], Materials 2019, 12, 4131; doi:10.3390/ma12244131 www.mdpi.com/journal/materials Materials 2019, 12, 4131 2 of 14 unorganized “biopaper” scaffolds [4] have been popular due to their ease of production and ability to recapitulate biochemical cues seen in vivo by using natural polymers and a nanoporous structure. However, these systems still lack critical microarchitecture especially when creating tissue analogues of more ordered nanofibrous structures [5]. The lack of systematically organized fibrils may result in insufficient mechanical properties of these artificial tissues. Some groups have designed bioreactors to physically stimulate cellular production of organized collagen structures but production times can be extensive [6]. Leveraging additive manufacturing techniques to produce more similar tissue-specific analogues may increase utility. Various groups have developed methods to finely pattern electrospun nanofibers into organized structures. Fibers have been directed onto spinning mandrel collectors to create fiber spools [7], micropatterned via insulator-collector geometries to induce specific shapes [8] and positioned in precise locations by rapidly translating the collector with an X-Y stage [9–12]. The latter technique, sometimes termed direct-write electrospinning (DWES) is a relatively new additive manufacturing approach to electrospinning into highly organized confirmations. DWES significantly lowers the required voltage to induce Taylor cone formation by decreasing the syringe tip to collector distance and incorporates a translating X-Y stage to pattern collected nanofibers. Li et al. successfully lowered spinning voltages to thresholds as small as 50 V, enabling them to electrospin living bacteria immersed in a polyethylene oxide-water solution [10]. Furthermore, they demonstrated the ability to electrospin directly onto hydrogels and PDMS films as well as electrospin freestanding fibers across two spin “initiators.” [8]. Polymer melts have been elecrospun in melt electro-writing (MEW) to create organized fibrous scaffolds, but they are incompatible with heat sensitive polymers like collagen [13–18]. Additionally, varying polymer solutions and custom equipment make it is necessary to re-optimize methods across laboratories. This drawback is the primary disadvantage preventing DWES from being a scalable method for scaffold manufacturing. Natural, synthetic, and even blended polymer biomaterials [2] have been employed in electrospinning to generate nanofiber constructs that imitate native extracellular matrix (ECM) for tissue regeneration [2]. Collagen is particularly interesting as a scaffolding material because it is highly abundant within mammalian ECM. Of the 29 types of native collagen, Type I makes up 70–90% of collagen within the human body and can be found in tightly organized fiber networks in tissues like the meniscus [19,20]. Despite this, instead of electrospinning pure collagen into organized nanofibrous collagen scaffolds, the majority of researchers have opted for hydrogels or nanofibrous scaffolds with random orientation. These scaffolds are suitable in many applications, however, in many tissues precise fiber organization results in unique in vivo properties like transparency in the cornea [21]. For this reason, DWES is a superior method for producing organized collagen scaffolds. Despite the extensive work to optimize parameters for traditional collagen electrospinning, organized collagen scaffolds have yet to be produced via DWES. Leveraging DWES as a 3D printing approach can help to further develop more complex and biomimetic scaffolds that mimic the native environment of cells or introduce bio-bandages that accelerate wound healing. A critical and often overlooked step in electrospinning is determining optimal environmental parameters for nanofiber production, as process temperature and relative humidity can directly impact fiber morphology. For this reason, it is critical to optimize a consistent production environment for repeatable fiber making in order to scale for large-scale manufacturing of scaffolds. Many prior groups have done exhaustive studies on solution characteristics such as solvent composition [22] but relatively few have done exhaustive studies on environmental parameters. To our knowledge, no such studies on environmental parameters have been performed for direct-write electrospinning of pure collagen. Here, we present a systematic approach to optimizing critical parameters for DWES of collagen employing an environmental chamber for strict monitoring of relative humidity during the electrospinning process. Our printer, capable of manufacturing highly ordered 3D printed scaffolds from nanofibrous collagen, was evaluated by optimizing electrospinning characteristics of collagen across an array of varied process parameters including spin voltage, relative humidity and acetic acid Materials 2019, 12, 4131 3 of 14 concentration. The print procedure was automated using custom LabVIEW software built to control translation of the print stage and import common G-code files. Additionally, process parameters were monitored in real-time to assess the impacts of small-scale voltage and humidity fluctuations effect fiber morphology and placement. Electrospun collagen fibers were examined to ascertain the morphology of2019, nanofibers produced Materials 12, x FOR PEER REVIEW and the quality of fiber placement which we have defined 3 of 14 as the spin quality ratio (SQR). Finally, to demonstrate their mechanical properties we have tensile tested the 96 monitored in real-timefiber to assess the impacts of small-scale voltage and humidity fluctuations effect manufactured nanofibrous mats. 97 fiber morphology and placement. Electrospun collagen fibers were examined to ascertain the morphology of nanofibers produced and the quality of fiber placement which we have defined as the 2. 98 Materials and Methods 99 spin quality ratio (SQR). Finally, to demonstrate their mechanical properties we have tensile tested 100Electrospinning the manufactured nanofibrous 2.1. Printer Setup fiber mats. 101An 2. Materials and Methods electrospinning 3D printer was designed for all collagen fiber production experiments. The electrospinning printer contains four main components: an X-Y stage, a syringe pump, a high voltage 102 2.1. Electrospinning Printer Setup DC power supply and a Z-positioner. Two DC servo linear translation stages (ThorLabs, Newton, NJ, 103 were bolted An electrospinning 3D printer was designed for all collagen fiber production experiments. USA) together to serve as the main X-Y positioner. Attached to the X-Y positioner,The a custom 104 electrospinning printer contains four main components: an X-Y stage, a syringe pump, a high voltage stage (3D printed with a Form2 from FormLabs, Somerville, MA, USA) houses an integrated ground 105 DC power supply and a Z-positioner. Two DC servo linear translation stages (ThorLabs, Newton NJ) plane gold-coated Thepositioner. ground plane is to connected to a metal microscope 106 made were from boltedatogether to serveglass as theslide. main X-Y Attached the X-Y positioner, a custom slide is clipped the negative terminal of a PS300 voltage powerground supply (Stanford 107 holder stagethat (3D printed withto a Form2 from FormLabs, Somerville MA)high houses an integrated plane 108 made from Sunnyvale, a gold-coatedCA, glass slide.An TheNE-1000 ground plane is connected to a Era, metalFarmingdale, microscope slide Research Systems, USA). syringe pump (New NY, USA) 109 holder that is clipped to the negative terminal of a PS300 high voltage power supply (Stanford was used to drive collagen solution from a 3 mL syringe to a 27-gauge needle tip mounted to a third 110 Research Systems, Sunnyvale An NE-1000 A syringe pumpInstruments (New Era, Farmingdale was used with screw-driven translation stage for CA). Z-positioning. National PXIe-1071NY) workstation 111 to drive collagen solution from a 3 mL syringe to a 27-gauge needle tip mounted to a third screwa custom LabVIEW program was used to synchronize the voltage supply, syringe pump and stage into 112 driven translation stage for Z-positioning. A National Instruments PXIe-1071 workstation with a a 113 coordinated routine. Within G-code can be supply, uploaded to print customprint LabVIEW program was the usedprogram, to synchronize the voltage syringe pumpcomplex and stagegeometries. into The entire print setup is housed in an ETS-5532 environmental chamber to control temperature and 114 a coordinated print routine. Within the program, G-code can be uploaded to print complex 115 geometries. The entire print setup is housed in an ETS-5532 environmental chamber to control humidity (Figure 1). 116 temperature and humidity (Figure 1). (A) A1.schematic depiction of the complete printer setup. (B)direct-write The direct-write 117Figure 1.Figure (A) A schematic depiction of the complete electrospinning electrospinning printer setup. (B) The 118printhead printhead and collector stage. Detail imageshowing showing single erupting fromfrom TaylorTaylor cone on and collector stage. (C) (C) Detail image singlefiber fiber erupting cone on 119surface of surface of the droplet meniscus depositingon onsubstrate substrate collector. the droplet meniscus andand depositing collector. Materials 2019, 12, 4131 4 of 14 2.2. Collagen Solution Formula Collagen solutions were made with acetic acid and evaluated for their ability to form aligned fibers during the electrospinning process. 500 mg of type I bovine dermis derived atelocollagen powder (Cosmo Bio USA, Carlsbad, CA) was vortexed in 2 mL of 40, 50 and 60% acetic acid (Sigma-Aldrich, St. Louise, MO, USA) diluted in deionized water until completely dissolved (approximately 30 min) to a concentration of 250 mg/mL. The solution was then preloaded into a 3 mL syringe and placed onto the syringe pump. From there, it was coupled to the dispensing needle using 24 Gauge polytetrafluoroethylene (PTFE) tubing with luer lock hubs from Hamilton (Reno, NV, USA). 2.3. Electrospinning Process Collagen fibers were electrospun directly onto glass slides pre-treated with dry film Teflon spray (Dupont, Midland, MI, USA). Up to 600 lines of collagen were spun onto samples 30 lines at a time with a pitch of 15 microns and an approximate syringe tip to substrate distance of ~0.580 mm. A two-step voltage application was employed where 1600 V was initially applied to induce fiber formation and the voltage was then lowered to a preset maintenance voltage after three seconds and held there for the duration of the run. Temperature, relative humidity, and maintenance voltage were all controlled via the custom LabVIEW software and parameters were varied to determine optimal spin parameters. Table 1 shows the experimental parameters tested. Optimal spin quality was determined by counting the number of organized, straight fibers drawn on the sample using image processing in ImageJ and Matlab. Table 1. The list of parameters surveyed for optimizing collagen nanofiber formation. Parameters [Acetic Acid] (%) Relative Humidity (%) Voltage (V) 40 20 1000 50 30 1100 60 40 1200 50 1300 1400 1500 2.4. Scanning Electron Microscopy (SEM) Electrospun collagen fibers were examined with scanning electron microscopy to ensure a fibrous morphology and to compare microstructural differences across multiple spinning parameters. Samples to be imaged were sputter coated with a thin layer of carbon (~30 nm) prior to loading in the SEM. Scanning electron microscopy images were acquired using a Hitachi SU-70 SEM (Tokyo, Japan). All electron microscopy was performed at the Advanced Imaging and Microscopy Laboratory (AIMLab) at the University of Maryland Nanocenter. 2.5. Definition of High-Quality Fibers The quality of fibers produced can vary drastically given the combination of chamber humidity, spin voltage, and acetic acid concentration. For this reason, a quantitative measure of spin quality was defined after preliminary investigations of direct-write electrospinning parameters. During each electrospinning experiment, a target number of straight collagen fibers was recorded along with the resultant number of straight fibers produced. Primarily, our use of direct-write electrospinning is as an additive manufacturing technique capable of producing highly organized electrospun scaffolds. As such, production of wavy or curly fibers reduce placement accuracy and predictability of the print technique, and hence, these fibers were excluded from the line counts (Figure 2). Here, we define the spin quality ratio (SQR) as Equation (1): SQR = (# of straight lines produced)/(# of lines attempted), (1) Materials 2019, 12, 4131 5 of 14 The SQR can be used as a plotted metric to estimate quantitatively the linearity of lines produced during process. Materials the 2019,electrospin 12, x FOR PEER REVIEW 5 of 14 159 160 161 Figure 2. A light microscopy image comparing straight fibers and curly fibers produced during the electrospinning fibers were detected usingusing an image processing algorithm and counted electrospinningprocess. process.Straight Straight fibers were detected an image processing algorithm and and curlyand fibers were filtered counts. counted curly fibers werefrom filtered from counts. 162 2.6. Fiber Counting Algorithm 2.6. Fiber Counting Algorithm Samples were imaged with a Nikon Eclipse Upright microscope (Tokyo, Japan) with a motorized Samples were imaged with a Nikon Eclipse Upright microscope (Tokyo, Japan) with a motorized X-Y stage and a composite image of the entire slide was constructed using Nikon Elements BR (Basic X-Y stage and a composite image of the entire slide was constructed using Nikon Elements BR (Basic Research) software (version 4.13.04, Tokyo, Japan). An ImageJ pre-processing macro was created Research) software (version 4.13.04, Tokyo, Japan). An ImageJ pre-processing macro was created to to enhance the contrast between the background and fibers in order to binarize images. A Matlab enhance the contrast between the background and fibers in order to binarize images. A Matlab algorithm was formulated to count the number of high-quality fibers deposited onto sample slides. algorithm was formulated to count the number of high-quality fibers deposited onto sample slides. The block diagram in Figure 3 outlines the procedures for (a) pre-processing images and (b) fiber The block diagram in Figure 3 outlines the procedures for (a) pre-processing images and (b) fiber calculations. First a single slide image was converted into an 8-bit format within ImageJ. Brightness calculations. First a single slide image was converted into an 8-bit format within ImageJ. Brightness and contrast were adjusted to 80 and 167, respectively. A rolling ball image background subtraction and contrast were adjusted to 80 and 167, respectively. A rolling ball image background subtraction was performed to improve contrast further (rolling ball = 5 light disable) and the image threshold was was performed to improve contrast further (rolling ball = 5 light disable) and the image threshold set to 230. Finally, the image was exported as a binary image file into Matlab. was set to 230. Finally, the image was exported as a binary image file into Matlab. In Matlab, a Hough Transform routine was used to approximate the number of organized In Matlab, a Hough Transform routine was used to approximate the number of organized horizontal fibers in the image. A 1000 × 1000-pixel subsection was first extracted along the centerline horizontal fibers in the image. A 1000 × 1000-pixel subsection was first extracted along the centerline of the parent image and the Hough transform was calculated for that sub-image. Peaks were found of the parent image and the Hough transform was calculated for that sub-image. Peaks were found within the transform image and used to identify horizontal lines within a given angular threshold. within the transform image and used to identify horizontal lines within a given angular threshold. The entire sequence was repeated for the entire parent image. The algorithm was refined and validated The entire sequence was repeated for the entire parent image. The algorithm was refined and via manual counts across several sample images. validated via manual counts across several sample images. 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 Materials 2019, 12, 4131 Materials 2019, 12, x FOR PEER REVIEW 6 of 14 6 of 14 179 180 181 182 Figure 3. Block diagrams of (A) an image pre-processing algorithm for binarizing composite images of Figure 3. Block diagrams of (A) an image pre-processing algorithm for binarizing composite images electrospun fibers and (B) a nanofiber counting algorithm that quantifies the number of straight fibers of electrospun fibers and (B) a nanofiber counting algorithm that quantifies the number of straight crossing the center of the imaged sample. Manual counting was performed to validate automated counts. fibers crossing the center of the imaged sample. Manual counting was performed to validate automated counts. 2.7. Tensile Testing Procedures 183 184 185 186 187 188 189 190 191 192 193 194 showcase ability to build collagen fiber constructs, 30 layers of fibers were electrospun 2.7. To Tensile Testingour Procedures (1600 V, 30% RH, and 28 ◦ C) with 15 µm spacing into 2.1 mm × 1.8 mm fiber mats and tensile tested. To showcase ourmats ability to build collagen constructs, 30 layers of fibers were electrospun For comparison, fibers were also spun from fiber a solution of hexafluoro-2-propranol (HFIP), another (1600V, 30% RH, and 28C) with 15 µm spacing into 2.1 mm x 1.8 mm fiber mats and tensile tested. solvent commonly used for collagen fiber spinning [22]. HFIP-collagen solutions were mixed to a For comparison, fibers mats were also spun from a solution of hexafluoro-2-propranol (HFIP), concentration of 8% w/w (138 mg/mL) and electrospun with the same parameters described above. Fiber another commonly used for collagen fiber spinning mixed mats weresolvent removed from Teflon coated glass substrates with a[29]. razorHFIP-collagen blade and the solutions thickness were of samples to a concentration of 8% w/w (138 mg/ml) and electrospun with the same parameters described was measured before loading into an Electrodynamic Testing System from Test Resources (Shakopee, above. Fiber mats were Teflon substrates with(3M, a razor blade and the MN, USA). Samples wereremoved secured from between twocoated pieces glass of P400 sandpaper Maplewood, MN, thickness of samples measured before loading into antoElectrodynamic Testing System fromand Test USA) and stretched at was a rate of 1.0 mm/minute until failure determine ultimate tensile strength Resources (Shakopee, MN). Samples were secured between two pieces of P400 sandpaper (3M, Young’s modulus. Analysis of the results was performed using Monotonic software (Test Resources) Maplewood MN) and stretched at a rate of 1.0 mm/minute until failure to determine ultimate tensile and plotted with Excel. strength and Young’s modulus. Analysis of the results was performed using Monotonic software 3.(Test Results Resources) and plotted with Excel. 195 3.1. Initial Characterization of Electrospun Collagen Fibers 3. Results 196 Collagen fibers were spun directly onto Teflon-coated glass slides to evaluate spin quality 3.1. Initial Characterization of Electrospun Collagen Fibers for a given set of critical electrospinning parameters. Voltage, relative humidity, and acetic acid concentration to establish parametersglass for direct-write electrospinning asfor ana Collagenwere fibersvaried were spun directly optimal onto Teflon-coated slides to evaluate spin quality additive manufacturing 30% acetic acid solution were also prepared and electrospun but was given set of critical method. electrospinning parameters. Voltage, relative humidity, and acetic acid not spinnable atwere relative humidity lower than 50% (results not shown). SEM images of fibers produced concentration varied to establish optimal parameters for direct-write electrospinning as an with 40% acetic acid display most fibers light in color, withwere clearalso shading. This and hintselectrospun at fibers being additive manufacturing method. 30% are acetic acid solution prepared but solid cylindricalat(Figure 4A,C). Figurelower 4B shows electrospun suboptimal was and not spinnable relative humidity than low 50%quality (resultsfibers not shown). SEMwith images of fibers parameters. Fibers here appear darkfibers in color appearing on the background. produced with 40% acetic aciduniformly display most arewith lightno in shading color, with clear shading. This hints at Closer higher magnifications Figure 4D) reveal fibers seem to havefibers a flat electrospun and planar fibers inspection being solidatand cylindrical (Figure(inset 4A and C). Figure 4B shows low quality morphology. Here, it is shownFibers that fiber can vary the electrospinning process. with suboptimal parameters. here morphology appear uniformly darkduring in color with no shading appearing on the background. Closer inspection at higher magnifications (inset 4D) reveal fibers seem to have a flat and planar morphology. Here, it is shown that fiber morphology can vary during the 197 198 199 200 201 202 203 204 205 206 Materials 2019, 12, 4131 Materials 2019, 12, x FOR PEER REVIEW 7 of 14 7 of 14 207 208 Organized fibers appear in linear bundles of vertical lines with minimal distortion while unorganized electrospinning process. Organized fibers in linear bundles of vertical lines with minimal fibers appear in loops that crowd the base of appear the substrate. distortion while unorganized fibers appear in loops that crowd the base of the substrate. 209 210 211 212 213 214 Figure4.4.Scanning Scanningelectron electronmicroscopy microscopy image image of of electrospun electrospun collagen Figure collagen nanofibers. nanofibers. Example Exampleimage imageof morphology and and fiber fiber alignment alignmentatatlow low(A) andhigh high ofelectrospun electrospunfibers fibers with with good good cylindrical cylindrical morphology (A) and magnification (C). (Inset C) Fibers appear three dimensional and are tightly packed into stacks. magnification (C). (Inset C) Fibers appear three dimensional and are tightly packed into stacks. Exampleimage imageofofelectrospun electrospunfibers fiberswith withpoor poormorphology morphologyand andrandom randomorientation orientationatatlow low(B) (B)and and Example highmagnification magnification (D). (Inset Fibers appear with a flat morphology randomly arranged high (D). (Inset D) D) Fibers appear with a flat morphology and and randomly arranged on theon the collector substrate. collector substrate. 215 3.2. 3.2.Optimization OptimizationofofCollagen CollagenElectrospinning ElectrospinningParameters ParametersBased BasedononSQR SQR Composite 216 Compositeimages imagesofofelectrospun electrospunsamples sampleswere wereanalyzed analyzedwith witha aMatlab Matlabimage imageprocessing processing sequence to detect fiber linearity. Spin quality ratio (SQR) was defined to quantitatively determine 217 sequence to detect fiber linearity. Spin quality ratio (SQR) was defined to quantitatively determine 218 the theorganization organizationofoffibers fibersproduced producedvia viadirect-write direct-writeelectrospinning. electrospinning.Figure Figure55shows showsthe theresulting resulting 219 data dataset setinina aseries seriesofofplots plotsofofRH RHvsvsSQR SQRfor forall allacetic aceticacid acidconcentrations concentrations(vertical (verticalaxis) axis)and andvoltages voltages (horizontal axes). Generally, as RH increased, SQR decreased for all data sets, resulting in an increase 220 (horizontal axes). Generally, as RH increased, SQR decreased for all data sets, resulting in an increase of fiber fiber linearity. linearity. 40%and 40% and 50% 2nd rows) 221 ofofdisorganized disorganizedfiber fiberspinning spinning and and aa decrease decrease of 50% (1st(1st andand 2nd rows) acetic acid produced the highest SQRs, peaking at 0.66 Fibers produced 222 acetic acid produced fibersfibers with with the highest SQRs, peaking at 0.66 and and 0.63.0.63. Fibers produced withwith these these conditions showed strong linearity when examined under light microscopy. High SQR indicates 223 conditions showed strong linearity when examined under light microscopy. High SQR indicates 224 well-controlled well-controlledfiber fiberformation formationwith withsignificant significantlinearity linearitymaking makingthese thesetwo twoconcentrations concentrationspreferable preferable 225 for forcontrolled controlledelectrospinning. electrospinning. 226 Further inspection of fiber spinning with 40% acetic acid shows that a lower relative humidity 227 produces fibers with the best linearity peaking at 20 and 30%. Notably, as the voltage reaches 1300 228 and 1500 the SQR peaks at 30% RH for 40% acetic acid. For 50% acetic acid, a more consistent trend 229 of peak SQR is found at 30% RH. 60% acetic acid consistently produced random and unorganized 230 fibers with lower SQR values that rarely surpassed 0.3. Interestingly, for 50% relative humidity the 231 calculated SQR is comparable for lower acetic acid concentrations. Generally, once the acetic acid 232 concentration reaches 60%, SQR decreases significantly and fiber formation becomes more chaotic 233 and less controllable. 234 For 40% acetic acid, as the voltage increases there is a slight increase in SQR, particularly at a 235 relative humidity of 30%, while no apparent trends seem to be present for 50 and 60% concentrations. 236 Microscopic inspection of samples (not shown) showed an increased presence of electrospun material Materials 2019, 12, x FOR PEER REVIEW 237 238 8 of 14 at higher humidity. As the RH increases, the fiber production rate also increases, resulting in fibers 8 of 14 piling up in an organized fashion as they exceed the XY motor translation speed. Materials 2019, 12, 4131 239 240 241 Figure 5. Data plot summary for relative humidity (RH) vs. spin quality ratio (SQR). The horizontal Figure 5. Data summary forincreasing relative humidity (RH)the vs.vertical spin quality ratio data (SQR). The horizontal axes show data plot electrospun with voltage and axes show electrospun with axes show data electrospun with increasing voltage and the vertical axes show data electrospun with increasing acetic acid concentration. increasing acetic acid concentration. 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 Further inspection of fiber spinning with 40% acetic acid shows that a lower relative humidity While lightwith microscopy readily identifies linearity, SEM images recorded order to produces fibers the best linearity peaking atfiber 20 and 30%. Notably, as thewere voltage reachesin1300 and closely fiber morphology as acetic the voltage and50% relative wasconsistent manipulated. 1500 the examine SQR peaks at 30% RH for 40% acid. For acetichumidity acid, a more trendFigure of peak6 illustrates the morphology for collagen fibers produced with 40% acetic acid solution and 30%RH SQR is found at 30% RH. 60% acetic acid consistently produced random and unorganized fibers with over voltages covering the entire investigated range. Fibers were solid with cylindrical morphology lower SQR values that rarely surpassed 0.3. Interestingly, for 50% relative humidity the calculated SQR high aspect ratio acetic acrossacid theconcentrations. range of attempted voltages. Collagen fiber diameter remained isand comparable for lower Generally, once the acetic acid concentration reaches consistent, measuring ~1–2 microns across the voltage range. Small cracks and surface defects 60%, SQR decreases significantly and fiber formation becomes more chaotic and less controllable.seen on images can be attributed to voltage defects in the carbon coating usedincrease for SEMininspection. These fibers For 40% acetic acid, as the increases there is a slight SQR, particularly at a exhibited both good morphology high SQR, demonstrating highlyfor organized fiber placement. relative humidity of 30%, while noand apparent trends seem to be present 50 and 60% concentrations. SEM images of samples electrospun at 1500V across all investigated humidities and acetic acid Microscopic inspection of samples (not shown) showed an increased presence of electrospun material concentrations are As shown in increases, Figure 7 the in fiber a more expansive of fiber resulting morphology and at higher humidity. the RH production ratestudy also increases, in fibers organization. At the lowest humidity (20RH) collagen fiber morphology generally appeared solid, piling up in an organized fashion as they exceed the XY motor translation speed. withWhile few flat and planar fibers present on thefiber surface. Fibers were well-organized, consistent with light microscopy readily identifies linearity, SEM images were recorded in order to the SQR values fiber foundmorphology previously.as 30RH revealed similar morphology and manipulated. arrangement for fibers closely examine the voltage and relative humidity was Figure 6 formed from 40 and 50% acetic acid solutions, while 60% acetic acid solutions produced illustrates the morphology for collagen fibers produced with 40% acetic acid solution and 30% RH overa combination of solid and flat fibers. Additionally, there is solid a significant increase morphology in the volume of voltages covering the entire investigated range. Fibers were with cylindrical and unorganized fibers. high aspect ratio across the range of attempted voltages. Collagen fiber diameter remained consistent, Once the relative humidity exceeded 30%, fiber morphology and organization degraded measuring ~1–2 microns across the voltage range. Small cracks and surface defects seen on images can significantly all acetic concentrations. Atfor 40RH, the acetic acid concentration increased be attributed across to defects in theacid carbon coating used SEMas inspection. These fibers exhibited both fibersmorphology became flatter morphology. At the highest tested relativefiber humidity, all fibers viewed took good andinhigh SQR, demonstrating highly organized placement. on a planar morphology. Under high magnification fibers appeared almost indistinguishable with the background and are distributed randomly across the sample space. This finding is consistent with the steep drop-offs in SQR found in the previous section. Materials 2019, 12, 4131 Materials 2019, 12, x FOR PEER REVIEW 264 265 266 267 9 of 14 9 of 14 Figure 6. Scanning electron microscopy images of collagen nanofibers. Fibers were electrospun from Figure 6. Scanning electron microscopy images of collagen nanofibers. Fibers were electrospun from 40% acetic acid at 30% relative humidity with varying voltages of (A) 1100 V, (B)1300 V, and (C) 1500 40% acetic acid at 30% relative humidity with varying voltages of (A) 1100V, (B)1300V, and (C) 1500V. V. Fibers have a clear cylindrical morphology and appear to have a high degree of fiber alignment Fibers all all have a clear cylindrical morphology and appear to have a high degree of fiber alignment on on substrate. thethe substrate. SEM images of samples electrospun at 1500 V across all investigated humidities and acetic acid concentrations are shown in Figure 7 in a more expansive study of fiber morphology and organization. At the lowest humidity (20 RH) collagen fiber morphology generally appeared solid, with few flat and planar fibers present on the surface. Fibers were well-organized, consistent with the SQR values found previously. 30 RH revealed similar morphology and arrangement for fibers formed from 40 and 50% acetic acid solutions, while 60% acetic acid solutions produced a combination of solid and flat fibers. Additionally, there is a significant increase in the volume of unorganized fibers. Once the relative humidity exceeded 30%, fiber morphology and organization degraded significantly across all acetic acid concentrations. At 40 RH, as the acetic acid concentration increased fibers became flatter in morphology. At the highest tested relative humidity, all fibers viewed took on a planar morphology. Under high magnification fibers appeared almost indistinguishable with the background and are distributed randomly across the sample space. This finding is consistent with the steep drop-offs in SQR found in the previous section. Materials 2019, 12, 4131 Materials 2019, 12, x FOR PEER REVIEW 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 10 of 14 10 of 14 Figure 7. SEM images of fibers electrospun at 1500 V for various relative humidity (shown in upper right of each image) and of acetic acid concentrations of (A) (B) 50%, andhumidity (C) 60%. (shown in upper Figure 7. SEM images fibers electrospun at 1500V for40%, various relative right of each image) and acetic acid concentrations of (A) 40%, (B) 50%, and (C) 60%. 3.3. Peak Loadand Young’s Modulus of Uncrosslinked Fibers 3.3. As Peak Loadand Young’s Modulus of Uncrosslinked a proof of concept, thick collagen nanofiberFibers mats were electrospun at 30% relative humidity from 40% (250 mg/mL collagen) and HFIP solutions (138 mg/mL collagen) to humidity form 30 As a acetic proof acid of concept, thick collagen nanofiber mats were electrospun at 30% relative layers of fibers. These nanofiber mats were mechanically tested to measure ultimate tensile strength from 40% acetic acid (250mg/ml collagen) and HFIP solutions (138 mg/ml collagen) to form 30 layers and Young’s modulus. Fiber mats were electrospun tested to a thickness of 30 layers. tensile Figure strength 8A,B show of fibers. These nanofiber mats were mechanically to measure ultimate and the resultant fiber mat loaded into the tensile testing setup at the beginning of an experiment. is Young’s modulus. Fiber mats were electrospun to a thickness of 30 layers. Figure 8A,B showIt the apparent from the image that fiber mats spun from HFIP solution are thicker than mats from acetic resultant fiber mat loaded into the tensile testing setup at the beginning of an experiment. It is acid solution. This consistent work showing electrospun frommats HFIPfrom solutions apparent from theis image thatwith fiberprevious mats spun from HFIP solution are collagen thicker than acetic having larger fiber diameters than acetic acid solutions [23–25]. Figure 8C,D compare the average acid solution. This is consistent with previous work showing electrospun collagen from HFIP peak load and young’s modulus with standard deviation. Although the peak load for HFIP-based solutions having larger fiber diameters than acetic acid solutions [23–25]. Figure 8C,D compare the fiber matspeak is significantly higher (p = 0.006)with thanstandard AA-based fiber mats, the young’s modulus no average load and young’s modulus deviation. Although the peak load show for HFIPstatistically significant difference (p = 0.268). Future work and studies will look at further categorizing based fiber mats is significantly higher (p = 0.006) than AA-based fiber mats, the young’s modulus the influence of crosslinking, layerdifference amount, and solventwork on material properties. show no statistically significant (p =spinning 0.268). Future and studies will look at further categorizing the influence of crosslinking, layer amount, and spinning solvent on material properties. Materials 2019, 12, 4131 Materials 2019, 12, x FOR PEER REVIEW 11 of 14 11 of 14 Figure 8. Example image of tensile testing system with collagen fiber mats made with (A) HFIP and (B) 40% acetic acid. Collagen mats testing were stretched in thecollagen above configuration untilwith failure theand (C) Figure 8. Example image fiber of tensile system with fiber mats made (A)and HFIP peak load (n = 4, p = 0.006) and (D) young’s modulus (n = 4, p = 0.268) were recorded for samples and (B) 40% acetic acid. Collagen fiber mats were stretched in the above configuration until failure and the were plotted. the averages (C) peak and loadstandard (n = 4, pdeviations = 0.006) and (D) young’s modulus (n = 4, p = 0.268) were recorded for 283 284 285 286 samples and the averages and standard deviations were plotted. 4. Discussion 287 4. Discussion Relative Humidity, Acetic Acid Concentration and Fiber Morphology 288 Direct-write exhibits potential as a method to produce nanofibrous scaffolds. Relative Humidity, electrospinning Acetic Acid Concentration and Fiber Morphology Precise control over fiber placement in the future will allow researchers to build precision matrix scaffolds electrospinning exhibits potential as a method produce scaffolds. muchDirect-write like native ECM. Furthermore, bioanalogs of ordered tissuetolike corneananofibrous and meniscus can be Precise control over fiber placement in the future will allow researchers to build precision matrix produced for regenerative medicine and transplantation [26,27]. To effectively incorporate DWES as a 3D scaffoldsmodality, much likeenvironmental native ECM. Furthermore, bioanalogs ordered tissue cornea and meniscus printing and process settings mustofbe optimized forlike reproducibility. Relative can be produced forenvironmental regenerative medicine [26,27]. To effectively incorporate humidity is a critical parameterand thattransplantation contributes to systematically electrospinning fibers DWES as a 3D printing modality, environmental and process settings must be optimized for into an ordered structure. In addition, selecting the correct solvent is paramount to maintaining high reproducibility. RelativeFor humidity is aancritical environmental parametertothat contributes to quality electrospinning. this reason, exhaustive study was performed extract the optimal systematically electrospinning fibers into an ordered structure. In addition, selecting the correct relative humidity and acetic acid concentration for direct-write electrospinning of collagen nanofibers. solvent is paramount to maintaining electrospinning. exhaustive Based on the presented study, 40%high aceticquality acid was confirmed to beFor thethis bestreason, solventan concentration study was performedcollagen to extractfibers. the optimal humidity and acetic acid for directfor electrospinning Purerelative acetic acid is well-known for concentration its ability to solubilize write electrospinning of collagen nanofibers. collagen but initially was unsuitable as a solvent for electrospinning due to its high affinity to Based onslow the presented study, acetic aciddemonstrations was confirmed of to collagen be the best solvent concentration collagen and evaporation rate 40% [28–30]. Early electrospinning utilized for electrospinning collagen fibers. Pure acetic acid is well-known for its ability to solubilize collagen but initially was unsuitable as a solvent for electrospinning due to its high affinity to collagen and slow evaporation rate [28–30]. Early demonstrations of collagen electrospinning utilized 289 290 291 292 293 294 295 296 297 298 299 300 301 302 Materials 2019, 12, 4131 12 of 14 hexafluoroisopropanol (HFIP) or trifluro-ethanol (TFE) as solvents to achieve a spinnable viscosity, but high toxicity of these fluoro-organic solvents make them unsuitable for everyday use [31]. Additionally, there are conflicting reports on how similar electrospun collagen produced from these solvents is to native collagen on a molecular level [32]. Acetic acid still shows promise as a solvent because it does not completely denature collagen in solution like HFIP and TFE [22]. Here, we analyzed the impact of acetic acid concentration on fiber morphology and organization. The highest concentration tested (60%) resulted in consistently poor fiber morphology and low control on fiber placement across all relative humidities. The lower concentrations (40 and 50%) resulted in solid, cylindrical fiber morphology and improved fiber placement, especially at relative humidities below 40%. From this we can deduce that diluting the acetic acid concentration with water increases the evaporation rate of the carrier solvent when the humidity is sufficiently low allowing fibers to dry prior to deposition on the collector. Evaporation of a solution is regulated by the temperature and humidity of the surrounding environment as well as the vapor pressure of the solution. Because the vapor pressure of water and acetic acid are very similar (2.5 kPa and 3.76 kPa, at 28 ◦ C respectively) [33] the relative humidity seems to highly impact the rate of spinning and fiber morphology. Liu et al. demonstrated a diluted 40% acetic acid solution as a suitable electrospinning solvent for type I collagen, however relative humidity and other acetic acid concentrations were left unreported. Polk et al. reported dissolving collagen in acetic acid solutions of 20–50% for compressed air-driven pneumatospinning but do not report environmental parameters or results for solutions other than 40% [25]. Despite its critical role in determining fiber morphology, few researchers have reported on the relative humidity required to produce electrospun collagen. This may be due to the prevalence of fluoro-alcohol use as a solvent. Our work fills a void in the literature showing the relationship between collagen electrospinning solvent, relative humidity and fiber morphology and organization. 5. Conclusions In summary, an exhaustive study was performed to ascertain ideal acetic acid concentration, relative humidity and voltage parameters for direct-write electrospinning of highly organized collagen nanofibers for scaffold production. A custom electrospinning printer was designed and built in order to produce organized single lines of collagen nanofibers with cylindrical morphology. Acetic acid concentration, relative humidity and spin voltage were all varied to establish best practices for producing single fibers of collagen with high linearity and control of fiber placement. An acetic acid concentration of 60% produced wet fibers that absorbed onto the substrate with an increasingly planar morphology with increasing humidity, despite showing a degree of control over fiber placement for 20% relative humidity. Acetic acid concentrations of 40 and 50% were found to consistently produce solid fibers with cylindrical morphology at relative humidity lower than 40% making them optimal concentrations for direct-write electrospinning of collagen. As a rule of thumb, the relative humidity should be kept ≤30% in order to maintain a solid fiber morphology. Additionally, SQR most consistently peaked at 20% and 30% relative humidity for 40% and 50% acetic acid, respectively. Finally, the mechanical properties of fiber mats electrospun with 40% acetic acid and HFIP as a solvent were tested to show the ability to electrospin large scaffolds of materials. The process parameters identified in this work can enable researchers to direct-write electrospin pure collagen scaffolds with specific morphologies that more accurately mimic native ECM in specific tissues. Author Contributions: Conceptualization, F.A.A.J., L.J., and K.P.; methodology, F.A.A.J. and K.W.; software, F.A.A.J. and L.J.; validation, F.A.A.J., L.J.; formal analysis, F.A.A.J.; investigation, F.A.A.J. and L.J.; resources, L.J.; data curation, F.A.A.J.; writing—original draft preparation, F.A.A.J.; writing—review and editing, K.W. and K.P.; visualization, L.J.; supervision, K.P.; project administration, K.P.; funding acquisition, L.J. and K.P. Funding: This research was funded by US Army Medical Research and Materiel Command (USAMRMC), award number W81XWH-17-C-003. The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation. Materials 2019, 12, 4131 13 of 14 Acknowledgments: The authors would like to acknowledge Linzie Wagner and Carolynn Nofsinger for their administrative support of this project. 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Available online: http://ddbonline.ddbst.de/AntoineCalculation/ AntoineCalculationCGI.exe (accessed on 9 December 2019). © 2019 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/). 

W4306924819.txt

Original Research 20 October 2022 DOI 10.3389/fmars.2022.873548 TYPE PUBLISHED OPEN ACCESS EDITED BY Jeremy Kiszka, Florida International University, United States REVIEWED BY Roberto Carlucci, University of Bari Aldo Moro, Italy Tiago Andre Marques, University of St. Andrews, United Kingdom Movements and diving behavior of Risso’s dolphins in the Southern California Bight Brenda K. Rone 1*, David A. Sweeney 1, Erin A. Falcone 1, Stephanie L. Watwood 2 and Gregory S. Schorr 1 Foundation for Marine Ecology and Telemetry Research, Seabeck, WA, United States, Ranges, Engineering and Analysis Department, Naval Undersea Warfare Center, Newport, RI, United States 1 2 *CORRESPONDENCE Brenda K. Rone brenda.rone@marecotel.org SPECIALTY SECTION This article was submitted to Marine Megafauna, a section of the journal Frontiers in Marine Science 10 February 2022 30 August 2022 PUBLISHED 20 October 2022 RECEIVED ACCEPTED CITATION Rone BK, Sweeney DA, Falcone EA, Watwood SL and Schorr GS (2022) Movements and diving behavior of Risso’s dolphins in the Southern California Bight. Front. Mar. Sci. 9:873548. doi: 10.3389/fmars.2022.873548 COPYRIGHT © 2022 Rone, Sweeney, Falcone, Watwood and Schorr. This is an openaccess 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. Risso’s dolphins (Grampus griseus), uncommon prior to the 1970’s, are now regularly observed within the Southern California Bight. During long-term cetacean monitoring programs on United States Navy range areas in the Southern California Bight from 2009–2019, we deployed 16 Argos-linked satellite tags on Risso’s to acquire objective, detailed depictions of their movements and behaviors. Individuals were tracked for a median of 10.7 days (range = 0.8 – 19.7). Kernel density estimation suggested individuals utilized the entire Southern California Bight with the 50% core use area centered around San Clemente and Santa Catalina Islands where most of the tag deployments occurred. Grand median dive depth was 101 m (max = 528) and dive duration was 5.6 min (max = 11.1). We used generalized mixed models to assess seasonal and environmental effects on distribution and diving behavior including month, distance to shore, time of day, lunar phase, sea surface temperature, and chlorophyll-a residuals. Animals were further from shore (including islands) during a full versus new moon and from the mainland during the last versus first quarter moon. Animals also tended to be closer to land in the fall and early winter months. Dives were deeper yet shorter during the night, during a full moon, and when animals were further offshore. Animals conducted nearly twice as many dives at night compared to day, though deep dives (> 500 m) occurred at all times of day. This study provides insights into Risso’s distribution and behavioral trends while identifying priorities for future research. KEYWORDS Risso’s dolphin, Grampus griseus, satellite telemetry, generalized linear mixed models, Southern California Bight Frontiers in Marine Science 01 frontiersin.org Rone et al. 10.3389/fmars.2022.873548 Risso’s have a primarily teuthophagus diet consisting of both squid and octopus (Clarke, 1996; Baird, 2002; Blanco et al., 2006; Jefferson et al., 2006; Bearzi et al., 2011; Yates and PalavecinoSepú lveda, 2011; Bloch, 2012; Milani et al., 2017; Luna et al., 2021), though they also feed on fish and crustaceans (Reeves et al., 2002; Bloch, 2012). With the exception of octopus in the western Mediterranean (Blanco et al., 2006), squid appear to be their preferred cephalopod prey (Clarke, 1996; Bearzi et al., 2011; Yates and Palavecino-Sepú lveda, 2011; Bloch, 2012). In California waters, some species of squid that have been identified as important prey for Risso’s, including jumbo squid (Dosidicus gigas) (Orr, 1966; Gilly et al., 2006) and market squid (Loligo opalescens) (Kruse, 1989), perform diurnal vertical migrations (Watanabe et al., 2006). These species serve as both prey and predators within the ecosystem, feeding on deep-, mid-, and shallow-water organisms such as krill and other crustaceans, small fish, and even other cephalopods, with observations suggesting they may feed at the surface in the early evening (Markaida and Sosa-Nishizaki, 2003). Jumbo squid in particular have demonstrated daytime depths exceeding 250 m, nearsurface depths at dusk, and variable depths throughout the night (Gilly et al., 2006). Our understanding of foraging behavior of Risso’s has advanced markedly in recent years. Initial observational studies suggested Risso’s rested or traveled during the day and foraged at night (Shane, 1995b). An increase in both click rates and bouts at night suggested Risso’s foraging activity is related to diurnal patterns in squid behavior (Soldevilla et al., 2010). Most recent work indicates that Risso’s perform foraging dives during the day as well as at night (Benoit-Bird et al., 2017; Arranz et al., 2018; Benoit-Bird et al., 2019) with maximum depths exceeding 500 m particularly common during the day (Benoit-Bird et al., 2019). They regularly switch predation tactics between a nearsurface generalist and deep-water squid specialist even within a single dive (Benoit-Bird et al., 2019). They plan foraging dives based on knowledge attained on previous dives (Arranz et al., 2018, Visser et al., 2021) and use a spin dive strategy to decrease transit time to exploit the diurnal mesopelagic layer (Visser et al., 2021). Reliable assessments of Risso’s movements and diving behavior are challenging yet essential for understanding population structure, stock boundaries, habitat use, and potential exposure to anthropogenic impacts. Satellite tags can provide a more objective, detailed depiction of individual movements and behavior than visual or acoustic surveys alone. As part of a long-term research effort to examine cetacean movements, habitat use, and ecology within the Southern Introduction Risso’s dolphins (Grampus griseus, hereafter referred to as “Risso’s”) are a medium-sized, cosmopolitan odontocete found in temperate and tropical waters. They typically occur between latitudes 60°N to 60°S where sea surface temperatures are greater than 10°C; however, the highest numbers of individuals are found between 30° and 45° latitudes, in waters 15–20°C (Kruse et al., 1999; Baird, 2002; Jefferson et al., 2014b). In the eastern Pacific, Risso’s are abundant and widely dispersed year-round (Leatherwood et al., 1980). They can be found as far north as the Gulf of Alaska and south to Tierra del Fuego (Baird, 2002; Jefferson et al., 2014b). Risso’s are most often associated with shelf-edge and upper continental slope habitat, in areas of steep bottom topography with average depths ranging from 100 to 1000 m (Kruse, 1989; Baumgartner, 1997; Kruse et al., 1999; Baird, 2002; Cañadas et al., 2002; Jefferson et al., 2006; Azzellino et al., 2008). Although highest densities are found along this shelf/slope region, they utilize a wide range of habitats, from shallow, coastal areas (Boer et al., 2013; Jefferson et al., 2014b) to offshore pelagic waters (Baird, 2016). Group size is typically 10–50, although singletons and groups upwards of 4,000 individuals have been documented (Leatherwood et al., 1980; Baird, 2002; Baird, 2016). Currently, the population abundance of Risso’s along the coasts of Washington, Oregon, and California is estimated at 6,336 (CV = 0.32) individuals (Carretta et al., 2020). Movement patterns within this population have been discerned through sightings and acoustic detections. It is thought that Risso’s in this region demonstrate broadscale seasonal shifts northward from California into Oregon/Washington in the spring/summer months coincident with rising water temperatures (Green et al., 1992). High inter-annual seasonal variability north of (Dohl et al., 1983; Kruse, 1989; Soldevilla et al., 2010), and yearround presence within, the Southern California Bight (Soldevilla et al., 2010), a productive oceanographic region off the coast of the southwestern United States (U.S.), have been documented. Long–term changes in the abundance and distribution of Risso’s have also been documented in the Southern California Bight. In the late 1950s, they were rarely encountered there (Norris and Prescott, 1961). Between 1972 and 1975, sightings increased in frequency as their distribution shifted inshore (Leatherwood et al., 1980). Starting in the El Niño years of 1982–83, occurrence further increased in the Bight and Santa Catalina Island was identified as particularly important habitat (Shane, 1995b). They continue to be regularly documented there both visually and acoustically (Soldevilla et al., 2010; Jefferson et al., 2014a; Bacon et al., 2017). Frontiers in Marine Science 02 frontiersin.org Rone et al. 10.3389/fmars.2022.873548 SPLASH10-A tags transmitted a record of diving behavior in the form of a behavior log (BL). BL data were received via the Argos satellite system and from a land-based Argos receiving station (Wildlife Computers Mote) installed on San Clemente Island in 2014 (Jeanniard-du-Dot et al., 2017). BL data consisted of summarized dive and surfacing events and included event start and end times and the maximum depth of dives. As the number of tag deployments grew, our understanding of Risso’s dive behavior improved. Thus, adjustments to tag programming were made during the course of study to maximize the quantity and relevance of BL data captured while minimizing data gaps. Dive events were defined as any submergence that exceeded either 30 m (6 tags) or 50 m (2 tags) depth and lasted more than 30 s (3 tags) or 1 min (5 tags) (Table 1). Depth accuracy for the pressure transducers in these tags was independently verified in a pressure chamber to 3,000 m, resulting in a maximum error of <+/- 2.5% of the recorded value (Schorr et al., 2014). Initially, start and end times were determined by crossing the 5 m depth threshold upon descent or ascent of a qualifying dive for the first two tags. After assessment of tag performance, the next six tags were programmed with start and end times determined by the transition from wet to dry when the tag broke the surface during a typical breath, and therefore this programming regimen California Bight, we remotely deployed 16 Argos-linked satellite tags on Risso’s between 2009 and 2019, and here report on habitat use, dive behavior, and movements of these tagged individuals. Materials and methods Data collection Tag data collection and processing Fieldwork was undertaken in the Southern California Bight, primarily off San Clemente and Santa Catalina Islands, from 2009 to 2019 (Figure 1). Satellite tags in the Low Impact Minimally Percutaneous External-electronics Transmitter (LIMPET) configuration were deployed on or near the dorsal fin using a Dan-Inject modified air rifle. Tags were affixed by two 3.4 cm medical-grade titanium barbed darts which were gas sterilized prior to attachment (Andrews et al., 2019). The tags contained either Wildlife Computers’ (Redmond, WA) SPOT5 location-only, or SPLASH10-A or SPLASH10-F location and dive reporting transmitters. The age class of each tagged individual was estimated based on the relative size and level of scarring (Kruse et al., 1999; Hartman et al., 2016). FIGURE 1 Maps of the study area with filtered locations of Risso’s dolphins tagged within the Southern California Bight annually. The inset shows the study area in a broader geographic context of the state of California. Range Polygons: Blue = Southern California Range Complex; Purple = Point Mugu Sea Range; Orange = Southern California Anti-submarine Warfare Range; Pink = Shore Bombardment Area. Frontiers in Marine Science 03 frontiersin.org Rone et al. 10.3389/fmars.2022.873548 TABLE 1 Details of Risso’s dolphin satellite tag deployments by individual. Tag ID Age class Tag type GgTag001 A GgTag002 Event Start/End time by Defined dive threshold Deployment date Locality Transmission duration (days) No. locations for analyses SPOT5 19Jul2009 East side of SCI 13.6 100 J SPOT5 11Nov2009 Santa Cruz I. 19.3 221 GgTag003* A SPOT5 24Jun2010 SOAR 19.7 239 GgTag004* A SPOT5 24Jun2010 SOAR 11.2 127 GgTag005 A SPOT5 08Jan2011 Catalina I. 6.7 82 GgTag006 A SP10-A 18Jun2011 San Diego 8.3 21 GgTag008 A SP10-A 5 m Depth 50m/1min 24Jul2011 East side of SCI 5.3 76 GgTag009 A SP10-A 5 m Depth 30m/1min 20Sep2011 Catalina I. 0.3 7 GgTag010 A SPOT5 20Jan2012 Catalina I. 10.1 175 GgTag011 A SP10-A Wet/Dry 30m/30s 26Mar2013 Catalina Basin 13.9 204 GgTag012 A SP10-A Wet/Dry 30m/30s 21May2013 Catalina Basin 8.0 100 GgTag015 A SP10-A Wet/Dry 30m/30s 26Feb2016 East side of SCI 11.7 167 GgTag016 S SP10-F 11Apr2016 Catalina I. 7.3 109 GgTag018 A SP10-A Wet/Dry 50m/1min 04Apr2017 San Nicolas Basin 18.6 354 Gg-2018112098368 A SP10-A Wet/Dry 30m/1min 20Nov2018 SOAR 7.6 120 Gg-20190719102468 A SP10-A Wet/Dry 30m/1min 19Jul2019 Catalina Basin 11.3 197 *Tagged within the same group. SCI, San Clemente Island; SOAR, Southern California Anti-submarine Warfare Range; For age classes, A, Adult; S, Sub-adult; J, Juvenile; for tag type, SP, SPLASH; A, Argos; F, Fastloc GPS. removed) were combined with the tag’s Douglas-filtered Argos data to provide the highest resolution location data available for this individual. The cumulative distance traveled during each tag transmission and the straight-line distance from the deployment location were calculated using all locations that passed the filter, regardless of quality. For dive behavior analyses, Douglas-filtered locations were fit by a continuous-time correlated random walk model (Johnson et al., 2008) that incorporated Argos ellipse errors (McClintock et al., 2015) using the crawl (v2.2.1) R package (Johnson and London, 2018; R Core Team, 2021) to estimate the animal’s position at the start time of every dive. Although standard errors ranged from 0 to 26 km for model-estimated locations, a function of Argos location qualities and time between consecutive Douglas-filtered locations, they were considered the best estimate of the animal’s position at the start time of each dive. Given these inherent sources of error or uncertainty in our spatial data, we exercised caution when interpreting results associated with data derived from both model-estimated and Douglas-filtered locations. allowed for a more accurate assessment of dive and surfacing times (Table 1). The single SPLASH10-F tag that was deployed during this study was programmed to collect only maximum dive depth histogram data (https://wildlifecomputers.com/ourtags/splash-archiving-tags/splash10-f/) to prioritize the collection of higher resolution Fastloc GPS location data in addition to Argos location data, and thus was excluded from dive analyses. Locations received via Argos satellite were re-processed by Argos using the Kalman filter to improve location accuracy (Lopez et al., 2014), uploaded to Movebank (www.movebank. org), and then processed using the Douglas Argos-Filter (v10.5.2) with the ‘distance-angle-rate’ filter (Douglas et al., 2012). Filtering parameters were set as follows: maximum-redundant-distance at 3 km, maximum sustainable rate at 15 km h-1, and the rate coefficient (the angle created by three subsequent points, Ratecoef) at the default of 25. Argos location classes 2 and 3 have an estimated error from Argos of < 500 m (Argos User’s Manual) and were automatically retained. Filtered SPLASH-10F tag Fastloc GPS locations (locations with residuals > 35 or time error > +/- 3 min Frontiers in Marine Science 04 frontiersin.org Rone et al. 10.3389/fmars.2022.873548 the cosine term describe phase shifts near new and full moons, while effects from the sine term describe phase shifts near the first and last quarters. Seasonal influence was investigated using month (for movement models) and sea surface temperature and residuals from the linear regression of chlorophyll-a against sea surface temperature (for dive models), referred to as chlorophyll-a residuals (Soldevilla et al., 2011). The calculation of chlorophyll-a residuals was performed using the glmmTMB (v1.1.3) R package (Brooks et al., 2017) and included a random effect to account for differing oceanographic measurements across tagged individuals due to different deployment dates and movements for each individual. Environmental data collection A series of ArcGIS layers were created containing bathymetric, remote sensing, land, and Navy range boundaries. Depth, slope, distances to nearest shore (either islands or mainland) and to the mainland, and assessment of locations within range boundaries were extracted by overlaying tag location data onto raster surfaces or in relation to polygons in ArcGIS v10.7.1 (NAD 1983 UTM Zone 11N) (ESRI, 2019). Less than 1% of location estimates were on land according to the raster surfaces—a result of poor-quality locations and Risso’s use of steep near-shore bathymetry; these were removed from subsequent movement and dive behavioral analyses. For analyses of depth and slope, only Douglas-filtered locations of qualities 2 and 3 were included, given the size of error ellipses associated with lesser quality Argos positions (Lopez et al., 2014) and the variable nature of depth and slope over a limited geographic area. Bathymetric data were obtained from the 2-min Gridded Global Relief Data (ETOPO2v2) (National Centers for Environmental Information, 2006). The slope raster was created using the slope surface tool available in ArcGIS Spatial Analyst. Remote sensing data from the CoastWatch ERDAP server (https://coastwatch.pfeg.noaa.gov/erddap) were extracted using the xtractomatic (v3.3.2) package (Mendelssohn, 2017) in R, and associated with each Douglas-filtered location for general habitat description and with each dive’s crawl-modeled location for dive models. Mean sea surface temperature (°C) was extracted from the National Oceanic and Atmospheric Administration (Optimum Interpolation Sea Surface Temperature 0.25˚ resolution) with the Advanced Very High Resolution Radiometer sensor which combines both satellite and in-situ data to produce daily gap-free composites (Reynolds et al., 2007). Mean chlorophyll-a concentration (mg m -3 ), an indicator of primary productivity, was obtained from the Moderate Resolution Imaging Spectroradiometer on board the Aqua satellite (West U.S., 0.0125˚ resolution); eight-day composites were used to fill in data gaps due to cloud cover. Median values are reported to minimize the effects of outliers. Solar elevation and lunar phase were calculated for Douglasfiltered and crawl-modeled locations using the oce (v1.4-0) package (Kelley and Richards, 2020) in R. A time-of-day classifier was assigned to each record: day (solar elevation ≥ -12˚ below the horizon, inclusive of nautical dawn and twilight) and night (< -12˚ below the horizon). Lunar phase was incorporated into each model as a pair of cyclical predictors using the sine and cosine of the calculated lunar phase to estimate the amplitude and phase angle of lunar effects (DeBruyn and Meeuwig, 2001). To do this, calculated lunar phases were converted into radians where a lunar phase of 0 was 0 radians, 0.5 was p radians, and 1 was 2p radians. Effects from Frontiers in Marine Science Data analysis Movements Areas of common use were approximated using 50% and 95% kernel density estimation based on the Douglas-filtered locations from all tags using the adehabitatHR (v0.4.19) package (Calenge, 2006) in R. The adehabitatHR ad hoc method (function input h = “href”) for computing the smoothing parameter was used, along with all other default function inputs. For days with more than one filtered location per individual, an average daily position was used to reduce the influence of autocorrelation between multiple locations in a day (Heide-Jørgensen et al., 2002). These kernel density isopleths were plotted in ArcGIS v10.7.1. Inshore/offshore movements were evaluated using two metrics: distance to shore (included mainland and islands) and distance to mainland (west coast only, excluded islands). These movements were modeled as a function of cyclical lunar phase, month, and time of day using generalized linear mixed models from the glmmTMB package (Brooks et al., 2017). Models were fit as gamma distributions with log link functions and used the maximum likelihood parameter estimation method. Two movement models were created: one with distance to shore as the response variable and the other with distance to mainland as the response variable. To account for autocorrelation within individuals, a random effect was included in all models specifying the tagged individual (or pair of tagged individuals if from the same group). We tagged two individuals within the same encounter, although these individuals were not observed to be closely associated at the time of tagging (tagged 32 min apart, group size = 45, group spread = 300 x 400 m). Following inspection of the animals’ movements, we used a random effect value indicating periods of associated movements to minimize autocorrelation within this apparent social group. 05 frontiersin.org Rone et al. 10.3389/fmars.2022.873548 Inspection of a plot of the residual autocorrelation function indicated that this random effect did not sufficiently reduce temporal residual autocorrelation. Thus, we incorporated secondary, nested random effects of different time increments (1, 2, 3, 4, 6, 8, and 12 h) within individuals to account for temporal autocorrelation, selecting the time period that minimized Akaike’s information criterion (AIC) for each model. After determining the nested effect that minimized AIC, separate models were created for each possible combination of lunar phase predictors (only cosine, only sine, both cosine and sine), and the model that minimized AIC was chosen as the best-fit model that most accurately described the lunar phase shift. Within each best-fit model, predictor significance was determined using a Type II Wald Chi-Square Test (a < 0.05) using the ANOVA function from the car (v3.0-12) R package (Fox and Weisberg, 2019). Model predictions were created for the average individual by setting all random effects to zero using the predict.glmmTMB function from the glmmTMB package (Brooks et al., 2017) in R. Prediction plots were created to display the effects of each significant predictor on the response variables using the patchwork (v1.1.1), ggplot2 (v3.3.5) and ggformula (v0.10.1) packages (Wickham, 2016; Kaplan and Pruim, 2020; Pedersen, 2020) in R. For these plots, as stated in each figure’s caption, the values for other predictors in each model were fixed at the median (continuous variables) or modal value (categorical variables). Dives Dive rates were calculated within each day and night period by dividing the number of dives recorded by the duration of BL messages received during the respective day/night periods. We used the maptools (v1.1-4) R package (Bivand and Lewin-Koh, 2022) to determine the times at which the sun crossed the -12 degrees threshold to separate the day and night periods. We then created a generalized linear mixed model of dive rates as a function of time of day (day or night) using the maximum likelihood parameter estimation method from the glmmTMB package (Brooks et al., 2017). This model included a random effect of tagged individual to account for autocorrelation in dive rates, and modeled data were limited to day/night periods during which less than 50% of the period included BL data gaps (n = 25 day and 32 night periods modeled). Dive duration and maximum depth were each modeled separately using generalized additive mixed models fit as gamma distributions with log link functions using the gamm4 (v0.2-6) and MuMIn (v1.43.17) R packages (Barton, 2020; Wood and Scheipl, 2020) and the maximum likelihood parameter estimation method. Dive duration and depth were both modeled as a function of time of day, cyclical lunar phase (as used in the movement models), distance to shore (including islands), sea surface temperature, chlorophyll-a residuals, and a smoothed term of either dive depth or duration, respectively, formed via shrinkage cubic regression splines with four A B FIGURE 2 Distribution of filtered locations (A, from 16 tags) and dives (B, from 8 tags) across months from satellite tags deployed on Risso’s dolphins in the Southern California Bight between 2009 – 2019. The number of tags that contribute to monthly data are denoted above each bar. Frontiers in Marine Science 06 frontiersin.org Rone et al. 10.3389/fmars.2022.873548 tagged near San Clemente or Santa Catalina Islands. Of the two remaining tags, one was deployed near San Diego and one south of Santa Cruz Island (Table 1; Figure 1). Individuals of three age classes were tagged: adult (n = 14), sub-adult (n = 1), and juvenile (n = 1). Animals were tracked for a median duration of 10.7 days (range = 0.3–19.7) with a combined total of 2,299 locations after Douglas filtering (Table 1). All Risso’s remained within the Southern California Bight throughout the duration of tag deployments (Figure 1). Cumulative straight-line distances between consecutive locations per tag ranged from 14.5 km with an average speed of 0.5 km h-1 to 1,836.2 km at 3.9 km h-1 (Table 2). Movements revealed by satellite tracking showed individual variation within the Southern California Bight: some animals ranged broadly throughout the Bight, while others remained more localized (Figure 1). Risso’s utilized most of the Bight with the 50% core use centered around San Clemente and Santa Catalina Islands and the surrounding basins (Figure 3), a pattern likely influenced by tagging location. Tagged dolphins utilized habitat within the boundaries of all four U.S. Navy ranges dimensions of the basis. Dive depth and duration models included the same implementation of nested random effects, and we used the same AIC selection strategy for detecting lunar phase shifts as was used for the movement models described above. Predictor significance was determined using Wald F Tests (a < 0.05) from the mgcv (v1.8-38) R package (Wood, 2011) and model prediction plots were created for the average individual by excluding all random effects using the predict.gam function from the mgcv package (Wood, 2011). Results Movements Location data were obtained from 16 Risso’s tagged within the Southern California Bight (Table 1; Figure 1). Location data were collected across nine years with data collected from every month except October (Figure 2). Fourteen individuals were TABLE 2 Summary of habitat characteristics at filtered Argos locations for individual Risso’s dolphins. Tag ID Distance from Cumulative deployment distance location (km) moved (km) Average daily distance traveled (km/day) median(range) Distance to shore (km) Distance to mainland (km) Bottom depth (m) Slope (˚) Chl (mg m-3) SST (˚C) median (range) median (range) median (range) median (range) median (range) median (range) GgTag001 57.0 (9.1–138.7) 767.3 56 16.8 (1.2–39.0) 82.6 (50.0–112.2) 948 (31–1,944) 4 (0.1–11) 0.4 (0.2–1.4) 19 (17–21) GgTag002 28.7 (1.4–80.4) 1,301.0 67 17.2 (3.4–35.6) 44.1 (11.9–85.0) 895 (150–1,944) 4 (0.1–11) 0.7 (0.2–2.3) 16 (15–17) 957 (57–1,969) GgTag003* 62.4 (6.9–156.0) 1,836.2 93 28.2 (2.2–72.3) 87.3 (13.4–154.9) GgTag004* 78.5 (7.4–132.6) 1,060.4 94 27.9 (5.9–47.9) 110.3 (43.3–150.1) 1082 (103–1,900) 2 (0–12) 0.4 (0.1–1.4) 16 (14–18) 3 (0.1–12) 0.6 (0.2–1.0) 14 (13–17) GgTag005 40.3 (2.8–63.9) 660.4 98 20.1 (1.1–46.6) 63.9 (30.2–100.3) 918 (135–1,257) 2 (0.1–7) 0.3 (0.2–18) 14 (14–15) GgTag006 41.2 (11.9–118.8) 204.2 24 39.9 (3.7–46.8) 47.6 (19.4–61.8) 893 (193–1,185) 2 (0.2–12) 0.3 (0.2–0.4) 18 (17–19) GgTag008 27.8 (3.2–78.9) 396.2 74 7.1 (1.05–26.7) 77.6 (34.6–95.7) 923 (24–1,307) 4 (0.1–13) 0.3 (0.2–0.4) 19 (18–20) GgTag009 6.8 (3.3–8.2) 14.5 NA 3.7 (1.6–6.5) 33.1 (27.0–34.5) 233 (129–518) 5 (2–7) 0.3 (0.2–0.4) 19 (18–19) GgTag010 19.2 (0.4–113.4) 615.3 61 1.8 (0.02–35.3) 44.4 (28.6–52.4) 114 (9–1,296) 3 (0–13) 0.7 (0–73) 15 (15–16) GgTag011 54.6 (2.0–95.9) 1,153.0 83 38.0 (8.5–68.2) 86.0 (27.7–131.7) 1,367 (363–2,006) 3 (0.1–14) 0.3 (0.2–0.8) 15 (14–16) GgTag012 43.2 (4.6–66.9) 590.2 74 16.4 (1.58–27.7) 26.1 (7.5–55.9) 714 (155–1,295) 2 (0–11) 0.3 (0.2–0.4) 18 (18–19) GgTag015 36.0 (7.1–73.5) 1,031.2 88 18.1 (1.2–52.1) 90.6 (22.6–130.0) 1,004 (108–1,914) 2 (0.1–11) 0.3 (0.2–0.7) 17 (15–18) 17 (15–18) GgTag016 33.8 (0.5–83.4) 804.5 110 18.8 (1.4–39.4) 39.3 (22.9–82.0) 711 (31–1,906) 3 (0–7) 0.9 (0.4–3.6) GgTag018 48.5 (2.0–108.0) 1,615.4 87 42.9 (0.3–93.0) 122.4 (44.3–178.0) 1,078 (71–1,890) 3 (0–13) 0.9 (0.3–6.6) 16 (16–18) Gg2018112098368 72.9 (6.9–83.6) 489.8 64 2.3 (0.09–41.0) 39.5 (29.5–106.3) 898 (9–1,715) 4 (0.3–12) 0.2 (0.1–1.3) 18 (18–19) Gg20190719102468 24.6 (2.5–60.9) 826.7 73 11.8 (0.37–27.8) 52.5 (5.0–95.8) 795 (63–1,323) 3 (0.1–12) 0.5 (0.3–1.0) 21 (18–22) *Tagged within the same group. Cumulative horizontal distances were calculated using straight-line distances between filtered locations. Positions that were located on land were removed for habitat values but retained for distance calculations. Only locations with Argos quality 2 and 3 were used for quantifying depth and slope. Chl, Chlorophyll-a; NA, not applicable; SST, Sea Surface Temperature. Frontiers in Marine Science 07 frontiersin.org Rone et al. 10.3389/fmars.2022.873548 FIGURE 3 Core use areas for 16 Risso’s dolphins tagged in the Southern California Bight from 2009–2019 as indicated by 50% (gray line) and 95% (black line) kernel density estimates of the combined location data across tags. Stars represent deployment locations. Kernel density isopleths were derived from one average daily position to minimize autocorrelation bias, and are overlayed on high resolution ETOPOv2 bathymetry in meters. Military training ranges are indicated by colored polygons as follows: Blue = Southern California Range Complex; Purple = Point Mugu Sea Range; Orange = Southern California Anti-submarine Warfare Range; Pink = Shore Bombardment Area. early winter (Figures 4B, C), with closest distances occurring near the islands during that time (Figure 4C). considered (Figure 1). All tags but one were deployed within either the Southern California Range Complex or Point Mugu Sea Range training areas, and 89% of locations fell within these areas, spanning all 11 months that tags transmitted. Fourteen percent of locations fell within the Southern California Antisubmarine Warfare Range and the Shore Bombardment Areas specifically, and dolphins utilized these areas during seven different months of the year. Details of movement model parameter estimation and significance testing are provided in Table 3. Distance from mainland was significantly predicted by time of day, month, and sine of lunar phase (Figures 4A, B, D), while distance from shore was significantly predicted by month and cosine of lunar phase (Figures 4C, E). Animals were found closer to the mainland during first quarter lunar phase and furthest during the last quarter phase (Figure 4D). Animals were predicted to be farther from shore during a full moon compared to during a new moon (Figure 4E), although 95% confidence intervals were large across all lunar phases. Time of day helped explain differences in distance to mainland, although minimally, with animals closer to mainland during the daylight hours versus night (Figure 4A). Seasonality influenced movements with some variability. Animals were generally closer to land (both mainland and shore) in fall and Frontiers in Marine Science Dives A total of 1,469 h of BL data were recorded (n = 8 tags, range = 0.87–428.3 h per tag) (Table 4). Dive data were collected in seven different months across five years from eight of the 16 tagged individuals (Table 1; Figure 2). These data were comprised of 3,071 dives with a grand median (median of all tag medians) depth of 101 m (max = 528 m) and a grand median dive duration of 5.6 min (max = 11.1 min) (Table 4). The maximum dive depth recorded for each dolphin ranged from 148–656 m (Table 4); the longest dive, 16.1 min, was down to 64 m at night. Dives beyond 500 m depth were conducted during both day and night hours, as demonstrated by a sample two-day dive trace (Figure 5A), with comparable frequency (7% of total dives in each period). Animals dove more frequently at night than during the day (p = 8.14e-07), with night and day dive rates at 5.21 (SE = 0.5) and 2.73 (SE = 0.5) dives per hour, respectively. Shallow dives (< 100 m) were more frequent during the day (63%, Figure 5B) versus night (35%, Figure 5B). 08 frontiersin.org Rone et al. 10.3389/fmars.2022.873548 TABLE 3 Model results including type II Wald chi-square (movement models) or Wald F (dive models) testing and parameter estimation with associated significance testing. Response variable Explanatory variable Chi-Square or F testing Model parameter estimation and Significance testing Chisq of F Value p Value Parameter Variable Distance to Mainland Time of Day 18.250 0.000 Month 36.492 0.000 sin (Lunar Phase) 18.748 0.000 Distance to Shore Time of Day 0.215 0.643 Month 22.855 0.011 cos (Lunar Phase) 9.060 0.003 Dive Depth Time of Day SST b b Std. Error z Value p Value Intercept [Day, April] 4.407 0.133 33.220 0.000 Night 0.022 0.005 4.270 0.000 January -3.550 0.227 -1.560 0.118 February -1.690 0.156 -1.090 0.276 March -0.290 0.100 -2.900 0.004 May -1.163 0.291 -4.000 0.000 June -0.406 0.195 -2.080 0.038 July -0.209 0.170 -1.230 0.218 August -0.355 0.230 -1.540 0.122 September -1.154 0.367 -3.140 0.002 November -0.686 0.225 -3.050 0.002 December -0.884 0.324 -2.730 0.006 sin (Lunar Phase) -0.144 0.033 -4.330 0.000 Intercept [Day, April] 3.311 0.291 11.375 0.000 Night 0.009 0.020 0.463 0.643 January -1.354 0.496 -2.731 0.006 February -0.621 0.324 -1.920 0.055 March -0.082 0.205 -0.399 0.690 May -0.835 0.643 -1.298 0.194 June -0.412 0.423 -0.974 0.330 July -0.506 -0.370 -1.367 0.172 August 0.083 0.483 0.172 0.863 September -1.716 0.778 -2.206 0.027 November -1.383 0.496 -2.791 0.005 December -1.398 0.692 -2.019 0.043 cos (Lunar Phase) -0.185 0.062 -3.010 0.003 Intercept [Day] 5.050 0.295 17.131 0.000 103.949 0.000 Night 0.242 0.024 10.196 0.000 3.069 0.080 SST -0.029 0.016 -1.752 0.080 Chlorophyll-a Residuals 0.007 0.935 Chlorophyll-a Residuals 0.002 0.027 0.081 0.935 Distance to Shore 11.793 0.001 Distance to Shore 0.003 0.001 3.434 0.001 cos (Lunar Phase) 20.951 0.000 cos (Lunar Phase) -0.112 0.025 -4.577 0.000 Dive Duration 22255 0.000 Dive Duration Dive Duration Effective degrees of freedom = 2.687 0.185 8.284 119.230 0.000 Night -0.142 0.013 -10.919 0.000 SST 2.426 0.119 SST 0.016 0.010 1.558 0.119 0.559 Time of Day Intercept [Day] 1.534 0.000 Chlorophyll-a Residuals 0.341 0.559 Chlorophyll-a Residuals 0.008 0.014 0.584 Distance to Shore 8.556 0.003 Distance to Shore -0.001 0.001 -2.925 0.003 cos (Lunar Phase) 8.405 0.004 cos (Lunar Phase) 0.038 0.013 2.899 0.004 Dive Depth 38290 0.000 Dive Depth Effective degrees of freedom = 2.898 Parameter information for categorical variables is relative to the categories listed in brackets next to each model’s intercept term. For the dive models, smooth term effective degrees of freedom are listed in place of model parameter information. m-3 (range = 0–73) with the lowest and highest concentrations in January 2012 (Table 2). Details of dive model parameter estimation and significance testing are provided in Table 3. Dive depth was significantly Across all years and months, Risso’s were documented in median sea surface temperatures of 17˚C (range = 13–22˚C), with the coolest temperatures in June 2010 and warmest in July 2019 (Table 2). The median chlorophyll-a concentration was 0.4 mg Frontiers in Marine Science 09 frontiersin.org Rone et al. 10.3389/fmars.2022.873548 A B C D E FIGURE 4 Prediction plots from the fitted models for distance to mainland (A, B, D) and distance to shore (C, E). Solid black lines/dots represent mean predicted values with shaded areas/error bars representing the 95% CI. Hash marks along the x-axis shows the spread of data. For the creation of these plots, values for other predictors in each model were set at the median or modal value as follows: time of day = day, month = April, sine of lunar phase = -0.370, cosine of lunar phase = -0.010. TABLE 4 Summary of dive parameters by individual of eight Risso’s dolphins satellite-tagged within the Southern California Bight. Whale ID Total behavior log data (h) Dives No. dives for analyses Duration (min.) median (range) Depth (m) median (range) GgTag008 81.8 116 3.7 (1.1–9.3) 59 (30–608) GgTag009 0.8 5 5.3 (4.4–5.9) 124 (42–132) GgTag011 299.5 405 6.2 (0.9–13.8) 152 (30–624) GgTag012 0.9 5 11.1 (5.4–11.4) 528 (90–560) GgTag015 259.8 195 5.8 (1.0–16.1) 74 (30–576) 232 (50–656) GgTag018 428.3 1285 6.4 (1.4–11.1) Gg-20181120-98368 169.5 470 4.0 (1.1–11.0) 72 (30–592) Gg-20190719-102468 228.4 590 5.2 (1.1–11.1) 78 (30–608) Frontiers in Marine Science 10 frontiersin.org Rone et al. 10.3389/fmars.2022.873548 A B FIGURE 5 (A) Sample two-day dive trace from a Risso’s dolphin in the Southern California Bight with the time of day denoted by dots for day (yellow) and night (blue). Blanks indicate periods where there are gaps in the data. Extended surface time is indicated by a straight horizontal line along 0 m. (B) Distribution of dive depth estimates for all dive tags during the day and night. For both plots, time of day was defined by a solar altitude (≥ -12 degrees for day and < -12 degrees for night), which was calculated for every dive start at the modeled location estimate closest in time. characteristic of these steep topographic areas (Kruse et al., 1999). Outside of the Southern California Bight, they are documented in various habitats ranging from shallow waters such as 2–5 m in Monterey Bay, CA (Jefferson et al., 2014b) to offshore pelagic waters of Hawai'i in depths exceeding 3,000 m (Baird, 2016). In the Azores around Pico Island, they can be found from 0–10 km from shore (Hartman et al., 2016) where steeply sloped nearshore topography results in water depths increasing from 200 m to 1000 m within 5 km (Hartman et al., 2008). While relatively short deployment durations of this study preclude any meaningful discussion of large-scale movements into and out of the Southern California Bight, they do provide new insights into localized movements. Changes in Risso’s density indicate latitudinal seasonal shifts occur along the U.S. west coast (Green et al., 1992; Forney and Barlow, 1998). In the Bight, Santa Catalina Island has repeatedly been identified as important habitat for Risso’s (Shane, 1995a; Shane, 1995b; Soldevilla et al., 2010), with acoustic detections occurring on 75% of the days at a site on the south end of the island (Soldevilla et al., 2010). The near year-round acoustic presence at Santa Catalina Island may suggest that an island-associated population exists and/or a greater Southern California Bight resident population, with possible seasonal influx from a larger migratory population (Soldevilla et al., 2010). In this study, all Risso’s were tagged in the outer waters (defined as waters offshore of the mainland coast) of the Southern California Bight, mainly in the greater surrounding waters of the outer predicted by dive duration, time of day, distance to shore, and cosine of the lunar phase (Figure 6). Dive duration was significantly predicted by dive depth, time of day, distance to shore, and cosine of lunar phase (Figure 7). Dives were deeper and shorter at night than during the day (Figures 6B, 7B), when dolphins were further offshore (Figures 6C, 7C), and during a full versus a new moon, respectively (Figures 6D, 7D). Wide 95% confidence intervals for dive depth and duration as a function of lunar phase reflect low sample sizes at some lunar phases (see hash marks along x-axis of (Figures 6D, 7D). Discussion As regular inhabitants of the Southern California Bight, Risso’s have been frequently documented on line-transect surveys throughout the region (e.g., Barlow and Forney, 2007; Jefferson et al., 2014a) and across seasons in recent years (e.g., Schorr et al., 2019). Habitat used by tagged dolphins in this study ranged from the deep waters of the numerous basins to the shallow waters inshore of San Clemente and Santa Catalina Islands along the mainland coast (9–2,006 m), with Argos locations sometimes clustered around steep slope features (Figure 1). Globally, densities of Risso’s appear higher over the outer continental shelf and upper regions of the continental slope (Jefferson et al., 2014b). The patchy distribution and local abundance of this species may be driven by enhanced productivity associated with currents and upwellings Frontiers in Marine Science 11 frontiersin.org Rone et al. 10.3389/fmars.2022.873548 A B C D FIGURE 6 Prediction plots of significant variables (A–D) from the models of dive depth. Solid black lines/dots represent mean predicted values with shaded areas/error bars representing the 95% CI. Hash marks along the x-axis shows the spread of data. For the creation of these plots, values for other predictors in each model were set at the median or modal value as follows: time of day = night, Chl residuals = -0.008, SST = 15.81, distance to shore = 24.68 km, sine of lunar phase = -0.736, cosine of lunar phase = -0.435, dive duration = 5.6 min. Chl, Chlorophyll-a (mg m-3); SST, Sea Surface Temperature (°C). despite demonstrating varied and often highly mobile movement patterns, and most remained relatively local to their tagging location (Figure 3), suggesting year-round residence within the Bight is plausible. Alternatively, if some of these individuals were members of the larger WashingtonOregon-California migratory stock, it is entirely feasible that the limited number of tags and their generally short durations simply did not capture movements out of this region. Unfortunately, durations like these are not uncommon for remotely deployed tags on small cetaceans, where factors including surface activity, interactions with conspecifics, and the challenge of achieving optimal tag placement often lead to premature detachments. The seasonality of inshore/offshore movements were most apparent when considering distance to any landmass, rather than to the mainland coast. According to our models, Risso’s were significantly closer to land, though this was typically an Channel Islands. Nearly all individuals spent at least some of their time within the shallower nearshore waters of these islands (Figure 1). Of the 16 Risso’s, 12 approached within 20 km of Santa Catalina Island (Figure 1), in all seasons and in eight different years. Of these 12, only half were tagged within 20 km of this island (min = 3 km). All but one of these six tags also left this island; the brief transmission for the sixth tag prevents further discussion of its movements. In fact, the only animal out of the 16 that demonstrated close association with Santa Catalina Island for most of its tag transmission was actually tagged in the San Nicolas Basin on the west side of San Clemente Island. While these tags may not confirm or refute the presence of island-associated individuals or populations, they do further demonstrate the importance of Santa Catalina Island for the broader, regional population throughout the year. While animals in this study were tagged in all but one month of the year, all remained within the Southern California Bight Frontiers in Marine Science 12 frontiersin.org Rone et al. 10.3389/fmars.2022.873548 A B C D FIGURE 7 Prediction plots of significant variables (A–D) from the models of dive duration. Solid black lines/dots represent mean predicted values with shaded areas/error bars representing the 95% CI. Hash marks along the x-axis shows the spread of data. For the creation of these plots, values for other predictors in each model were set at the median or modal value as follows: time of day = night, Chl residuals = -0.008, SST = 15.81, distance to shore = 24.68 km, cosine of lunar phase = -0. 435, dive depth = 123.5 m. Chl, Chlorophyll-a (mg m-3); SST, Sea Surface Temperature (°C). While earlier research in the Bight suggested that Risso’s foraged at night (Shane 1995a; Soldevilla et al., 2010) with slow travel/rest the predominant daytime behaviors (Shane, 1995b; Smultea et al., 2018), more recent studies have documented daytime foraging excursions (Arranz et al., 2018; Arranz et al., 2019; Benoit-Bird et al., 2019). Dives in excess of 500 m recorded on these tags occurred across all times of day (max = 607 m day, 656 m night) (Figure 5B) and are comparable to results from Benoit-Bird et al. (2019). Dives were predicted to be 1.27 times deeper (eb; b = 0.24, SE = 0.02, p-value < 2e-16) and 4.70 times shorter (eb; b = -0.14, SE = 0.01, p-value < 2e-16) at night versus day (Figures 6B, 7B), and to occur nearly twice as often. This depth difference is potentially driven by both a greater number of dives at < 100 m depth during the day (63% day versus 35% night) and > 100 m at night (65% night versus 37% day) (Figure 5B). These results would align with increased nighttime foraging activities that have been described island, from September through January (p < 0.05). Although market squid spawn in the Southern California Bight year-round (Navarro et al., 2018), concentrated spawning aggregations occur nearshore over shallow sandy substrate in southern California along the mainland coast and the Channel Islands (Zeidberg et al., 2012; Van Noord, 2017) from October through April/May (Fields, 1965). Past observational research suggested that both Risso’s and short-finned pilot whales (Globicephala macrorhynchus), another squid-eating species common within the Southern California Bight prior to the 1982–83 El Niño event, shift into the Channel Islands during the winter months to feed on spawning market squid (Sinclair, 1992; Shane, 1995b). Our data suggest this seasonal, prey-driven shift has continued in the decades since. These may be fine-scale, localized shifts by animals that remain within the greater Southern California Bight throughout the year, those of migratory animals shifting into the Bight, or both. Frontiers in Marine Science 13 frontiersin.org Rone et al. 10.3389/fmars.2022.873548 (Clarke and Denton, 1962). Deep scattering layer species seek lower light levels to avoid predators, and are thus found at deeper depths during brightly moonlit nights (Boden and Kampa, 1967), increasing use of near-surface waters at night as the moon wanes (Herná ndez-Leó n et al., 2002). It may be a cost-effective strategy for Risso’s to pursue prey at deeper depths around a full moon—as suggested by our model—when layers may provide additional biomass to target. The question also should be raised whether changes in dive behavior associated with lunar phases are driven by illumination levels themselves, or whether they are the result of an endogenous signal that is associated with the 29.5 day lunar cycle, or a combination of both. It seems plausible that light may have significant biological influence on marine systems with predominantly clear water and skies but in geographic locations where waters are more turbid and skies are obscured by clouds for at least part of the year, as they are in the Southern California Bight (e.g., Rastogi et al., 2016), an endogenous cue may play a more significant role. In (Benoit-Bird et al. 2009), lunar phase accounted for more variability in micronekton migration than surface irradiance, suggesting that lunar effects other than light levels effect these prey, and in turn, the predators that pursue them. Results from this study suggest seasonal trends in movements, demonstrate year-round presence within the Bight, and further describes lunar and diel influences on behavior. We have presented our statistical findings and suggest biological explanations based on findings in the literature. However, we must acknowledge that statistical significance according to model output should be interpreted with caution from a biological perspective, as these data have limitations. Although several of these tags lasted nearly three weeks, the kernel density analysis was likely biased by deployment location. In addition to sample size limitations, some of these data included gaps from failed transmission and from short tag durations that didn’t span across seasons and lunar phases. The effects of Argos location quality and resolution, and the resulting prediction errors from modeled animal tracks based on Argos data, further limit interpretation of these results. While we filtered Argos locations to reduce error before using them in standardized movement models, predicted locations had standard errors ranging from 0 to 26 km. There were occasional adjustments to tag programming to optimize data collection that may have affected some statistics. When looking at environmental variables such as sea surface temperature and chlorophyll-a, in-situ measurements collected around an animal is the only way to provide accurate, real-time representation of that variable with high resolution, but this was not available for these tags. Remote sensing can capture patterns in these data over a broad spatial region, but cloud cover is an inherent limitation. Although we were able to combine a remote previously (Soldevilla et al., 2010; Arranz et al., 2019; BenoitBird et al., 2019). While Benoit-Bird et al. (2019) documented elevated Risso’s echolocation in shallow waters at night, we are unable to make further comparisons due to the limited temporal and spatial coverage of acoustic data and the lack of nighttime tag data from that study. Described as dynamic foragers, Risso’s target different prey layers, switching between shallow water generalist and deep water specialist strategies (Benoit-Bird et al., 2019) and appear to consider optimal energy investments and tradeoffs prior to the next dive (Arranz et al., 2018; Visser et al., 2021). Jumbo squid are at depths > 250 m during the day, travel to near-surface waters at dusk, and then demonstrate highly variable dive behavior until they presumably find a zone to exploit for food (Gilly et al., 2006). In Hawai'i, the mesopelagic layer migrates from the 400–700 m depth during daylight hours to 0–400 m at night, though pilot whales appear to target the deeper 400 m layer at night, potentially in search of higher caloric prey (Owen et al., 2019). In the Azores, Risso’s target two deep scattering layers, a broad one at 500–700 m and a narrow one at 400 m, with both layers performing diel vertical migration (Visser et al., 2021). Off Santa Catalina Island, Risso’s were detected in three layers: a deep static layer at 450 m, a mid-water migrating layer at 100–300 m, and an intermittent shallow layer at 50 m along with scattered patches consisting of fish and squid (Benoit-Bird et al., 2019). It’s possible that Risso’s utilize the photic advantage of daylight to capitalize on the low energetic costs of shallow layer exploitation in between bouts of deep layer explorations (the switching behavior documented in Benoit-Bird et al., 2019) and thereby conserve energy for nighttime hunts that are directed at preferential prey, such as jumbo squid, that while vertically migratory, are not necessarily reaching shallow waters throughout the nighttime hours. Lunar phase has been linked to changes in zooplankton biomass (Herná ndez-Leó n et al., 2002) and depth (Boden and Kampa, 1967) and horizontal displacement in the mesopelagic layer in Hawaiʻi (Benoit-Bird et al., 2009). In this study, lunar phase predicted both horizontal and vertical shifts, albeit with large confidence intervals. Risso’s were located 1.45 times further away from shore (including islands) (Figure 4E) during a full moon than a new moon, a pattern similar to that observed in Hawaiian pilot whales (Owen et al., 2019). Lunar phase has been associated with changing use of the water column in Cuvier’s beaked whales (Ziphius cavirostris) (Barlow et al., 2020) and jumbo squid (Gilly et al., 2006), and like the Risso’s tagged in this study, pilot whales (Owen et al., 2019), Galá pagos fur seals (Arctocephalus galapagoensis) (Horning and Trillmich, 1999), and bigeye tuna (Thunnus obesus) (Musyl et al., 2003) all appear to use deeper waters during a full moon. Full moon light levels at 100–150 m are equivalent to mid-day light levels at ~500 m Frontiers in Marine Science 14 frontiersin.org Rone et al. 10.3389/fmars.2022.873548 sensing and an in-situ dataset to derive sea surface temperature at modeled locations, we had to create 8-day averaged composites for chlorophyll-a to create gap free composites. This mismatch in resolution between animal location estimates and environmental variables is another potential source of error. It should be noted that remote sensing can underestimate values for chlorophyll-a and sea surface temperature (Gomez et al., 2020; Keates et al., 2020), and absolute values should be interpreted with caution. These aforementioned factors may have influenced the significance of these predictors in the dive models. We believe one of the greatest values of these analyses are their ability to aid formulation of new hypotheses and research directions. Additional targeted data collection will strengthen model results and allow for more concrete biological interpretations of these findings. Deployments in other regions of the Southern California Bight as well as during specific periods where data gaps currently exist [e.g., gaps in month (Figure 2) and lunar phase (Figure 6)] would provide a more comprehensive regional dataset. The short duration of these tags highlights the need for more tag development to improve attachment while continuing to minimize impact. A regional photo-identification catalog, genetic analyses, and the use of medium-duration, dart attached archival acoustic tags (e.g., Sweeney et al., 2022) along with prey mapping would provide meaningful connections between the existing research within this region. It is our hope that this work will foster purposeful discussions and ideas for future work to manage and protect this species. Funding Funding for fieldwork and tags was provided by the U.S. Navy, Pacific Fleet (N66604-14-C-0145, N66604-18-P-2187) and via the Californian Cooperative Ecosystems Studies Unit (N62473-19-20025), the Chief of Naval Operations Environmental Readiness Division (N45) via a grant from the Naval Postgraduate School (Grant No. N000244-10-1-0050) and the Environmental Security Technology Certification Program (N66604-14-C-2438). Additional support was provided by the Office of Naval Research and the Living Marine Resources program. Acknowledgments We would like to thank the U.S. Navy, Pacific Fleet, N45, the Environmental Security Technology Certification Program, the Living Marine Resources program, and the Office of Naval Research for support of this research. We thank all participants from the Marine Mammal Monitoring on Navy Ranges program at the Naval Undersea Warfare Center, Jeff Foster for contributions in the field, and the Naval Postgraduate School and Californian Cooperative Ecosystems Studies Unit for contract facilitation. We thank Stacy DeRuiter for assistance with analyses and Chip Johnson, Jessica Chen, and Shannon Coates and three reviewers for their thoughtful comments and suggestions. This study was conducted under U.S. National Marine Fisheries permits No. 540-1811, 16111, 15330, 20605. Tagging was approved by the Cascadia Research Collective Institutional Animal Care and Use Committee. Data availability statement Conflict of interest The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Ethics statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The animal study was reviewed and approved by Cascadia Research Collective Institutional Animal Care and Use Committee. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Author contributions BR, EF, GS, and SW conducted the fieldwork. BR, GS, and DS conducted the data analysis. All authors contributed to the article and approved the submitted version. Frontiers in Marine Science 15 frontiersin.org Rone et al. 10.3389/fmars.2022.873548 References Andrews R. D., Baird R. 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Review The Relationship between the Gut Microbiota and Exercise: A Narrative Review Diego Domínguez-Balmaseda and Guillermo García-Pérez-de-Sevilla * Faculty of Sport Sciences, Universidad Europea de Madrid, 28270 Madrid, Spain * Correspondence: guillermo.garcia@universidadeuropea.es Abstract: The intestinal microbiota plays an important role in the homeostasis of the intestinal tract, and the fact that exercise could have an impact on its functionality is currently the subject of various studies. Exercise is used as a strong preventive and treatment strategy in numerous chronic diseases, influencing metabolism, immunity, and physiological functions. Some recent studies provide information on exercise as a factor that could favor the growth of beneficial species and increase the diversity of the intestinal microbiota. However, the difficulty in determining the true effect of exercise is only due to the confounding influence of lifestyle and dietary habits. Regardless, there is still interest in the implications of the gut microbiota for elite sports because the details are considered crucial for success in different competitions. This narrative review tries to collect the information available in the literature on the role that exercise plays in the modulation of the balance of the intestinal microbiota. Keywords: gut microbiota; exercise; health; athletes; sports 1. Gut Microbiota Citation: Domínguez-Balmaseda, D.; García-Pérez-de-Sevilla, G. The Relationship between the Gut Microbiota and Exercise: A Narrative Review. Hygiene 2022, 2, 152–162. https://doi.org/10.3390/ hygiene2040014 Academic Editor: Daniela Haluza Received: 29 August 2022 Accepted: 22 September 2022 Published: 28 September 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). The main objective of the narrative is to analyze the association between the intestinal microbiota and exercise and to conclude if there is sufficient evidence to consider new approaches for intestinal recovery and protection as a new important factor in performance. Joshua Lederberg, an American biologist who received the Nobel Prize in Medicine in 1958, stated that the microorganisms found in humans protect us symbiotically in our own body. These communities of microorganisms, known today under the term microbiota, consist of a variety of microorganisms that includes eukaryotes, archaea, bacteria, and viruses, constituting about 1.3% of the body mass, and are essential to maintain optimal health. These microorganisms inhabit different parts of the body, colonizing the mucosa within a given anatomical niche, such as the respiratory system, the walls of the digestive system or also the urogenital system, among other surfaces [1]. More diversity of the microbiota is found in the intestinal tract and mouth, while the skin contains less diversity, followed by the vagina, although a consensus has not yet been reached on the number of species that inhabit it [2]. The microbiota is known as the set of microorganisms residing in each ecosystem, with a symbiotic relationship and with adaptive properties and rapid renewal, forming a large metabolic unit. The intestinal microbiota is a set of microorganisms made up of approximately 100,000 million bacteria that live in our intestine. The microbiota is responsible, among other functions, for maintaining the well-being of the intestinal mucosa, helping us digest food and converting harmful elements into less toxic substances. In 2014, the term “intestinal flora” was coined to describe the intestinal microbiota, although later, it was changed to gut microbiota [3]. Some gut microbiota bacteria attach to the cells’ external wall and to specific receptors called adhesins. They can adapt to the specific conditions of the environment, such as humidity, temperature, or pH, being able to activate defense mechanisms in case of interaction, for example, with a harmful virus. The homeostasis of the intestinal tract depends Hygiene 2022, 2, 152–162. https://doi.org/10.3390/hygiene2040014 https://www.mdpi.com/journal/hygiene Hygiene 2022, 2 153 on the balance between the microbiota, intestinal permeability, and local immunity; in the event of any non-adaptive alteration, it can produce negative effects on the host itself. When these alterations occur, it is known as intestinal dysbiosis, which can affect digestion, nutrient absorption, vitamin production, and the control of harmful microorganisms [3]. Most of the bacteria that make up the gut microbiota live in the colon [4]. The colon is described as a potentially active organ due to the extensive activity of the gut microbiota and has been compared to the liver due to its high metabolic capacity [5]. The microbiota is dominated by four main phyla: Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria [6]. Although there are also bacteria in smaller proportions that are vital for the function of the microbiota, Firmicutes and Bacteriodetes stand out due to their proportion throughout the intestinal tract, as they represent 60% and 20%, respectively, of all the gut microbiota, while the Proteobacteria phylum covers 5% and Actinobacteria 3%, approximately [7]. 2. The Gut Microbiota Function The functions of the gut microbiota include the following: 2.1. Protection Function in the Intestinal Barrier The gut microbiota plays an important role in helping to maintain the integrity of the intestinal barrier, thus protecting the host from pathogens. In turn, the microbiota develops defense mechanisms together with the epithelial cells, which are responsible for establishing an effective and harmonious intestinal barrier, preventing the passage of harmful substances, antigens, toxins, and microbial products and, on the other hand, the absorption of nutrients benign [8]. The main elements that form the intestinal barrier are the mucus, the tight junctions between enterocytes, antimicrobial peptides, and immunoglobulin A (IgA) secretion [9]. The intercellular junctions of the intestinal barrier separate the internal environment from the external environment, making it impermeable to certain pathogenic substances and permeable to nutrients [10]. The mucosa interacts with the microbiota to maintain intestinal homeostasis; in addition, the intestinal microbiota induces the biosynthesis of antimicrobial peptides by mucosal Paneth cells, such as cathelicidins, lectins, and defensins, by activating their membrane receptors. Bacteriodetes thetaiotaomicron and Lactobacillus innocua are two of the main agents that stimulate Paneth cells [11]. Another reinforcement of this barrier function by the microbiota of the intestinal lumen is the production of lactic acid by Lactobacillus, which reinforces the ability of human lysozyme released in the lumen to destroy peptidoglycan [12]. Short-chain fatty acids (SCFAs) released by bacterial fermentation also stimulate the production of the antimicrobial peptide cathelicidin. These SCFAs acidify the intestinal lumen and prevent colonization by pathogens sensitive to this pH, such as Salmonella, Escherichia, and Clostridium difficile [9]. The homeostasis of the barrier function would result in an optimal symbiosis; otherwise, if the function of the intestinal barrier does not favor an adequate mechanism, it will have an exaggerated or abnormal immune response that would lead to poor control of intestinal permeability, leading to what we know as intestinal dysbiosis [13,14]. 2.2. Function on the Development of the Immune System The gut microbiota contributes to gut immunomodulation in conjunction with the innate and adaptive immune systems [9]. In line with the previous protection function, the immune system has co-evolved to maintain a symbiotic relationship with the commensal species of the microbiota while keeping other species considered pathogenic in this intestinal ecosystem under control. About 70% of the lymphocytes in the human body live in the digestive system, which gives us an idea of the importance of this function in the development [15]. Components and cell types of the immune system involved in the immunomodulatory process include gut-associated lymphoid tissues (GALT), effector and regulatory T cells, IgA- Hygiene 2022, 2 154 producing B (plasma) cells, Group 3 innate lymphoid cells, resident macrophages, and dendritic cells in the lamina itself [16]. The role of the gut microbiota in forming a functional GALT is implicit in the poor development of Peyer’s patches and isolated lymphoid follicles that are marked by an abundance of IgE B cells rather than IgA B cells [17]. The absence of the intestinal microbiota causes the plasma cell populations of the intestinal mucosa to differentiate mainly towards greater production of IgE, instead of IgA, which is associated with an increased risk of allergies [14]. Effector T cells are regulated in the mucosa by Th17 cells. Some bacteria, such as Bacillus fragilis, belonging to the Bifidobacterium or Clostridium genera, activate Treg lymphocytes via Toll-like receptors (TLR) that are part of the innate immune system, inducing systemic anti-inflammatory effects, which helps to develop tolerance to antigens present in the diet and some bacteria in the lumen [16]. This cross-communication between the microbiota and the epithelial cells is also responsible for the release of IgA and for stimulating the production of glycoproteins to form more mucus. The main stimulators of epithelial IgA production are Gram-negative species such as Bacteroides, which activate dendritic cells in the mucosa so that they induce IgA production by plasma cells (activated lymphocytes) in the submucosa. This IgA prevents microorganisms from activating pro-inflammatory pathways, as they cannot bind when coated by these antibodies, to receptors such as TLRs. In turn, IgA collaborates in capturing antigens and prevents their penetration through the intestinal barrier, limiting the movement of pathogens and neutralizing toxins throughout the lumen [18]. The presence of high populations of Suturella, on the other hand, reduces IgA production in the lumen, since this bacterium releases IgA-degrading proteases [15]. The complexity of the intestinal microbiota, per se, is a defense against the colonization of this ecosystem by other types of microorganisms [18]. This is especially important in adults who have a much more stable intestinal microbiota than children or subjects with intestinal dysbiosis. The production of bacteriocins by the resistant microbiota is responsible for this resistance to colonization of the lumen by other bacteria. Similarly, the bacteriophages in the intestinal lumen can prevent the establishment of susceptible bacterial species. All these mechanisms prevent the bacterial translocation factor through the intestinal epithelium and therefore prevent a systemic immune response to a possible infection [19]. 2.3. The Intestinal Microbiota Performs a Metabolic Function Contributing to the digestion of nutrients from the diet [9]. The gut microbiota obtains its nutrients mainly from carbohydrates in the diet. Fermentation of carbohydrates that escape proximal digestion and indigestible oligosaccharides are metabolized by bacteria such as Bacteroides, Roseburia, Bifidobacterium, Faecalibacterium, and Enterobacteria, resulting in the synthesis of SCFAs such as butyrate, propionate, and acetate, which are rich sources of energy for the host [20]. This metabolic process is favored by the more acidic pH of the right colon. In contrast, in the left colon, microorganisms grow more slowly due to the low arrival of food with a more neutral pH [21]. Members of the genus Bacteroides are the predominant organisms involved in carbohydrate metabolism, achieved by expressing enzymes such as glycosyltransferases, glycoside hydrolases, and polysaccharide lyases. The best example among these organisms is Bacteroides thetaiotaomicron, which is endowed with a genome encoding more than 260 hydrolases [22]. The gut microbiota also has an efficient protein metabolizing machinery that functions through microbial proteinases and peptidases in conjunction with human proteinases. Various amino acid transporters in the bacterial cell wall facilitate the entry of amino acids from the intestinal lumen into the bacteria, where they convert the amino acids into small signaling molecules and antimicrobial peptides (bacteriocins) [23]. Hygiene 2022, 2 is Bacteroides thetaiotaomicron, which is endowed with a genome encoding more than 260 hydrolases [22]. The gut microbiota also has an efficient protein metabolizing machinery that functions through microbial proteinases and peptidases in conjunction with human proteinases. Various amino acid transporters in the bacterial cell wall facilitate the entry of amino 155 acids from the intestinal lumen into the bacteria, where they convert the amino acids into small signaling molecules and antimicrobial peptides (bacteriocins) [23]. Another metabolic function of great importance is described in the literature, such as Another metabolic function of great importance described in the the synthesis of vitamins such as vitamin K and vitamin B, is and the function ofliterature, lipid regu-such as the synthesis of vitamins such as vitamin K and vitamin B, and the function of lipid lation. In addition, members of the genus Bacteroides synthesize conjugated linoleic acid regulation. In addition, members of the genus Bacteroides synthesize conjugated linoleic (CLA), which has antidiabetic, antiatherogenic, antiobesogenic, hypolipidemic, and imacid (CLA), which has antidiabetic, antiatherogenic, antiobesogenic, hypolipidemic, and munomodulatory properties [24]. The gut microbiota has also been shown to positively immunomodulatory properties [24]. The gut microbiota has also been shown to positively impact lipid metabolism by suppressing the inhibition of lipoprotein lipase (LPL) activity impact lipid metabolism by suppressing the inhibition of lipoprotein lipase (LPL) activity in adipocytes [25]. in adipocytes [25]. 3. Factors ThatThat Modify the Gut Microbiota 3. Factors Modify the Gut Microbiota Many factors could modify the gut microbiota. The The onesones that that stand out are or or Many factors could modify the gut microbiota. stand out diet are diet eating habits, probiotics, prebiotics and polyphenols, pharmacological treatments, and eating habits, probiotics, prebiotics and polyphenols, pharmacological treatments, and physical exercise [26].[26]. physical exercise Other factors involved, apartapart fromfrom thosethose mentioned above, are stress, genetics, age, age, Other factors involved, mentioned above, are stress, genetics, types of birth, and exercise [26], (Figure 1). types of birth, and exercise [26], (Figure 1). Figure 1. Factors modifying the gut [26]. [26]. Figure 1. Factors modifying themicrobiota gut microbiota Exercise and Gut Microbiota There are several potential mechanisms by which exercise could modify the gut microbiota. Several studies by Hoffman Goetz and colleagues [27,28] have reported that exercise alters intraepithelial lymphocyte gene expression, downregulating proinflammatory cytokines and up-regulating anti-inflammatory cytokines and antioxidant enzymes. These immune cells reside close to microbial communities and produce antimicrobial factors that are essential for mediating host-microbial homeostasis [29]. Similarly, exercise can affect the integrity of the intestinal mucus layer, which plays an important role in preventing pathogens from adhering to the intestinal epithelium and serves as an important substrate for certain mucosa-associated bacteria, such as A. Hygiene 2022, 2 156 muciniphila. Exercise raises core temperature and causes heat stress, particularly when performed for prolonged periods or in a hot environment [27]. Exercise can also reduce intestinal blood flow by more than 50%, with significant intestinal ischemia occurring within 10 min of high-intensity exercise [28]. At rest, the splanchnic bed undergoes rapid reperfusion. Although the intestine is an anaerobic environment, intestinal epithelial cells primarily use oxidative metabolism, and high-intensity exercise is known to transiently impair intestinal barrier function [28]. Therefore, exercise-induced heat stress and ischemia may produce interactions between the intestinal mucosal immune system and the microbiota residing in the intestinal lumen. Evidence for the role of exercise in modifying the human gut microbiota first emerged from cross-sectional studies in 2014 [29]. Clarke et al. found that the gut microbiota of professional rugby players had a higher alpha diversity and a higher relative abundance of 40 different bacterial taxa than the gut microbiota of the control group, being sedentary subjects [29]. More recently, Bressa et al. compared active women with sedentary controls and found that women who engaged in at least 3 h of exercise per week had increased levels of Faecalibacterium prausnitzii, Roseburia hominis, and Akkermansia muciniphila [30], which are producers of butyrate [31], while A. muciniphila has been associated with a lean body mass index (BMI) and better metabolic health [32]. Several studies have also attempted to correlate the composition and metabolic capacity of the microbiota with cardiorespiratory fitness, with Durk et al. showing a higher ratio of Firmicutes to Bacteroidetes in subjects with better metabolic health, and was significantly correlated with maximal oxygen consumption (VO2max ) [33]. Estaki et al. found in younger adults that microbial diversity and abundance of butyrate-producing bacterial taxa were positively correlated with cardiorespiratory fitness [34], while Barton et al. found, through metagenomic analyses differences in fecal microbiota between athletes and sedentary controls. Athletes showed relative increases in different metabolic pathways, such as amino acid biosynthesis and carbohydrate metabolism, as well as increased production of SCFAs, such as acetate, propionate, and butyrate produced by the microbiota [35]. However, all these studies were limited by their cross-sectional design and their difficulty in controlling diet effects on the gut microbiota. There is considerable interindividual variability in the microbiota composition, and active individuals tend to eat differently from sedentary ones [36]. For example, Clarke and colleagues found that increased protein intake by elite rugby players explained many of the observed differences in gut microbiota [29]. These limitations suggested the need to analyze longitudinal studies to determine whether exercise independently alters the gut microbiota in humans. Recently, Allen and colleagues obtained results in the first controlled longitudinal study with a strict diet to evaluate the effects of exercise on the gut microbiota. Various bacterial taxa were differentially altered by exercise depending on the subjects’ BMI. For example, exercise increased Faecalibacterium species in lean subjects but reduced their abundance in obese subjects, Bacteroides species decreased in lean subjects and increased in obese subjects. Six weeks of exercise also increased the abundance of butyrate-producing taxa and fecal acetate concentrations, but only in lean subjects. Interestingly, most bacterial taxa and SCFAs that increased with exercise subsequently decreased during the 6-week sedentary rest period, indicating that the effects of exercise on the microbiota were transient and reversible [37]. Similarly, Cronin and colleagues sought to determine whether a short-term exercise regimen, with or without whey protein supplementation, could alter gut microbial composition and function in predominantly overweight or obese adult men and women. The post-intervention evaluation revealed no significant changes in taxonomic composition or metabolic pathways in either exercise group compared to baseline. However, a trend for increased bacterial diversity was observed in the exercise and exercise plus whey protein groups compared to the group receiving whey protein alone. Metagenomic and metabolomic analyzes revealed only modest alterations in microbial metabolism. Although the study had a large sample size, the authors note that self-reported maintenance of usual Hygiene 2022, 2 position and function in predominantly overweight or obese adult men and women. The post-intervention evaluation revealed no significant changes in taxonomic composition or metabolic pathways in either exercise group compared to baseline. However, a trend for increased bacterial diversity was observed in the exercise and exercise plus whey protein groups compared to the group receiving whey protein alone. Metagenomic and metabolomic analyzes revealed only modest alterations in microbial metabolism. Although 157 the study had a large sample size, the authors note that self-reported maintenance of usual dietary intake and a wide BMI range may have prevented the detection of more significant changes [38]. and a wide BMI range may have prevented the detection of more significant dietary intake Munukka changes [38]. and colleagues conducted a similar study to determine whether resistance Munukka colleagues conducted in a similar study to determine whetherwomen. resistance exercise could and affect the gut microbiota previously sedentary overweight Six exercise affect the gut microbiota in previously sedentarythe overweight women. Six weeks ofcould light-to-moderate intensity exercise cycles increased relative abundance of weeks of light-to-moderate intensity exerciseProteobacteria. cycles increased relative abundance of A. muciniphila and a decrease in the phylum Whatthe is most interesting is that A. muciniphila and a of decrease in the phylum Proteobacteria. What is most is only the microbiota about half of the subjects responded significantly to interesting exercise. Metthat only the microbiota of about of thetraining subjectsdecreased respondedthe significantly agenomic analysis revealed that half exercise abundancetoofexercise. several Metagenomic revealed that exercise training decreased the abundance of several genes related analysis to fructose and amino acid metabolism [39]. genesThese related to fructose and amino acid metabolism [39]. effects on the gut microbiota, findings suggest that exercise has independent These findings suggest thator exercise hasmay independent effects on the significant gut microbiota, but training of longer duration intensity be necessary to induce taxobut training of longer duration or intensity may be necessary to induce significant taxonomic and metagenomic changes. In addition, the microbiota of lean people may respond nomic and metagenomic In addition, the lean people may(Figure respond differently to an exercisechanges. intervention than that ofmicrobiota overweightofor obese people 2). differently to an exercise intervention than that of overweight or obese people (Figure 2). Figure2.2.Proposed Proposedmodel modelof ofhow howexercise exercisemodulates modulatesdifferent differentorgans organsand andthe thegut gutmicrobiota microbiotaand and Figure epithelium[40]. [40]. epithelium Likewise, function, there will bebe a lower capacity Likewise,whenever wheneverthere thereisisa areduced reducedimmune immune function, there will a lower capacin strength, coordination, or speed, so a decrease in mucosal immunity is associated itymuscle in muscle strength, coordination, or speed, so a decrease in mucosal immunity is assowith anwith alteration in the in athlete’s efficiency. Although it occurs moremore commonly in enciated an alteration the athlete’s efficiency. Although it occurs commonly in durance exercises than in team sports [41].[41]. Altered gut gut motility or enteric nervous system endurance exercises than in team sports Altered motility or enteric nervous sysactivity is another mechanism by which may influence the gut microbiota. Exercise tem activity is another mechanism by exercise which exercise may influence the gut microbiota. reduces transit time in the large intestine and has been shown to speed the movement of gas through the gastrointestinal tract [42,43]. Exercise impacts the autonomic nervous system by increasing general vagal and sympathetic tone [44]. However, regional, or global changes in gastrointestinal transit are likely to deeply affect the intestinal pH, mucus secretion, biofilm formation, and nutrient availability to the microbiota. Mechanical forces increase in the abdomen during most types of aerobic exercise, which could potentially influence intestinal motility or increase the mixing of intestinal contents [44]. Physical training can also alter the enterohepatic circulation of bile acids [45]. Bile acids are potent regulators of gut microbiota community structure, and the absence of these molecules is associated with significant alterations in gut microbial communities [46]. Hygiene 2022, 2 158 We have seen how physical exercise can modulate the intestinal microbiota, we also see that adaptations to exercise can also be influenced by the intestinal microbiota, playing an important role in the production, storage, and expenditure of energy obtained from the diet, as well as in inflammation, oxidative reactions, and hydration status [47]. The intestinal microbiota promotes the digestion and absorption of food to produce energy by the host, in this process the colon metabolizes complex carbohydrates and later they are fermented into SCFA such as butyrate, acetate and propionate [48]. Propionate and acetate are transported in the bloodstream to different organs in the human body where they are used as substrates for energy metabolism, particularly by hepatocyte cells, which use propionate for gluconeogenesis [49]. Given the energy requirements during physical exercise, the intestinal microbiota exerts positive effects on the performance of athletes by providing energy substrate to the muscle through the production of SCFA [48,50]. One of the main physiological adaptations to physical exercise is the modulation of oxidative and nitrosativo stress to avoid tissue damage from intestinal permeability and bacterial translocation [51]. It has been described that endurance athletes have a high prevalence of upper respiratory tract infections and gastrointestinal problems, including increased permeability of the gastrointestinal epithelial wall, also called “leaky gut”, altered mucosal thickness, and higher bacterial translocation rates. Athletes exposed to highintensity exercise show a higher incidence of gastrointestinal problems, symptoms such as diarrhea, bloating, nausea, and even bleeding [52]. Ismaeel et al. recently described that ROS can be decisive for the cell adaptation process, acting as redox signaling molecules. The intestinal microbiota regulates the maintenance of the gastrointestinal redox state and mitochondrial morphology, through the production of SCFAs and secondary bile acids, which will improve the functioning of the intestinal barrier and the immune response of the mucosa [53]. Recently, the intestinal microbiota has been related to the maintenance of adequate hydration during exercise. The phylum Bacteroidetes has been shown to reduce plasma sodium levels, while the Clostridium genus reduced plasma osmolality levels in healthy young individuals. These results suggested that the microbiota influences cellular solute transport across the intestinal mucosa and contributes to hydration status, while reducing plasma osmolality. In the same experiment, high numbers of Bifidobacterium influenced T cell levels, reflecting interaction with the host’s immune response. Because good hydration status and a properly functioning protective gut barrier are essential for athletic performance, and because some athletes do not meet their fluid needs during exercise, it is important to understand the role of the microbiota in the water transport and changes associated with the intestinal mucosa [54]. 4. Methods Used to Study the Microbiota Currently, the analysis of the intestinal microbiota is carried out with the stool samples of the subjects participating in the studies, before DNA extraction, it can be considered to lyophilize the samples, collected in the sterile container, in which, in this way, the percentage of water can be obtained. Next, the most widely used standardized technique is DNA extraction using the IHMS (International Human Microbiota Standards) standard operating protocol. The standardized IHMS “Q” protocol is the one recommended by the International Microbiome Consortium [55]. The homogenizer (Bullet Blender Storm, Next Advance, NY, USA) was used using glass balls. DNA quantification was performed with the PicoGreen dsDNA Quant iT kit (ThermoFisher Scientific, Waltham, MA, USA) and the FP 8300 spectrofluorometer (Jasco, Tokyo, Japan) and DNA purity estimation was performed with a spectrophotometer, using Nanodrop 1100 (Thermofisher Scientific, Waltham, MA, USA). The product of the extraction is used for massive sequencing. For sequencing, libraries are generated by amplifying the V3 and V4 hypervariable regions of the 16S rRNA gene, which are present in all bacterial species. Hygiene 2022, 2 159 The primers used for library generation included the double index sequence needed for multiplexing along with heterogeneity spacers to increase library complexity. The product was a 459 bp amplification which is visualized on a Gelred stained 0.8% agarose gel (Merck KGaA, Darmstadt, Germany). DNA amplifications are typically sequenced on a MiSeq Illumina platform (Illumina, San Diego, CA). Sequence results can be analyzed using Quantitative Insights into Microbial Ecology (QIIME2), v. 18.2, [56]. Reads are assigned to an Organizational Taxonomic Unit (OTU) when sequence identity was greater than or equal to 97%. Sequences that did not match any reference are discarded. The analysis of beta diversity allows us to know the distribution of each microbiota sample in space, considering the distance matrix that exists between the different OTUs that make up the community, so that the microbiota samples that are close in space they are more like each other. Four indices are commonly used to determine beta diversity: • • • Bray Curtis: this index is not affected by null values (when a species is not detected, or its value is 0). It is a quantitative index because it considers the abundance of species and does not consider phylogenetic distances. This index is between 0 and 1. Jaccard: measures the degree of similarity, its range is from 0 to 1, it does not consider the abundance of the species but only their presence or absence, so it is affected by null values. Weighted Unifrac and Unweighted Unifrac: Unifrac methods consider information about the abundance of species and their phylogenetic distance. The Unweighted parameter is qualitative because it only considers the presence or absence of individuals, while the Weighted parameter considers the abundances. The analysis of alpha diversity allows us to know quantitatively, how diverse the microbiota is, how many different species there are. Four indices are commonly used to terminate alpha diversity: • • • • Observed OTUs: this index allows us to know at a quantitative level the number of OTUs in each sample. Pielou index (evenness): this index is indicative of how the species are distributed, of their uniformity in the microbiota, if there are a few species that dominate the microbiota (low evenness) or the abundance of the species is similar (high evenness). Shannon index: the Shannon index is an index that considers the richness of species (richness) and the scale considering the distribution of abundance of each species (evenness). Faith’s index: it is an index that takes taxonomy into account, that is, it is a phylogenetic index that measures the distances of the branches that separate one OTU from another, the more different the 2 OTUS are phylogenetically, the higher the Faith’s index, the greater the Phylogenetic separation of all OTUS within Faith’s largest index sample. 5. Conclusions The key component that determines the correct function of the intestinal barrier is the gut microbiota. However, more studies are needed to understand how the gut microbiota composition can influence physical performance. Given that details and small gains are recognized as crucial for competitive success in athletes, it is reasonable to consider the gut microbiota as a potential factor that could modulate physical efficiency and would need further study to determine its importance and to find strategies that can mediate the effects of exercise. Author Contributions: D.D.-B.: writing and review of the drafts of the manuscript and acquisition, analysis, and interpretation of data. G.G.-P.-d.-S.: design and conceptualization of the study, writing and review of the drafts of the manuscript, and supervision. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Hygiene 2022, 2 160 Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no potential conflict of interest with respect to the research, authorship, and/or publication of this article. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. Brüls, T.; Weissenbach, J. The human metagenome: Our other genome? Hum. Mol. Genet. 2011, 20, 142–148. [CrossRef] [PubMed] Prats, G. 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Chan et al. Trials (2016) 17:460 DOI 10.1186/s13063-016-1582-z STUDY PROTOCOL Open Access A home-based exercise intervention for caregivers of persons with dementia: study protocol for a randomised controlled trial Wai Chi Chan1* , Nicola Lautenschlager2,3,4, Briony Dow5, Suk Ling Ma6, Corine Sau Man Wong1 and Linda Chiu Wa Lam6 Abstract Background: Family members, who provide the majority of care for persons with dementia, are especially vulnerable to developing depression. Interventions targeting their depressive symptoms have been proposed but their efficacies vary considerably. It has been suggested that interventions carried out in the home setting and involving both caregivers and care recipients are more efficacious. This study aims to compare the efficacy of a home-based structured exercise programme involving both persons with dementia and their caregivers with nonexercise social contact control in treating depression among caregivers. Methods/design: This is a parallel-group, assessor-blind, randomised controlled trial. A total of 136 caregiver-carerecipient dyads (i.e. 272 participants in total) will be recruited and randomly allocated to either a home-based structured exercise (sitting Tai Chi) group or a social contact control group. The trial comprises a 3-month intervention phase followed by an extended observation phase of another 3 months. All participants will be assessed at baseline, 6th week, 12th week and 24th week. The primary outcome will be the reduction in depression among caregivers as measured by the Hamilton Rating Scale for Depression. The secondary outcomes will be burden, quality of life, cognitive performance and balance ability of the caregivers, as well as the neuropsychiatric symptoms, cognitive function, balance and functional abilities of the persons with dementia. We will also examine whether the brain-derived neurotrophic factor gene modulates mood changes in response to exercise. Discussion: The findings offer a potential avenue of intervention by providing a low-cost, safe and effective treatment for depression among dementia caregivers, which may in turn also benefit the care recipients. Trial registration: ClinicalTrials.gov Identifier: NCT02132039, registered on 28 April 2014. Keywords: Home-based exercise, Tai Chi, Caregivers, Dementia, Depression, Randomised controlled trial Background Dementia affects over 47 million people worldwide [1]. It causes decline in cognitive function, impairment in activities of daily living, and is associated with significant behavioural and psychological symptoms among the persons affected. Since the majority of dementia care is provided by family members [2], it is not surprising that they suffer from a high level of psychological distress * Correspondence: waicchan@hku.hk 1 Department of Psychiatry, The University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong Full list of author information is available at the end of the article and burden. It is now evident that informal caregivers of persons with dementia are at an elevated risk of developing depression and anxiety, and report poorer quality of life [3–5]. They also experience more stress than those who care for someone with physical disorders [6]. In a local study interviewing family caregivers of older adults with dementia in Hong Kong, they suffered significant stress and reported various negative emotions, conflicts between social roles, and difficulty in providing daily care and managing the care recipients’ behaviours [7]. Dementia caregiving has, therefore, emerged as a pressing health care issue. © 2016 The Author(s). 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. Chan et al. Trials (2016) 17:460 In view of this, different interventions have been developed to alleviate depressive symptoms among caregivers of dementia persons. Recent reviews have analysed the efficacies of educational [8] and social support (e.g. befriending and peer support, family support and social network interventions, and support groups) [9], and psychological [10] and Internet-based interventions [11] for caregivers of persons with dementia. Though clinically meaningful, these interventions achieved only small effects [8, 11] or inconsistent results [9]. This may be partly because the programme goals and target groups were not clearly defined. Therefore, it has been suggested that research should focus on subgroups of caregivers such as depressed caregivers [12]. Among interventions for depression, exercise therapy has drawn much attention from researchers and practitioners. Exercise is a subset of physical activity that is planned, structured, repetitive and purposeful, and regular exercise compares favourably to antidepressants as treatment for milder depression [13]. Though its antidepressant mechanism is not yet clear, evidence suggests that exercise improves mood by normalising brainderived neurotrophic factor levels [14], modifying serotonin function and releasing endogenous opioids [15]. More recently, exercise is reported to reduce atrial natriuretic peptide and brain natriuretic peptide, and to enhance copepetin and growth hormone among depressed individuals [16]. In addition, exercise improves one’s psychological and social sense of wellbeing [15]. It is, therefore, not surprising that a recent meta-analysis showed that exercise had a large and significant effect on depression (standardised mean deviation adjusted for publication bias = 1.11) with a fail-safe number of 1057 [17]. There has been emerging evidence supporting the effectiveness of exercise in treating depression in dementia caregivers. In a recent study, 17 caregivers participating in regular exercise of moderate intensity experienced less sense of burden and fatigue, and reported better sleep when compared with those in the control group [18]. Encouraging results have also been reported with endurance exercise [19], yoga [20], walking [21], and a combination of various physical activities [22]. However, the number of exercise studies for caregivers remains limited, and the sample sizes involved are usually small. This is probably related to the difficulty for dementia caregivers of participating in centre- or communitybased exercise interventions because they are unable to find someone to take care of the care recipients, and lack time for additional activities [23]. In some previous studies, caregivers also voiced a preference for a programme that both caregiver and care recipient can participate in [22]. Home-based programmes involving both caregivers and care recipients may offer a possible solution [24]. Not Page 2 of 8 only does the home-based dyadic intervention improve treatment adherence [25], it is also more successful in enhancing care-recipient function and reducing caregiver sense of burden [26]. We therefore propose to conduct the following assessor-blind randomised controlled trial of a homebased exercise intervention for both dementia caregivers and their care recipients. Objectives The objectives of the study are as follows: 1. To evaluate the effect of a home-based exercise programme on: (a). caregivers’ depression (b).caregivers’ burden, quality of life, cognitive performance and balance ability (c).neuropsychiatric symptoms, cognitive function, and balance and functional abilities among persons with dementia 2. To assess whether the brain-derived neurotrophic factor (BDNF) gene modulates mood changes in response to exercise intervention Methods/design Design This is a parallel-group, assessor-blind, randomised controlled trial, which comprises a 3-month intervention phase followed by an extended observation phase lasting for 3 months. Participants will be randomly assigned to either the home-based structured exercise (intervention) or the standard nonexercise social contact (control) group. The study is registered with ClinicalTrials.gov (NCT02132039), and conducted in accordance with the Declaration of Helsinki. Ethics approval has been sought from respective Institutional Review Boards. All results will be reported according to the Consolidated Standards of Reporting Trials (CONSORT) 2010 Statement [27]. A CONSORT flow diagram of the study is depicted in Fig. 1. Participants Caregiver-care-recipient dyads will be recruited from psychogeriatric and geriatric outpatient clinics and various community centres for elders in Hong Kong by convenience sampling. Potential participants will be identified by the research team and screened for eligibility based on the inclusion and exclusion criteria. Informed consent will be obtained from all eligible participants before enrollment. Inclusion criteria Caregivers: Chan et al. Trials (2016) 17:460 Page 3 of 8 Fig. 1 Consolidated Standards of Reporting Trials (CONSORT) flow diagram of the home-based exercise trial  Aged 50 years or above  Informal caregivers, i.e. unpaid family members who offer a substantial amount of care for the persons with dementia  Geriatric Depression Scale (GDS) [28] score >0 but <8 (i.e. the local cut-off score for clinically significant depression)  Understand Chinese  Receiving antidepressant treatment on a steady dose for at least 3 months (if applicable) Care recipients:  Aged 60 years or above  Dementia diagnosis confirmed by a physician  Dependence in at least one of the activities of daily living (ADL)  Mini Mental State Examination (MMSE) [29] score >10 Exclusion criteria Both caregivers and care recipients:  No regular (i.e. at least three times/week) Tai Chi practice or other forms of mind-body exercise such as yoga, qigong, or mindfulness training in the past 6 months  Presence of any condition which renders participants unsuitable for physical training, for example, severe psychotic symptoms, imminently suicidal, significant orthopaedic problems, or unstable medical conditions Randomisation and blinding Participants will be randomly assigned to either the intervention or the control group using a computergenerated block randomisation sequence (block sizes of 4). Randomisation will be performed by an independent Chan et al. Trials (2016) 17:460 team member who will not be involved in the recruitment, assessment, or delivery of the intervention. The allocation sequence will be concealed from the study investigators and research staff in sequentially numbered, opaque, and sealed envelopes. The assessor for clinical parameters will be blinded to the randomisation status, and the trainer who conducts the intervention will be blinded to the assessment results. Intervention The intervention consists of eight home-based sessions of sitting Tai Chi. Tai Chi is a traditional Chinese exercise that combines rhythmic and flowing patterns of bodily movements with coordinated breathing and mindful meditation [30]. It is a moderate-intensity aerobic exercise at about 3 to 6 metabolic equivalents [31]. It has been successfully implemented in the local older population [32] and, as a low impact exercise, it is associated with a lower risk of musculoskeletal injury making it a suitable form of exercise for older adults. Since the current study includes persons with significant cognitive impairment, we adopt sitting Tai Chi which is a simpler version of Tai Chi developed by Hong Kong Polytechnic University [33]. The exercise consists of 12 steps, which include weight shifting in different sitting positions, trunk and upper limb movements, and alternate thigh lift in a smooth and coordinated manner [33]. Four weekly exercise sessions will be arranged over the first 4 weeks, and four more biweekly sessions over the next 8 weeks. Each session will last for 1 h, which starts with 10 min of warm-up activities followed by 40 min of sitting Tai Chi practice and, lastly, 10 min of cool-down exercises. The caregiver-care-recipient dyads will practise Tai Chi in their home under the instruction of a Tai Chi teacher with more than 20 years of teaching experience, and a trained research assistant. In order to monitor the progress and address participants’ concerns, biweekly phone contacts will be made during the study period (12 scheduled phone contacts in 24 weeks). On completion of the eight home-based Tai Chi sessions, participants will be given a manual and training video with detailed instructions of the Tai Chi poses to facilitate their continuation of Tai Chi practices. A standard nonexercise social contact will be provided to the participants in the control group. Since social contact may be beneficial to one’s mood and may contribute to any improvement observed in the intervention group, we provide participants in the control group with a level of social contact equivalent to the intervention group. They will be visited by the research assistant eight times over 12 weeks. The visits involve a series of conversations related to neutral topics that are designed according to the principles of a befriending programme [34]. These topics are structured and standardised, and Page 4 of 8 do not involve any topic concerning physical activity. Similar to the intervention group, 12 biweekly phone calls will be made during the study period to offer a comparable level of social contact. Primary outcome Participants will be assessed at baseline, 6th week, 12th week and 24th week for their responses to intervention. The assessment schedules for caregivers and care recipients are described in Table 1. The primary outcome of the study is the proportion of participants who are classified as responders by the Hamilton Rating Scale for Depression (HAM-D-17) [35]. HAM-D-17 is a widely used and reliable measure of depressive symptoms. The scores range from 0 to 52, with higher scores indicating greater depression severity. Response to the intervention is defined as a reduction of the HAM-D-17 total score by ≥50 % from baseline to endpoint. Assessors will follow the structured interview guide for the Hamilton Depression Rating Scale (SIGH-D) when administering the HAM-D-17 [36]. Assessment for caregivers only: 1. The Zarit Burden Interview (ZBI) [37] is a 22-item self-administrated questionnaire specially designed to measure the stresses experienced by caregivers of persons with dementia. Caregivers will assess the impact of the patient’s disabilities on their life 2. The SF-12 Health Survey (SF-12) [38] measures the health-related quality of life and gives two component scores: a physical component summary (PCS) and a mental component summary (MCS) 3. The Executive Interview (EXIT25) [39] is a 25-item screening instrument developed to assess executive cognitive dysfunction 4. The International Personality Item Pool (IPIP) [40] consists of 50 items assessing the ‘Big Five’ personality traits: openness to experience, conscientiousness, extraversion, agreeableness, and neuroticism Assessment for care recipients only: 1. The Cornell Scale for Depression in Dementia (CSDD) [41] is a clinician-rated instrument measuring depressive symptoms in persons with cognitive impairment 2. The Neuropsychiatric Inventory (NPI) [42] assesses the frequency and severity of neuropsychiatric symptoms across 12 domains including psychotic features, mood changes, activities disturbances and vegetative symptoms 3. The Disability Assessment for Dementia (DAD) [43] evaluates functional abilities in both basic and Chan et al. Trials (2016) 17:460 Page 5 of 8 Table 1 Assessment schedule for caregivers and care recipients Baseline assessment Week 6 Week 12 Week 24 Caregiver only GDS (screening only) √ HAM-D-17 √ √ √ √ ZBI √ √ √ √ SF-12 √ √ √ EXIT25 √ √ √ IPIP √ √ √ Care recipients only CSDD √ √ NPI √ √ √ DAD √ √ √ MBI √ √ √ MMSE √ √ √ Digit span √ √ √ Delayed recall √ √ √ CVFT √ √ √ BBS √ √ √ FR √ √ √ TUG √ √ √ Logbook √ √ √ √ Pedometer √ √ √ √ BDNF genotype √ Body weight and height √ √ √ Blood pressure and pulse √ √ √ Caregivers and care recipients BBS Berg Balance Scale, BDNF brain-derived neurotrophic factor, CSDD Cornell Scale for Depression in Dementia, CVFT Category Verbal Fluency Test, DAD Disability Assessment for Dementia, EXIT25 Executive Interview, FR Functional Reach, GDS Geriatric Depression Scale, HAM-D-17 Hamilton Rating Scale for Depression, IPIP International Personality Item Pool, MBI Modified Barthel Index, MMSE Mini Mental State Examination, NPI Neuropsychiatric Inventory, TUG Timed Up and Go, ZBI Zarit Burden Interview instrumental activities of daily living in people with dementia 4. The Modified Barthel Index (MBI) [44] measures individuals’ performance on 10 activities of daily living: eating, personal hygiene, bathing, toileting, dressing, bowel control, bladder control, transfers, walking, and stair climbing Assessment for both caregivers and care recipients: 1. Sociodemographic information includes age, gender, education level and marital status 2. Lifestyle factors such as smoking, drinking, and sleeping habits. Health-related information like chronic health problems and medications will also be recorded. Resting blood pressure and heart rate will be taken. Body height and weight will also be measured to calculate Body Mass Index 3. Global cognitive function will be measured by the MMSE [29], digit span, delayed recall, and the Category Verbal Fluency Test (CVFT) [45] 4. Balance ability and functional mobility will be measured by the Berg Balance Scale (BBS) [46], the Functional Reach (FR) test [47], and the Timed Up and Go (TUG) test [48] 5. Physical activity and programme adherence will be assessed using logbook recordings. Before each assessment, participants will be asked to wear a pedometer for 1 week. Number of walking steps, history of falls, frequency, duration, and type of exercises during the week will be recorded in the logbook 6. Physiological markers – to examine the role of the BDNF Val66Met polymorphism in the antidepressant effect of exercise, buccal swabs will be collected at the baseline assessment Sample size calculation The sample size is calculated by the proportion of caregivers who respond to the intervention as indicated by the HAM-D-17. Taking reference of the previous study [49], we expect a 30 % reduction in the proportion of participants rated as depressed in the control group with social contact only. Assuming that a further 30 % reduction is achieved by sitting Tai Chi, we need 56 caregivercare-recipient dyads (i.e. 112 participants) in each arm to identify a significant effect at a two-tailed alpha of 0.05 and 80 % power. Allowing for 20 % dropout, the number of dyads in each arm will rise to 68. A total number of participants of 272 will be recruited. Statistical analyses Baseline differences in demographic and clinical characteristics between the intervention and control groups will be evaluated by chi-square and independent sample t tests. All data will be analysed using the intent-to-treat principle. For outcome measures that are not normally distributed (e.g. the HAM-D-17), log transformation will be applied to perform the analyses. Mixed-effects models will be fitted for each outcome measure using baseline, 6th, 12th and 24th week follow-up data. Possible confounding factors such as sex, age, and use of antidepressant medication will be treated as time-dependent covariate in the mixed-effects models. If applicable, subgroup analysis will be carried out to estimate whether there is any Chan et al. Trials (2016) 17:460 Page 6 of 8 gender difference (female versus male caregivers) regarding the effect of the intervention in alleviating depressive symptoms. Fixed effects for time, intervention group, and their interactions will be examined and unstructured covariance structure will be employed to account for the within-subject correlation over time. Levels of significance will be set at p < 0.05. All analyses will be performed using SPSS version 20.0 (SPSS Inc., Chicago, IL, USA). Abbreviations ADL: Activities of daily living; BBS: Berg Balance Scale; BDNF: brain-derived neurotrophic factor; CONSORT: Consolidated Standards of Reporting Trials; CSDD: Cornell Scale for Depression in Dementia; CVFT: Category Verbal Fluency Test; DAD: Disability Assessment for Dementia; EXIT25: Executive Interview; FR: Functional Reach; GDS: Geriatric Depression Scale; HAM-D17: Hamilton Rating Scale for Depression; IPIP: International Personality Item Pool; MBI: Modified Barthel Index; MCS: Mental component summary; MMSE: Mini Mental State Examination; NPI: Neuropsychiatric Inventory; PCS: Physical component summary; REC: Research Ethics Committee; SF-12: SF-12 Health Survey; SIGH-D: Structured interview guide for the Hamilton Depression Rating Scale; TUG: Timed Up and Go; ZBI: Zarit Burden Interview Discussion Since the prevalence of dementia doubles every 5 years after the age of 60, population ageing inevitably increases the number of persons with dementia. Hong Kong is no exception to this trend. It is estimated that around one in 10 local older persons are now suffering from dementia [50]. Caregiving for persons with dementia is demanding and challenging. Evidence shows that it adversely affects caregivers’ physical [51] as well as mental wellbeing [3–5], including a vulnerability to developing depression. In a review of 10 studies involving 790 caregivers of persons with dementia, more than one fifth of them suffered from a depressive disorder [3]. The prevalence of depressive symptoms among caregivers is even higher [14]. This study will investigate the efficacy of a structured exercise programme in alleviating depressive symptoms among caregivers of persons with dementia. We focus on caregivers with depressive symptoms instead of depressive disorders as the former constitute the largest group of depressed caregivers. Besides, because of ethical concerns, we will refer those who suffer from clinically significant depression to appropriate agencies for psychiatric assessment and treatment. The intervention is tailor-made for family caregivers in three ways. First, the programme is delivered at participants’ homes to facilitate their participation. Second, both caregivers and care recipients will be involved in the study. Third, a moderate-intensity aerobic exercise that has been successfully implemented in the local older population (i.e. sitting Tai Chi) is chosen as the intervention. Sitting Tai Chi is a safe, low-impact and low-cost exercise intervention. If it is shown to improve caregivers’ mood, it will offer a potentially effective treatment alternative for depression among caregivers of persons with dementia. In addition, we expect that home-based exercise will also benefit the care recipients. Acknowledgements We would like to express our sincere thanks to our Tai Chi teacher Mr. Charles Lee for agreeing to take part in the study. We would also like to thank the social centres and clinics for their valuable support in participant recruitment. In addition, we are grateful to Mrs Eleanor Chan and Mrs Miranda Tung for their advice on the assessment methods. Trial status The study is still recruiting at the time of submission. Funding The study is supported by the Health and Medical Research Fund of Food and Health Bureau of the Hong Kong Special Administrative Region Government (Ref: 11121441). Availability of data and materials Not applicable. Authors’ contributions WCC conceived the idea for the study, obtained funding, and took primary responsibility for study coordination and manuscript preparation. NL, BD, SLM and LCWL obtained funding and contributed to the concept and design of the study, intervention and outcome assessment. CSMW contributed to the coordination of the study, statistical analysis, and manuscript preparation. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate Ethics approval has been sought from the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (UW 13-453), Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee (2014.254-T), New Territories West Cluster Clinical and Research Ethics Committee (REC) (NTWC/CREC/ 1302/14), Kowloon West Cluster REC (KW-EX-15-076-86-12TCM), and Hong Kong East Cluster REC (HKEC-2015-045). Participation in the study is completely voluntary. Participants will be able to withdraw from it at any time. The decision to participate or withdraw will not affect their existing treatment or service received. Caregivers who are found to have significant depressive symptoms or other mental health problems will be referred to appropriate health services for follow-up. All personal information collected will be kept strictly confidential and used for research purposes only. Research team members will be responsible for safekeeping the personal data. Only the principal investigator and designated research staff will have access to the interim results and final trial dataset. Informed consent will be obtained from all eligible participants before enrollment. Author details 1 Department of Psychiatry, The University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong. 2Academic Unit for Psychiatry of Old Age, Department of Psychiatry, University of Melbourne, Melbourne, VIC, Australia. 3 NorthWestern Aged Mental Health, Royal Park Campus, Parkville, VIC, Australia. 4School of Clinical Neurosciences and the Western Australia Centre and Health and Ageing, University of Western Australia, Perth, WA, Australia. 5 National Ageing Research Institute, The University of Melbourne, Melbourne, Chan et al. Trials (2016) 17:460 VIC, Australia. 6Department of Psychiatry, The Chinese University of Hong Kong, Tai Po Hospital, Tai Po, Hong Kong. Received: 7 May 2016 Accepted: 27 August 2016 References 1. World Health Organization. The epidemiology and impact of dementia: current state and future trends. Geneva; 2015. Available from: http://www. who.int/mental_health/neurology/dementia/dementia_thematicbrief_ epidemiology.pdf. 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Academic Journal of Science and Technology ISSN: 2771-3032 | Vol. 2, No. 3, 2022 The Application of New Energy Materials in New‐energy Vehicle Yuting Qin1, a, Junying Fang2 1Xiamen University Malaysia, Jalan Sunsuria, Bandar Sunsuria, 43900, Sepang, Selangor Darul Ehsan, Malaysia University of Technology.69 Hongguang Dadao, Banan District, Chongqing, China aCorresponding Author’s Email: ege1909292@xmu.edu.my 2Chongqing Abstract: Climate change is becoming one of the biggest environmental challenges in the 21st century. New-energy vehicle produce less greenhouse gases as compared to the traditional vehicles with internal combustion engines. New energy vehicles have great potential to successfully address the issue of climate change. Additionally, new energy materials can also be used in automotive lightweight technology. This paper discussed the currently lightweight materials including high-strength low-alloy steel (HSLA), carbon fiber composite material, and modified plastics. According to the discussion in this paper, high-strength low-alloy steel can be used in automotive safety parts, chassis, and body. Carbon fiber composite materials can be used in car bodies, chassis, roofs, doors, head covers, hoods, rear wings, center consoles, trim strips, drive shafts, leaf springs, frames, brake pads, interior and exterior accessories. Modified plastics can be mainly used in exterior decorative parts, interior decorative parts, functional parts, and structural parts. However, three are still many drawbacks of application of new energy materials in newenergy vehicle. The current new energy materials used in automotive lightweight technology are usually costly and timeconsuming. Moreover, the characteristics of the new energy materials are still not clear. It could have a negative impact on the environment. Before this technology can be widely used in new-energy vehicles, more research is needed regarding the new energy materials. Keywords: New energy material, New-energy vehicle, Climate change, High-strength low-alloy steel (HSLA), Carbon fiber composite material, Modified plastic, Greenhouse gases. scale of the auto parts market continues to increase. The scale of China's auto parts market has grown from 3.46 trillion yuan in 2016 to 4.57 trillion yuan in 2020, with an average annual compound growth rate of 7.2%; the market size is expected to reach 4.9 trillion yuan in 2021. In recent years, new energy materials have wide applications in a variety of technological fields such as thinfilm solar cells, light-emitting device, and photodetector. Many new energy materials have been introduced to the newenergy vehicle industry and has produced some promising outcomes. Clean energy technologies of new-energy vehicles usually require a wide range of minerals1 and metals. The type and volume of mineral needs are very different based on the clean energy technologies (Table 1) [5]. In the current new-energy vehicle industry, reduction in weight is one of the development directions of the automotive industry in the future. The reduction in weight directly means an increase in cruising range. It is found that for the pure electric vehicles, the vehicle weight can be reduced by 10 kg, and the driving range can be increased by 2.5 km. As a result, the GHG emissions could be significantly reduced in the same diving range. In the lightweight industry chain of new-energy vehicles, the main raw materials for lightweight include highstrength steel, aluminum alloy, magnesium alloy, carbon fiber composite materials and modified plastics. These materials could constitute chassis, engine, seat, luggage rack, gear lever, interior, exterior and steering wheel, etc. Few studies have been conducted in this field due to the rapid growth of new energy material technology. The objectives of this paper are to: 1) present the classification of new energy materials and the application in new-energy vehicle; 2) discuss the future vision of new energy materials applied in new-energy vehicle. 1. Introduction Climate change is one of the biggest challenges of our times[1]. It is believed that one of the major reasons of climate change is human activity such as fossil fuel combustion. When these fossil fuels are combusted, greenhouse gases are released into the Earth's atmosphere. As a result, the heat from the sun was trapped and global temperatures to rise. Based on climate models, the Intergovernmental Panel on Climate Change (IPCC) predicts that the global temperatures will rise by about 1.4-5.8 degrees Celsius (2.5-10.4 degrees Fahrenheit) by 2100 [2]. According to this forecast, global temperatures will undergo dramatic changes not seen in the past 10,000 years, with potentially significant impacts on the global environment. To reduce global warming, many countries have agreed to reduce their greenhouse gas (GHG) emissions into the atmosphere to 1990 levels. Generally speaking, transportation sector produces one of the largest proportion of GHG emissions. It also has a negative impact on public health and the environment. Vehicle electrification has great potential to reduce air pollutions and tackle climate change [3]. Compared with the internal combustion engine vehicles, new-energy vehicles hardly generate greenhouse gases during driving. If it can completely replace internal combustion engine, new energy vehicles could effectively solve the climate change problem. According to data released by the China Association of Automobile Manufacturers, the production and sales of newenergy vehicles reached 397,000 and 383,000 respectively in October 2021, an increase of 1.3 times year-on-year. From January to October, the production and sales of new-energy vehicles reached 2.566 million and 2.542 million respectively, a year-on-year increase of 1.8 times [4]. At the same time, the 111 Table 1. Critical mineral needs for clean energy technologies Copper Nickel Chromium Solar photovoltaic High Low Low Bioenergy High Low Low Nuclear Moderate Moderate Moderate Electricity networks High Low Low New-energy vehicle and battery storage High High Low Hydrogen Low High Low Aluminium High Moderate Low High High Moderate Automotive lightweight technology is mainly divided into three aspects: structural optimization design, lightweight material application and advanced manufacturing technology. Among them, the use of lightweight alternative materials is generally recognized in the industry. The currently used lightweight materials mainly include high-strength low-alloy steel (HSLA), carbon fiber composite material, and modified plastics. Table 2 shows the mass reduction for different new energy materials. 2. New Energy Materials and the Application in New-energy Vehicle 2.1. Classification of New Energy Materials At present, new energy materials has been widely implemented in automotive lightweight technology. With the development of this technology in the last decade, research has begun to utilize new energy materials' properties. Table 2. Mass reduction for different new energy materials Lightweight materials Mass reduction Magnesium 30-70% Carbon fiber composites 50-70% Aluminum 30-60% Titanium 40-55% Advanced high strength steel 15-25% High-strength low-alloy steel (HSLA) refers to steel with cold rolling of 340 MPa and hot rolling of 490 MPa or more [6]. High strength steel has a better weight reduction effect. Compared with the traditional 340 MPa material, the theoretical weight reduction potential of the 600 MPa grade steel is about 20%, and the weight reduction potential of the 800 MPa material will increase to more than 30%. At present, high-strength steel is mainly used in automotive safety parts, chassis, and body. Carbon fiber composite materials can be used in car bodies, chassis, roofs, doors, head covers, hoods, rear wings, center consoles, trim strips, drive shafts, leaf springs, frames, brake pads, interior and exterior accessories etc. In 2019, the domestic demand of carbon fiber composite materials in China was 38000 tons (Figure 1) [7]. In the future, with the increase of China's scientific research capabilities and the steady development of the carbon fiber composite material industry, the domestic demand will continuously increase. Figure 1. China’s domestic demand of carbon fiber composite materials in in past decades Modified plastics are the plastic products that are processed and modified by filling, blending, strengthening and other methods based on general-purpose plastics and engineering plastics to improve the properties of flame retardancy, strength, impact resistance, toughness, etc. At present, modified plastics are mainly used in exterior decorative parts, interior decorative parts, functional parts, and structural parts. In 2019, the output of modified plastics of industrial enterprises above designated size in China reached 19.55 million tons. In 2021, China's domestic demand for modifies 112 roofs, doors, head covers, hoods, rear wings, center consoles, trim strips, drive shafts, leaf springs, frames, brake pads, interior and exterior accessories. Modified plastics can be mainly used in exterior decorative parts, interior decorative parts, functional parts, and structural parts. However, three are still many drawbacks of application of new energy materials in new-energy vehicle. The current new energy materials used in automotive lightweight technology are usually costly and time-consuming. Moreover, the characteristics of the new energy materials are still not clear. It could have a negative impact on the environment. Before this technology can be widely used in new-energy vehicles, more research is needed regarding the new energy materials. It can be seen that China's new-energy vehicle industry is splendid. But as far as the future is concerned, if new-energy vehicle is a solution for reduction of air pollutions and climate change, then automotive lightweight technology is the core technology. The new cutting-edge materials continuously incubated by research and development institute will become the basis and source of inspiration for the vigorous development of energy innovation and intelligent technology. It can provide infinite possibilities for the future of the newenergy vehicle industry. plastics of industrial enterprises above designated size is 21.1 million tons and the sales scale exceed three trillion RMB. 2.2. Future Vision of New Energy Materials applied in New-Energy Vehicle In December 2017, the Ministry of Industry and Information Technology issued the "Three-Year Action Plan for Promoting the Development of the New-Generation Artificial Intelligence Industry (2018-2020)", which pointed out to establish an architecture that supports vehicle intelligent computing platform, vehicle intelligent chips, and automatic driving operations. Research and development of key technologies and products such as systems and vehicle intelligent algorithms, and build a vehicle intelligent platform that integrates software, hardware, and algorithms. Electric energy, electronic control systems, and lightweight vehicle bodies play an important role in sensor interconnection, automated driving, and safety assurance of new-energy vehicle which will be the development trend of future transportation. The development of the new energy material industry will become the infrastructure supporting this trend. For example, the new-energy vehicle bodies can be built by plexiglas glass, which could be a god alternative for automotive glass. It has many advantages such as high transparency and high strength compared to the currently widely used metal and glass materials. The current new energy materials used in newenergy vehicle can be implanted with sensors. The manufacturing of special functional parts can be completed through 3D printing. The application of these new materials in the automotive industry will also be the development trend of future transportation. Last but not least, the existence of 5G signals will serve for truly intelligent urban transportation, and new-energy vehicle will become a new entrance to the Internet in the era of everything connected. References [1] Seymour, F., & Busch, J. (2016). Why forests? Why now?: The science, economics, and politics of tropical forests and climate change. Brookings Institution Press. [2] Milcarek, R. J., Turner, S., Zhang, R., Ahn, J., & Zhang, J. (2017). Predicting envelope and micro cogeneration design conditions for future climates. ASHRAE Transactions, 123(1). [3] Jacobson, M. Z. (2009). Review of solutions to global warming, air pollution, and energy security. Energy & Environmental Science, 2(2), 148-173. [4] Laitimes. (2021). China's New Energy Vehicle Production and sales lead the World (Expert Interpretation). laitimes. Retrieved July 10, 2022, from https://www.laitimes.com/en/article/18udh_1a483.html 3. Conclusion New energy material is being developed at an exponential rate, and it will revolutionize the new-energy vehicle industry. Compared with the internal combustion engine vehicles, newenergy vehicles hardly generate greenhouse gases during driving. If it can completely replace internal combustion engine, new energy vehicles could effectively solve the climate change problem. In addition, new energy materials have a great potential to implement in automotive lightweight technology due to their properties. According to the discussion in this paper, high-strength low-alloy steel can be used in automotive safety parts, chassis, and body. Carbon fiber composite materials can be used in car bodies, chassis, [5] IEA (2021), The Role of Critical Minerals in Clean Energy Transitions, IEA, Paris, Retrieved July 10, 2022, from https://www.iea.org/reports/the-role-of-critical-minerals-inclean-energy-transitions. [6] Yan, X., Xia, Y., Blum, H. B., & Gernay, T. (2020). Elevated temperature material properties of advanced high strength steel alloys. Journal of Constructional Steel Research, 174, 106299. [7] Li, Z., Feng, Z., Xu, L., Lv, C., Li, L., & Zhu, S. (2020) Research on the development strategy of high-performance fibers and their composite materials in my country[J]. China Engineering Science, 22(5):28-36. 113 

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International Orthopaedics (2020) 44:911–918 https://doi.org/10.1007/s00264-020-04494-0 ORIGINAL PAPER The column procedure preserves elbow stability on biomechanical testing Andrzej P. Podgórski 1,2 & Bartłomiej Kordasiewicz 1 & Stanisław Pomianowski 1 Received: 1 June 2019 / Accepted: 30 January 2020 / Published online: 12 February 2020 # The Author(s) 2020 Abstract Purpose The effect of open release of a post-traumatic elbow contracture on the stability of the joint has not been so far studied in vivo. Resection of elbow joint capsule, the key element of surgery, was reported to have no effect on the stability of cadaveric elbows. The joint capsule is yet known to participate in maintaining elbow stability as one of secondary stabilizers. Methods We assessed elbow joint laxity in 39 patients who underwent an open contracture release via the ‘column procedure’ described by B. Morrey and P. Mansat within the preceeding three to nine months. The measurements were taken with an apparatus designed particularly for this experiment according to the predetermined protocol. A preliminary part of the experiment showed that there was no significant difference between laxity of two elbow joints in healthy volunteers. Laxity of the operated elbows could be then compared with the contralateral joints. Results Mean absolute difference of laxity between healthy and operated elbows was 1.55° (0.1°–4.1°, SD = 1.1) being significantly lower than 2°, p = 0.0056. The difference of the joint laxity between the operated and healthy elbows did not differ statistically significantly by more than 0.6° from the difference of the laxity of two healthy elbows and, therefore, is not clinically noticeable. Conclusions Our experiment confirmed that the ‘column procedure’ is a safe procedure which does not compromise the stability of the elbow joint. Keywords Elbow stability . Elbow trauma . Elbow contracture . Column procedure Introduction Elbow stability results primarily from the integrity of relevant anatomical structures which maintain physiological laxity of the joint. Laxity can be defined as range of motion of the joint in the coronal plane and differs broadly between individuals [1, 2]. Physiological amounts of elbow laxity provide stability of the joint, which is a clinically assessed feature and implies This article does not contain any studies with animals performed by any of the authors. * Andrzej P. Podgórski apodgors@poczta.onet.pl; andrzej.podgorski@wim.mil.pl 1 Department of Traumatology and Orthopaedic Surgery, Adam Gruca Clinical Hospital, Center for Postgraduate Medical Education, Otwock, Poland 2 Department of Neurosurgery, Military Institute of Medicine, ul. Szaserów 128, 04-141 Warsaw, Poland correct biomechanics of the joint. Following biomechanical testing of cadaver elbows, Nielsen [3] and Dos Remedios [4] independently concluded that resection of elbow joint capsule, which is the key element of surgery for a post-traumatic contracture, does not affect the joint laxity, hence would not lead to instability in clinical setting. Those findings have not been confirmed in vivo even though the joint capsule is reported to participate in maintaining the stability of the elbow [2]. In our study, we assessed elbow joint laxity in 39 patients who had undergone an open release of a post-traumatic contracture. The measurements were taken using a specially constructed apparatus which allowed full and safe immobilization of the upper limb and precise biomechanical testing. Elbow stability derives from the congruence of the articular surfaces in roughly 50%, while the remaining half depends on the integrity of ligaments, capsule, interosseous membrane and, to a lesser degree, muscles of the arm and forearm which act as dynamic stabilizers [2, 5, 6]. Those structures are also classified as either primary or secondary stabilizers; the former are those whose injury leads directly to increased laxity of the 912 International Orthopaedics (SICOT) (2020) 44:911–918 joint, and the latter are the structures whose damage would increase laxity only after the relevant primary stabilizers had also been injured. Primary stabilizers include the anterior bundle of the medial collateral ligament, lateral collateral ligament complex and the congruence of the ulnohumeral joint [1, 7–9]. Important secondary stabilizers comprise the congruence of the radiohumeral joint, joint capsule and muscular attachments of the pronator and flexor muscles of the forearm and wrist to the medial humeral epicondyle and extensor muscles to the lateral epicondyle, respectively. An important part of the lateral collateral ligament complex, the lateral ulnar collateral ligament, serves as a primary stabilizer acting against posterolateral rotatory instability of the elbow [10]. Morrey and An in their biomechanical experiments examined the amount of contribution of the particular anatomic structures to the joint stability depending on the position of the elbow [2]. They noted that the articular capsule is responsible for 30% of valgus stability in full extension and that its importance diminishes with the flexion of the joint, dropping to near-zero values in 90° of flexion. The capsule also provides 32% and 13% of varus stability in elbow extension and 90° of flexion, respectively. It serves as the main stabilizer against joint distraction forces, providing 85% of resistance in extension and 8% in 90° of flexion. There is also an important relation between forearm rotation and elbow laxity, which is minimal in full supination, as demonstrated by Pomianowski [11]. The post-traumatic contracture of the elbow is a common complication of fractures and dislocations around the elbow caused by thickening and scarring of the articular capsule, altered shape and incongruence of bony surfaces, osteophyte formation, presence of intra-articular loose bodies and heterotopic ossification. Its occurrence is related to initial damage to the joint structures, intra-articular haematoma formation and individual propensity but can usually be effectively prevented by correct treatment and particularly by avoiding immobilization of the joint for longer than three weeks. Indications for Table 1 Initial lesion of the elbow in patients who subsequently underwent an open release of its contracture in our series surgical release include failure of physiotherapy after four to six months or presence of a surgical pathology, e.g. loose bodies within the joint, ossifications or a radioulnar synostosis. There are also patients with the so-called ‘stiff elbow’ who do not benefit from physiotherapy and who should be treated surgically without the usual delay. Our experience involves 279 patients who have undergone an open release of post-traumatic elbow contracture in our centre since 2003 (Table 1). The relatively high percentage of patients who developed a contracture following a radial head fracture was mainly due to prolonged immobilization of the joint and lack of adequate physiotherapy. Our surgical technique follows the outline of the ‘column procedure’ described by B. Morrey and P. Mansat [12]. The joint is approached via the anterolateral route between the extensor carpi radialis longus and brevis and the extensor digitorum communis anteriorly to the lateral collateral ligament. The articular capsule is bluntly dissected and excised until the anterior aspect of the ulna is visualized. The posterior compartment of the joint can be opened through the same incision if needed, and posterior capsulectomy can also be performed. Scar tissue, osteophytes and intra-articular loose bodies are removed. If deemed necessary, the procedure can involve other steps, e.g. radial head excision, interpositional arthroplasty, excision of synostosis or ulnar nerve transposition via a medial approach. Although there is no agreement on the superiority of the radial head replacement over simple excision [13], we always replaced the radial head with a prosthesis (KPS) designed by the senior author. Release of the ulnar nerve was consistently combined with its anterior transposition and was never performed prophylactically despite recent reports underlining its benefit [14–16]. Arthrolysis of the elbow can be performed using other approaches, most often Kocher approach, as well as combined with exposing the joint from the medial or posterior aspect [17–20] depending on the underlying pathology [21]. Sequelae of complex injuries, e.g. terrible triad of the elbow, are particularly demanding. Protocols Lesion Number of cases Percentage Distal humeral fracture Radial head fracture Proximal radius and ulna fractures Elbow dislocation Elbow dislocation and radial head fracture Elbow dislocation and other or multiple fractures Olecranon fracture Radius and ulna shaft fractures Multiple or other fractures Trauma to elbow with no fracture All 58 62 13 18 19 20 16 2 35 36 279 20.8 22.2 4.7 6.4 6.8 7.2 5.7 0.7 12.5 13.0 International Orthopaedics (SICOT) (2020) 44:911–918 913 Table 2 Additional procedures performed during the surgical release of elbow contracture in our patients Materials and methods Radial head replacement (KPS bipolar endoprosthesis) Radioulnar synostosis resection Lateral collateral ligament repair Assessment of elbow joint stability was performed using an apparatus designed particularly for this experiment, which measured the laxity of the joint by pivoting the forearm of the immobilized upper limb into valgus and varus alignment. The combined deviation angle was subsequently calculated by the device and was established as the physiological laxity of the examined elbow joint. The apparatus consisted of a chair equipped with an extending arm and a moveable frame containing the elbow and wrist immobilizers, the measuring module and the control panel (Figs. 1 and 2). The examined limb was immobilized in a padded two-part holder covering the distal part of the arm above the humeral epicondyles, which was tightened after the limb had been placed in the correct position. Padding increased the comfort of the patient and prevented the arm from rotating in the holder. The axilla was additionally fixed by an adjustable-length strap. The tested limb was positioned in 30° of elbow flexion to increase the contribution of the joint capsule to the stability of the elbow. The distal part of the forearm and the wrist was blocked in full supination to reduce the inherent laxity of the joint, as we assumed that any increase in the laxity would be more evident in this position (Fig. 3). The maximum valgus and varus forearm deviation was recorded based on the analysis of the increase in the moment of resistance. The measuring module recorded the current angle of the forearm deviation and the instantaneous torque to monitor the value of the torque derivative (Fig. 4). Deflection of the forearm would be stopped when either the torque itself 9 6 12 involving extensive arthrolysis, radial head excision and temporary external fixation have been elaborated [22, 23]. Due to the complexity of elbow injuries, optimal treatment of their sequelae varies among different centres according to the individual experience [23–25]. The results of the operative treatment vary from excellent to poor being closely dependent on the pre-operative status of the elbow, namely, on bone alignment and articular incongruence, as reported in other series [26–28]. An analysis of the 213 cases in which full medical record was available including the final assessment 12 months after the operation showed that mean MEPI score following surgery was 86.3 points compared to 63.2 points preoperatively (mean gain 22.9 points). If the articular surfaces were intact or healed in anatomic alignment, the results were usually spectacular with a significant operative gain regarding both the range of motion and the antalgic effect. Mean gain in flexion-extension range of movement after 12 months was 29° (− 10 to 95°), and mean improvement of forearm rotation was 26.8° (0 to 140°). In 27 cases, the surgical procedure involved other steps listed in Table 2. Seven patients required re-operation due to recurrence of the contracture, and two patients developed a deep infection. Fig. 1 Technique of ‘column procedure’. a Development of interval between the extensor carpi radialis longus and brevis and the extensor digitorum communis. b Blunt retractor is passed exposing anterior surface of distal humerus. c Exposure and incision of lateral capsule. d Final operative view after anterior capsulectomy and exposure of coronoid process 914 Fig. 2 The adjustable frame of the measuring device was equipped with a padded arm holder (1), elbow support (2), wrist immobilizer (3), electronic module (4) and safety switch (5) allowing steady and comfortable positioning of the examined limb. See text for details or torque derivative reached the maximum value defined by the investigators. These values had been predetermined experimentally to ensure that the forearm was deflected strongly enough, but the applied force did not cause the pain or arm movement in the immobilizing holder (Fig. 3). Minimum value of the torque derivative was also adjusted, which prevented the position of the maximum deviation from being recorded too early, e.g. due to muscle tightening by the examined patient. The selected parameters were identical for all measurements in the entire study. According to the established protocol, the measuring apparatus performed a cycle consisting of six transitions from maximum valgus to maximum varus deviation. At each extreme position, the angular value was recorded for the difference between neutral position and extreme deviation. Two results – the highest valgus and varus deviations – were rejected. The remaining values were used to calculate the mean joint laxity, being the sum of the mean valgus and varus deviations. High reproducibility of results was obtained; the difference between International Orthopaedics (SICOT) (2020) 44:911–918 the results rarely exceeded 0.5° for individual deviations in the same direction. The aim of our study was to determine if a correctly performed ‘column procedure’ leads to an increase in the physiological laxity of the joint, which in turn could result in instability of the elbow. In our reasoning, we assumed that the laxity of the joint following the operation could be assessed by comparing it with the contralateral normal elbow. The goal of the preliminary part of the experiment was therefore to define if there is any difference in laxity between the two elbow joints in a healthy individual. A series of measurements was taken on a group of volunteers recruited from 52 healthy individuals (29 males, 23 females) of mean age of 34 years (24 to 68) [29]. The selection criteria were normal anatomy and function of both elbow joints with no history of trauma to upper limbs or rheumatologic disease. Owing to the construction of the measuring apparatus, also individuals of exceptionally sturdy or slender build as well as those in whom their natural valgus angle of elbows exceeded 20° could not participate in the study. The results led to a conclusion that although the range of physiological elbow laxity defined as the deviation from the maximal valgus to maximal varus position varies significantly (10.6°–26.5°, mean value 17.8°), the difference in the laxity between two elbows of the same volunteer was only slight, amounting to a mean value of 1.19° (0.1°–3.8°). Moreover, there was no correlation between the side of the elbow with greater laxity and the dominant limb. We therefore concluded that the contralateral elbow could be used as a reference to assess a possible change of the joint laxity following surgery. The main part of the experiment consisted of examination of 39 patients who had undergone a standard open release of a post-traumatic elbow contracture in our centre during the preceeding 12 months. The patients were recruited according to precise criteria (Table 3). The measurements took place after completion of the physiotherapy and reaching the optimal range of motion of the elbow. The proposed time frame of three to nine months between the surgery and the examination was short enough to ensure that the results would not be influenced by any degenerative changes in the joint. There were 39 patients of mean age of 33 years included in the study, 25 (64.1%) of whom were males and 14 (33.9%) females. Thirty-six patients (92.4%) were right-handed, and three patients were left-handed (7.6%). The most prevalent injuries to the elbow were radial head fracture (11 cases – 28.2%) and distal humeral fracture (9 cases – 23%), which was in accordance with our overall experience. The mean flexion and extension of the elbow joint before the surgery were 117.4° and 44°, respectively, compared to 125.7° and 19.4° after the surgery. The resulting mean increase in the range of motion with respect to the flexion and extension arc was 32.8°. The mean increase in the rotational International Orthopaedics (SICOT) (2020) 44:911–918 915 Fig. 3 The measuring device conducted a cycle of transitions of the forearm between maximal valgus and varus deviations according to the predetermined protocol movements of the forearm was 13°. The average MEPI score was 67 points pre-operatively and 91 points post-operatively. In 24 patients (61.5%), the greater range of physiological laxity was found in the intact elbow joint and in 15 (38.5%) patients in the operated joint. Results Fig. 4 Illustrative graph showing mean relation between torque values exerted by the measuring device and the forearm deviation. Two dotted horizontal lines represent minimal (lower) and maximal (upper line) torque values predetermined by the investigators. Vertical arrow shows range of torque values within which measurements could be registered. Two coloured dots show mean valgus and varus deviation value. It can be appreciated that the valgus dot is located on the maximal torque line, while the varus dot is slightly lower. This shows that whereas the valgus deviation was registered upon reaching maximal torque value, the varus deviation was recorded when maximal torque derivative was reached For each patient, total range of laxity was calculated separately for the healthy and operated elbow. The absolute difference between the laxity ranges of both elbows was then calculated. 916 Table 3 Inclusion and exclusion criteria for biomechanical testing of the elbow stability in operated patients Inclusion criteria 1. Standard open release of a posttraumatic elbow contracture (‘column procedure’) 2. Surgery within the preceeding 3 to 9 months Exclusion criteria 1. Permanent damage to articular surfaces of the elbow a. Malunited intraartricular fractures b. Defect of an articular surface, e.g. excision of the radial head c. Radial head replacement 2. Surgical repair of a collateral ligament 3. Patients undergoing multiple surgeries for a contracture 4. Injury or other pathologies involving the contralateral elbow (except nerve entrapment or asymptomatic tendinopathy) 5. Technical limitations or problems with adhering to the protocol a. Patients of particularly sturdy or slender build b. Persistent flexion contracture limiting elbow extension to more than 30° c. Individual valgus alignment of elbow of more than 20° International Orthopaedics (SICOT) (2020) 44:911–918 The comparison between the two groups showed that the difference in the elbow laxity range between the operated and healthy elbows in the operated patients did not differ statistically significantly from the difference in the elbow laxity range between two healthy elbows in the healthy group (p > 0.1). The collected data allowed for detection of a difference of 0.3° in the healthy group and 0.46° in the group of operated patients with a statistical power of 80%. With 52 and 39 observations in the healthy and operated groups respectively, the mean value of 1.19° in a group of 52 healthy volunteers and the standard deviation in both groups of 1°, a difference between means of 0.6° could be considered as statistically significant. It has been found that the difference in the total deviation between the operated and healthy elbows does not differ statistically significantly by more than 0.6 from the difference in the total deviation of the two healthy elbows and, therefore, is not clinically noticeable. Discussion As stated earlier, analogous data had been previously obtained in healthy volunteers. Wilcoxon signed-rank test was used to verify the hypotheses concerning mean values of absolute differences between healthy elbows and between healthy and operated elbows by comparing the mean differences with the values of 1° and 2°. The equality of the absolute differences in the laxity of elbows between the healthy and operated groups was checked using the Mann-Whitney U test. Statistical significance was assumed at p < 0.05. The difference of 1° between laxity of the two elbows was considered as clinically noticeable. The results of previously performed examinations assessing range of laxity in healthy subjects showed that the mean elbow valgus deviation was 11.2° (6.4°–16.1°) and mean elbow varus deviation was 6.6° (3°–10.7°). The laxity range of the elbow was 17.8° (10.6°–26.5°). The mean difference in laxity between the two opposite elbows in the same person was 1.19° (0.1°–3.8°, SD = 0.84) and was significantly lower than 2° (p < 0.0001). It should be noted that there is no correlation between the side with greater elbow laxity and the dominant side. Although there were 94.2% right-handed patients in the healthy group, only in 57.6% of the volunteers the right elbow was the joint with a greater range of motion in the coronal plane than the left one. The results of the study in patients after surgical release of elbow joint contraction indicated that the mean absolute difference in the laxity range between healthy and diseased elbows was 1.55° (0.1°–4.1°, SD = 1.1) and was also significantly lower than 2°, p = 0.0056. Column procedure is a well-established and efficient technique in elbow surgery associated with favourable results. Although joint instability is not recognized as a common complication of this type of surgery, this issue has received little attention. The aim of our study was to analyse in an objective manner whether column procedure affects elbow laxity, which could in turn lead to joint instability. The vast experience of our centre allowed us to operate in a reproducible manner and to obtain surgical results concomitant with those of other authors, which additionally validates our conclusions. According to our knowledge and thorough search through the available medical databases, there were only two reports involving similar studies conducted in cadaver labs. Nielsen et al. examined the effect of anterior and posterior capsulectomy on elbow laxity in seven cadaver specimens, finding no convincing influence. Dos Remedios et al. in another study came to the same conclusions. These experiments were not, however, continued in vivo, and we suppose that the clinical value of these studies was limited as some of the biomechanical aspects, e.g. dynamic forces exerted by muscles around the elbow, could not have been well reproduced. As far as we are concerned, our experiment was the first biomechanical assessment of elbow laxity following column procedure with the use of a dedicated measuring device. The main limitation of our study is its narrow field. The study was not designed to analyse operative results of a widely used procedure nor to present improvements to the surgical technique but to prove using an impartial approach and sophisticated equipment that joint laxity is not violated. International Orthopaedics (SICOT) (2020) 44:911–918 Conclusion Our experiment confirmed that the ‘column procedure’ is a safe procedure which does not compromise the stability of the elbow joint. Acknowledgements We would like to acknowledge the technical help of Hugon Rudnicki, a design engineer and a programmer who had previously collaborated with our institution on the development and construction of the measuring device used during the experiment. Funding information There were no funding nor other sources of support to be disclosed. Neither the authors nor any members of their families have received any financial remuneration related to the subject of the article. Compliance with ethical standards Conflict of interest There was no conflict of interest. Ethical approval All procedures were in accordance with the ethical standards of the institutional research committee (Centre of Postgraduate Medical Education) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The measuring apparatus had been approved as a diagnostic device in living subjects by the Polish Ministry of Health (decision number 2/10/PLW). Informed consent, as stated above, was obtained from all individual participants included in the study. Statement of Informed consent Informed consent was obtained from all individual participants included in the study. 917 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 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Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 457326, 2 pages http://dx.doi.org/10.1155/2015/457326 Editorial Chemotherapy-Induced Nausea and Vomiting Bernardo Leon Rapoport,1 Alexander Molasiotis,2 Haralambos Raftopoulos,3 and Fausto Roila4 1 The Medical Oncology Center of Rosebank, Saxonwold, Johannesburg 2196, South Africa School of Nursing, The Hong Kong Polytechnic University, Hong Kong 3 Merck & Co., Rahway, NJ 07065, USA 4 Medical Oncology, Santa Maria Hospital, Terni, Italy 2 Correspondence should be addressed to Bernardo Leon Rapoport; brapoport@icon.co.za Received 15 June 2015; Accepted 15 June 2015 Copyright © 2015 Bernardo Leon Rapoport 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. In the past two decades, significant advances have been made in the management of chemotherapy-induced nausea and vomiting (CINV). These advances are primarily due to a greater understanding of the physiological and molecular pathways underlying CINV, which resulted in major progress in the management of patients with CINV. In the early 1990s, CINV treatment consisted of dexamethasone [1]. Improvements in the management of CINV control were achieved with the discovery of 5hydroxytryptamine (5HT3 ) receptor and the development of 5HT3 receptor antagonists (RA). This pathway is primarily involved in the acute phase of CINV. Subsequent studies demonstrated that the usage of the combination of 5HT3 RA and dexamethasone resulted in additional improvements in CINV control [2, 3]. Over the last decade, the discovery of the neurokinin-1 receptor antagonists (NK1 -RA) and its role in the pathogenesis of delayed phase of CINV has led to significant developments in the management of this complication of anticancer treatment. More importantly, these milestone achievements are significant and resulted in an improvement in anticancer treatment compliance, as well as an improvement in the quality of life of patients diagnosed with cancer. Despite these achievements, nausea, in particular, and vomiting remain a clinically significant problem for patients receiving both highly emetogenic chemotherapy (HEC) and moderately emetogenic chemotherapy (MEC). Seventy percent of patients treated with cisplatin-based HEC will achieve an overall antiemetic complete response when managed with a triple therapy consisting of a NK1 RA aprepitant in combination with a 5HT3 RA and corticosteroids prophylaxis [4, 5]. The current antiemetic guidelines (MASCC/ESMO, ASCO, and NCCN) endorse triple therapy treatment for patients receiving cisplatin- and AC-based chemotherapy regimes [6– 8]. The current special issue includes several reviews including the biology and pharmacology of the NK1 receptor and substance P, the antiemetic management of germ cell tumor patients undergoing multiple days’ chemotherapy treatment, radiotherapy induced nausea and vomiting (RINV), CINV induced by oral cytotoxic agents and targeted therapies in patients undergoing treatment for solid tumors, adherence to CINV guidelines and the benefits of NEPA (a new agent consisting of a combination of netupitant and palonosetron), and the treatment of breakthrough and refractory chemotherapyinduced nausea and vomiting. Additionally, 2 original clinical papers are presented investigating the ramosetron and olanzapine in the management of CINV. The review article by S. Garcia and P. Gascon provides an extensive overview of the basic knowledge of the NK1 receptor and substance P biology and the pharmacological basis of the usage of NK1 receptor antagonists in the management of delayed phase of CINV. The 5-HT3 receptor antagonists play an important role in the pathogenesis of the acute phase of CINV. K.-R. Kim et al. presented a pilot study with the potential usage of ramosetron, a tetrahydrobenzimidazole derivative structurally independent of the previously developed 5-HT3 receptor antagonists, such as ondansetron, granisetron, and tropisetron. Patients with germ cell tumor undergoing 5 days of cisplatin-based chemotherapy have a different mechanism 2 BioMed Research International and pattern of CINV compared to those receiving single day chemotherapy. The efficacy of antiemetic drugs, as observed in single day chemotherapy, is therefore not applicable. In this issue, P. Ranganath et al. from Indiana University discuss current recommendations and future directions for patients undergoing multiple day treatment. Depending on the site of irradiation, dosing, fractionation, irradiated volume, and radiotherapy techniques, the incidence of nausea and vomiting after radiotherapy is approximately 50–80%. RINV is a very important and not well researched area often underestimated by physicians. K. Jordan et al. include an overview of RINV and current guidelines recommendations as well as future directions. The treatment of nausea and vomiting caused by oral antineoplastic agents is primarily empirical, consisting of the administration of daily oral antiemetic therapy. The level of evidence of prophylactic antiemetics recommended for these agents is low. A. L. Costa et al. discuss the management of CINV induced by oral cytotoxic agents and targeted therapies in patients undergoing treatment for solid tumors. This article highlights the differences in the classification of emetogenic potential of oral antineoplastic agents between the different international guidelines, as well as different recommendations for prophylactic antiemetic treatments. NEPA is a new oral single fixed combination agent, containing a highly selective NK1 RA, netupitant with palonosetron. This agent is a pharmacologically and clinically distinct 5-HT3 RA. Palonosetron has a longer half-life compared with older 5-HT3 RAs. Palonosetron works synergistically with netupitant and has the potential to improve the efficacy in the prevention of the delayed phase of CINV when used in combination. P. J. Hesketh et al. discuss the use of NEPA in the context of how NEPA may overcome some of the barriers interfering with adherence to the various antiemetic guidelines. Several studies have shown that the antipsychotic agent olanzapine is effective in the management of CINV. The pharmacological mechanism of action consists of the blocking of neurotransmitter receptors including dopaminergic at D1 , D2 , D3 , and D4 brain receptors, serotonergic at 5-HT2a , 5-HT2c , 5-HT3 , and 5-HT6 receptors, catecholamines at alpha1 adrenergic receptors, acetylcholine at muscarinic receptors, and histamine at H1 receptors. In this special issue, R. M. Navari discusses the treatment of breakthrough and refractory chemotherapy-induced nausea and vomiting with special reference to olanzapine in this setting. In another paper, M. Abe et al. retrospectively analyze the role of olanzapine in 50 gynecologic cancer patients receiving cisplatinbased chemotherapy who had nausea despite the use of standard therapy. The treatment of CINV is evolving and this special issue highlights some of the recent developments as well as some of the controversies in this important field of oncology. Bernardo Leon Rapoport Alexander Molasiotis Haralambos Raftopoulos Fausto Roila References [1] M. S. Aapro and D. S. Alberts, “Dexamethasone as an antiemetic in patients treated with cisplatin,” The New England Journal of Medicine, vol. 305, no. 9, article 520, 1981. [2] G. Falkson and A. J. van Zyl, “A phase I study of a new 5HT3receptor antagonist, BRL43694A, an agent for the prevention of chemotherapy-induced nausea and vomiting,” Cancer Chemotherapy and Pharmacology, vol. 24, no. 3, pp. 193–196, 1989. [3] D. B. Smith, E. S. Newlands, O. W. Spruyt et al., “Ondansetron (GR38032F) plus dexamethasone: effective anti-emetic prophylaxis for patients receiving cytotoxic chemotherapy,” British Journal of Cancer, vol. 61, no. 2, pp. 323–324, 1990. [4] P. J. Hesketh, S. M. Grunberg, R. J. Gralla et al., “The oral neurokinin-1 antagonist aprepitant for the prevention of chemotherapy-induced nausea and vomiting: a multinational, randomized, double-blind, placebo-controlled trial in patients receiving high-dose cisplatin—the aprepitant protocol 052 study group,” Journal of Clinical Oncology, vol. 21, no. 22, pp. 4112–4119, 2003. [5] S. Poli-Bigelli, J. Rodrigues-Pereira, A. D. Carides et al., “Addition of the neurokinin 1 receptor antagonist aprepitant to standard antiemetic therapy improves control of chemotherapyinduced nausea and vomiting: results from a randomized, double-blind, placebo-controlled trial in Latin America,” Cancer, vol. 97, no. 12, pp. 3090–3098, 2003. [6] National Comprehensive Cancer Network, “NCNN Guidelines 1.2012: antiemesis 1.2015,” Tech. Rep. 1.2015, 2015. [7] F. Roila, J. Herrstedt, M. Aapro et al., “Guideline update for MASCC and ESMO in the prevention of chemotherapyand radiotherapy-induced nausea and vomiting: results of the Perugia consensus conference,” Annals of Oncology, vol. 21, supplement 5, pp. v232–v243, 2010. [8] E. Basch, A. A. Prestrud, P. J. Hesketh et al., “Antiemetics: American Society of Clinical Oncology clinical practice guideline update,” Journal of Clinical Oncology, vol. 29, no. 31, pp. 4189– 4198, 2011. 

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Zulfadli Abdul Aziz, Masrizal Mahmud, Dista Nurhasanah: Women’s Languange… AL-TA’LIM JOURNAL, 29 (3), 2022, (253-266) (Print ISSN 1410-7546 Online ISSN 2355-7893) Available online at http://journal.tarbiyahiainib.ac.id/index.php/attalim Women’s Language Used in ‘Birds of Prey and the Fantabulous Emancipation of One Harley Quinn’ movie Received: 05th April 2022; Revised: 06th November 2022; Accepted: 19 th November 2022 Permalink/DOI: http://dx.doi.org/10.15548/jt.v29i3.821 Zulfadli Abdul Aziz *) Universitas Syiah Kuala, Banda Aceh, Indonesia. E-mail: zulfadli.aziz@unsyiah.ac.id Masrizal Mahmud Universitas Syiah Kuala, Banda Aceh, Indonesia. E-mail: masrizal@unsyiah.ac.id Dista Nurhasanah Universitas Syiah Kuala, Banda Aceh, Indonesia. E-mail: distaanurhasanah@gmail.com Abstract: The purpose of this study is to analyse the major features of women’s language used by the female characters in “Birds of Prey and the Fantabulous Emancipation of One Harley Quinn” movie. This study investigates the types of women speech features found in the movie. A qualitative approach employing a content analysis method was used in this study. The data were taken from female characters’ utterances. The data were then identified and analysed based on the classifications of women’s speech features proposed by Lakoff. Findings of this study indicate that six of the features are used by the female characters: lexical hedges/fillers, empty adjectives, tag questions, super polite forms, avoiding strong words, and emphatic stress. Rising intonation, precise colour terms, intensifiers, and hypercorrect grammar have not been found in the current research data. Keywords: Language and gender; women’s language; women’s speech features. How to Cite: Aziz, Z., Mahmud, M., & Nurhasanah, D. (2022). Women’s Language Used in ‘Birds of Prey and the Fantabulous Emancipation of One Harley Quinn’ movie. Al-Ta lim Journal, 29(3). doi:https://doi.org/10.15548/jt.v29i3.821 INTRODUCTION empirical research, women's language based on the definition of Lakoff is used to describe women and the language they use (Bell & Gibson, 2011; Pourkalhor & Esfandiari, 2017; Swann & Deumert, 2018). Gender differences in language are often just one aspect of more persuasive linguistic differences in the society reflecting social status or power differences, if a community is very hierarchical, for instance, and within each level of the hierarchy men are more powerful than women, then linguistic differences between the speech of women and men may be just one dimension of more extensive differences reflecting the social hierarchy as a whole (Campbell-Kibler, 2010; Holmes, 2013; Jinyu, 2014). For example, men always try to take control in every conversation, sort out the topic, and interrupt Many people still believe in stereotypes about the difference in speech between female and male that are always related to power and status (Holmes, 2013; Holmes & Wilson, 2001; Kafi & Motallebzadeh, 2016). Montgomery (1995) however, suggests that even though there are some variations in speech differences between males and females, 'the differences of speech are not clear-cut and that a series of universal differences does not exist.’ Early studies that regard power as a main factor in speech differences between different genders can be attributed to Lakoff’s (2004, 2017). Although relying heavily on personal observations, and later Lakoff's definition of 'women's language' was criticized for feminist bias and a lack of 253 © 2022 by Al-Ta’lim. This work is licensed under (CC-BY-SA) 254 Volume 29, Number 3, November, 2022, Page 253-266 when they are talking. While women are more careful in choosing the language they use, they don’t have power to interrupt conversation, and also because the status of women is lower than men (Fishman, 2019; Sandberg, 2015). This present research aims to find out the types of women’s speech feature found in two movies: “Birds of Prey and the Fantabulous Emancipation of One Harley Quinn”. In these movies, the female characters are depicted as strong characters which had super powers and not the type of women who are just purely obedient to men. This movie is interesting to analyse because the female characters sometimes show their power towards the male characters, attracting us to further investigate the women’s language used by the female characters. This research seeks the answer types of women’s speech features found in “Birds of Prey and the Fantabulous Emancipation of One Harley Quinn” movie”. The researchers use Lakoff’s (1975) theory about the types of women’s language features. A language that signifies the characteristics of a woman's speaking style can be seen, for example, from relying more on an adjustment that conveys uncertainty and avoids bold and direct speech. A woman will question themselves if they feel unsure of their own beliefs, this can happen if they are unsure about a problem, that's why many problems faced by women appear with question marks (Chalupnik et al., 2017; Garcés-Conejos Blitvich & Sifianou, 2017). When women use more standard forms than men, it can be seen as women’s linguistic behaviour (Holmes, 2001). In many speech communities, when women use more of a linguistic form than men, it is generally the standard form which overtly prestigious form that women favour. Holmes also adds that the reasons women use more standard form because of the social class that is related to their status which is wrongly regarded as a subordinate group and their role in society relating to the function of speech expressing masculinity. As women are serving as models for children’s speech, the society expects that women use standard form commonly associated with more formal and less personal interactions. The role of modelling correct behaviour in the community is usually designated for women. Women may use different linguistic forms such as word-shape (morphology), for example, brunch or travelogue, pronunciation which women tend to pronounce more clearly, and different affixes due to the fact that women and men speak differently. Women also use more formal variant in the pronoun system, for example the use of the pronoun I do we, and they use vocabulary items differently (Holmes, 2001). Lakoff (1975) has proposed that there are linguistic differences between men and women. He strengthens that there are ten linguistic features which characterize women's speech: lexical hedges or fillers, tag questions, rising intonation on declaratives, 'empty' adjectives, precise colour terms, intensifiers such as just and so, hypercorrect grammar, super polite forms, avoidance of strong swear words, and emphatic stress. Holmes (2001) asserts that there is a distinction between ‘fillers’ and ‘hedges’, with sort of classified as a hedge, while ‘well’ and ‘you see’ are described as ‘meaningless particles’ and assigned to the same category as ‘pause fillers’ such as uh, um, and ah. Women tend to use this feature more than the men do. They use this feature to keep their femininity and as a protection when they are making a certain statement but they are not sure about their statement. When a woman attempts to describe a book for her friend, for example, she doubts that the book is a kind of book that is either interesting to read or boring for some people because she is unsure what kind of book is preferred by her friend. For this reason, a hedge is used to mitigate the meaning, as in the following examples. (a) The book is kind of well to read. (b) You know, this novel is good. The examples illustrate the speaker’s uncertainty about what is being uttered. She is © 2022 by Al-Ta’lim. This work is licensed under (CC-BY-SA) Zulfadli Abdul Aziz, Masrizal Mahmud, Dista Nurhasanah: Women’s Languange… using a lexical hedge to weaken the strength of an assertion that may cause offense. Furthermore, women also use tag question to express uncertainty (Lakoff, 1975). Holmes (2001) says that “tag may also express affective meaning. They may function as facilitative or positive politeness devices, providing an addressee with an easy entrée into a conversation”. For women, using tag question helps them overcome their uncertainty on a subject when they were trying to start a conversation (a). In this case, they demand a confirmation of their statement from others. However, in some cases, it is just a tool to start a small conversation that emphasizes a reality (b). (a) She’s very wonderful, isn’t she? (b) Today is very hot, isn’t it? Lakoff (1975) also argues that there is a special use of syntactic rules in female intonation patterns. They tend to use over-thetop expression because they anticipate to not being taken seriously by their counterparts, e.g., it’s really good? When women have to give a definite answer on a question, they will say it in a declarative tone by raising their intonation. (a) Who will you marry? (b) Uhm ... my bias, Yoon Dowoon ...? The example shows lack of specific answer to the question and uncertainty thus reflects on doubt of the character hence why women words are not treated seriously. In addition, women incline to use empty adjective. It is an adjective only used by women as opposed to that natural adjective commonly used by men. It is used to express a remarkable state of circumstances (Hall & Bucholtz, 2012). For example, men will say ‘great’ for something they think is really good, but women will say ‘adorable’ instead. There is, for instance, a group of adjectives that do not only have specific and literal meaning, but also have another use that indicating the speaker’s admiration for something. Some of these adjectives are neutral to both men and women, but another set seems to be confined to women’s speech. Representative lists of both types are below: Neutral Great Terrific Cool Neat women only adorable charming sweet lovely Men barely used some words listed in the right column because they believe it will damage their reputation, while women are free to use both. In expressing colour, women have more colour vocabulary than men because women's words are related to their interests. Women have a special term for colours like baby blue, maroon, navy etc (Romaine, 2003, 2017). (Blommaert, 2015) said that such a discrimination is absent from that of most men. As an example of precise colour terms, a woman may say: ‘The door is navy’ instead of blue. Women make the distinction of colour terms to indicate the level of importance whereas men use it to imitate women in a sarcastic way. Women also use intensifier when they want to express and emphasize what they want to say. Intensifier is a word that can emphasize a statement in order to make the listener surer of what the speaker is saying. When a woman use intensifier, it means she wants to express her opinion with a felling and to convey the emotional message as opposed to accurately phrasing a cognitive statement (Carli & Bukatko, 2012; Edwards & Hamilton, 2004). For example: (a) I feel so unhappy (b) That movie made me so sick Furthermore, women do not speak harshly and use hypercorrect grammar. They use subtle speech with standard verb to sound more polite (Lakoff, 1975). The standard verb form is used to avoid words or sentences that are not good and to reinforce politeness, e.g. I would be very thankful if you can show the way. The reason on why women use more correct grammar in their speech is that they tend to be more status conscious. Women adhere to social norms because they’re afraid to be judged when they speak so they aspire to exhibit “better” social behaviour than men. For © 2022 by Al-Ta’lim. This work is licensed under (CC-BY-SA) 255 256 Volume 29, Number 3, November, 2022, Page 253-266 example, women use more -ing pronunciations and fewer -in pronunciations than men, e.g. women usually use singing rather than singin’ (Garimella et al., 2019; Wolfson, 2014). To avoid an unpleasing situation, women use super polite form or speak indirectly so that to the audience do not feel offended or embarrassed by what they are saying and to sound more polite, e.g., she passed away (she died) (Lorenzo-Dus & BouFranch, 2003). Svendsen (2019) also argues that women tend to avoid offensive expressions because they are believed to be the experts of euphemism. Women also required to use particular polite forms such as” please” and “thank you”. Here is an example: (a) Come here. (b) Will you please come here? From the example above, (a) shows a less polite than a request “will you please come here?” because the addressee is not being forced to respond a command. Using an order as a question by suggesting the addressee seems more polite than an overt order. Additionally, unlike men, women avoid using swear words as they do not want to be seen as rude and disrespectful (Holmes, 2001). They prefer to use the word "oh dear" rather than “shit”. For the example: (a) oh goodness you take those things in wrong way again! (b) Damn it you take those things in wrong way again! The expression in (a) uses weaker swear word while expression (b) uses strong swear word. Because women tend to speak in more polite way, people will expect that expression (a) is spoken by a woman and expression (b) is spoken by a man. However, women use strong swear words more and more nowadays, but women’s language is not adopted by men. The differences between expression (a) and (b) are in how strong those words evoke the negative feeling on the addressee. In addition, women typically use a greater range of pitch than those used by men. For example, “what a beautiful dress!” and “it was brilliant performance”. When the speaker says those sentences, the speaker verbally highlights the words beautiful and brilliant by using pitch (usually higher), elongation (making the vowels last longer) or variation in dynamic volume (loudness). The speaker uses emphatics stress to emphasize the most important word in a sentence. METHOD The present study qualitatively analysed and examined the types of women’s women speech features found in the Birds of Prey and the Fantabulous Emancipation of One Harley Quinn movie. The subject of this research was the Harley Quinn’s Birds of Prey film, whereas the object of the research was the women's languages used in the movie. The data were gathered from the female characters’ utterances. They include words, phrases, and sentences that contain types of female speech features related to the topic. In the present study, the researchers used Miles et al. (2014) framework for data collection and analysis, which are commonly known as data reduction, data display, and drawing conclusion/ verification. There are nine steps of data reductions introduced by Miles and Huberman (2014), but the researchers only used two steps; sorting out the data and coding. The first step was used to collect the data and the second was used to analyse the data. Three steps of data collection were involved in the present study; The first has to do with identifying the conversations carried out by all the female characters in the movie. In the next step, we compare the transcript-data with subtitles. At the final stage, we organize the speech, particularly all utterances collected from the female characters. This made it easier for the researchers to analyse the language used by the female characters in the film. We also eliminated similar data that were considered redundant, so there is no repetition of data. The researchers selected the main points, and only focused on the important points while simplifying the data. © 2022 by Al-Ta’lim. This work is licensed under (CC-BY-SA) Zulfadli Abdul Aziz, Masrizal Mahmud, Dista Nurhasanah: Women’s Languange… Finally, the data were analysed based on the classifications of women’s speech features originally proposed by Lakoff’s (2004) and re-introduced by Holmes (2013): namely lexical hedges or fillers, tag questions, rising intonation on declaratives, ‘empty' adjectives, precise colour terms, intensifiers such as just and so, hypercorrect grammar, super polite forms, avoidance of strong swear words, and emphatic stress. The researchers used an observation checklist to record utterances containing particular language features currently being investigated. During this stage, the researchers underline and change the colours of the selected utterances based on Lakoff's (2004) theory of women's language. The researchers also used different letter symbols to distinguish each character to help with the analysis process. Techniques of Data Analysis After collecting the data, the researchers analysed the data using content analysis techniques. First of all, the types of previously mentioned women's speech features used by the female characters were identified. To ease the data analysis process, the following initials were used for each character; Haley Quinn as (HQ), Huntress as (H), Black Canary as (BC), Rene Montoya as (RM) and Casandra Cain as (CC). These were used in the observation checklist, as seen in the following table. Table 1. The observation checklist Features Lexical Hedges/Filler Tag Questions Empty' Adjectives Precise Color Terms Intensifiers Hypercorrect' Grammar Super polite' Forms Avoidance of Swear Words Emphatic Stress HQ H BC RM CC Secondly, the researchers determined which data were considered the dominant types and the dominant patterns of the women's language for each character. Afterwards, each of women’s speech features was investigated in order to find its functions. The results were analysed pragmatically based on the speaker’s meaning that is related to the context. At this stage, we provide an explanation of how the characters in the movie used women's language in their speech. The researchers then discussed the use of women's language by the characters based on social conditions. The final step is drawing conclusions, where the researchers use data display technique proposed by Miles et. al. (2014). RESULT AND DISCUSSION The researchers used Lakoff's (2004) theory to determine the types of women's speech features found in Birds of Prey and The Fantabulous Emancipation of One Harley Quinn movie. This theory was used as the reference in analysing the findings in which each classification was classified in the observation checklist. After carefully evaluating the movie conversations and its transcripts, the speech features used by women characters in the movie can be seen in the following table. Table 2. Women’s Speech Features in the Movie Features Lexical Hedges/Filler Tag Questions Rising Intonation on Declarative Empty Adjectives Precise Color Terms Intensifiers Hypercorrect Grammar Super polite Forms Avoidance of Swear Words Emphatic Stress Total Features HQ H 3 1 RM C 2 1 5 1 1 3 2 3 2 2 1 17 3 6 BC 1 2 1 1 2 3 11 2 From the data, we can see that there are eleven features spoken by Harley Quinn. She uses three lexical hedges/fillers (‘kinda hard’, © 2022 by Al-Ta’lim. This work is licensed under (CC-BY-SA) 257 258 Volume 29, Number 3, November, 2022, Page 253-266 ‘kinda nice’, ‘you know’), five tag questions (aren’t you), three empty adjectives (‘nice baby’, ‘sentimental value’, ‘pretty great’), three super polite form (‘gone’), two avoidance swear words (‘oh god’, ‘Jesus Christ’) and one emphatic stress (‘whatever’). Hunter uses six utterances of women’s speech features. She uses one filler (‘mhmm’), two super polite forms (‘I need your help’, ‘gone’), three emphatic stresses (‘it is not’, ‘a fucking’, and ‘I don’t have’). Black Canary uses one super polite from (‘lady’), and one avoidance swear word (‘thank God’). Rene Montaya uses 11 utterances of women’s speech features. She uses two lexical hedges/fillers (‘hmm’, ‘well you know’), one tag questions, (‘isn’t it’), two empty adjectives (‘sweetheart’ ‘amazing’), two super polite forms (‘loss’, ‘gone’), one avoidance swear word (‘Jesus’), and three emphatic stresses when she verbally highlights the words ‘spot’, ‘relationship’ and on ‘herself’. Casandra Cain only uses one filler which (‘mhmm’) and one tag question (‘aren’t they’). Lexical hedges/filler Hedges/filler is an expression that is used as a protection to the lack of confidence about the speaker’s statement. There are seven hedges/fillers found in the movie: HQ: “…. like out of a plane without a parachute, right in your fucking dumb face kinda hard.” HQ: “It was kinda nice having the kid around.” Harley utters the word "kinda" two times in different ways. The first one is when she talks about how she feels when she falls in love with Mr. J. She describes her emotions by saying that falling in love with Mr. J is like falling from a plane without a parachute. The second statement shows that Harley is not sure about her feelings for the kid. She is not sure whether the kid is really nice or bad. In both utterances, she uses "kinda" to express her uncertainty about her statement. To cover it up, she uses the word as protection. RM: “Well you know, the DA office is building a case against him too.” The use of hedges in Rene's utterance is a form of uncertainty about her statement. When Rene talks with her boss, she uses the phrase "well you know" as a meaningless particle. She uses it to cover up her uncertainty about the DA's office, who built a case against Sionis. She is not sure about her statement, but she still wants her boss to believe that the DA's office would build a case against Sionis. H: “Mhmm… I really like how you were able to kick so high in those tight pants.” RM: “Yes he is uhmm.. I uhmm he no longer there but-.” CC: “Uhmm thanks for the car.” Hunter, Rene, and Cassadra use fillers in their speech when they are uncertain about their statement. All of them use the fillers "Mhmm" and "Uhmm". When Hunter uses filler in her utterance, she is not sure if she could praise Black Canary or not. She says her compliments after using the filler, so she will feel more protected because of the pause filler. The same is applied to Rene and Cassandra’s utterances. They both use pause filler to cover up their lack of confidence when talking to someone who has a higher position than they do. Rene uses the filler when she is not sure if her partner is still in the Sionis’ army or not. For Cassandra’s utterance, the use of pause filler is also as a protection for her lack of confidence. When the toy car that Hunter lent her is able to relive her fear, she was very grateful. Cassandra is shy to say "thank you" to Hunter, so she uses the filler "Uhmm" before it to cover up her uncertainty. Tag Questions When they’re uncertain about something, women use tag questions to make the addressee agree with their statement. Here are the examples of tag question used in the movie: © 2022 by Al-Ta’lim. This work is licensed under (CC-BY-SA) Zulfadli Abdul Aziz, Masrizal Mahmud, Dista Nurhasanah: Women’s Languange… RM: “Isn’t Ms Yee?” HQ: “You are not gonna run, aren’t you?” CC: “They are all here for me, aren’t they?” Rene, Cassandra and Harley use tag questions for confirmation. Rene uses the word "isn't" because she wants an agreement from Ms. Yee. She also wants Ms. Yee to support her statement. Rene uses "isn't" because she is not sure if the spy is still in the building or not. She also wants Ms. Yee to help her convince their boss about her statement. The word "aren't you" is used in Harley's utterance when she talks with Cassandra in a shopping mall. "You're not gonna run, aren't you?" the tag question "aren't you" is used because she is not sure about what she is saying. She knows Cassandra prefers to be with her, but she is not really sure about it. She pauses for a moment after saying, "you are not gonna run," after she realizes that she is noy sure about what she is saying. She uses the tag question as a form of confirmation from Cassandra because she is not confident with her words. When Harley uses the words "nice baby", "sentimental value", and "pretty great", she wants the addressee to know about her feelings. She uses the word "nice baby" when she gives her pet a treat and talks with her. She uses the word "nice baby" as a form of admiration towards her pet. Harley knows her pet is not a baby, but calls it as such because she wants the pet to know that she loves her very much even though her pet does not understand what she is saying. Haley also uses two empty adjectives in her speech, "Oh no" and "pretty great". She uses the phrase "sentimental value" when Rene wants to use her baseball bat. She wants the addressee to know that the baseball bat meant a lot to her, which is why she uses an empty adjective to convey her feelings to the addressee. The phrase "pretty great" is used to express her feelings, that she feels pretty good despite the unexpected bad luck. RM: “Woahh.. I hate to be the one to break your heart sweetheart, but you are not done.” RM: “I mean we are amazing there.” Cassandra also uses tag question to make sure that the addressee agrees with her statement. "They are all here for me, aren't they?" when she is saying that, she knows for sure that Sionis's army is there to catch her because of the diamond in his stomach. She is not questioning the situation, but more of a confirmation from others. She is calling Hunter "sweetheart" so that Hunter knows that she feels sorry for Hunter. The word "sweetheart" is usually used to address children, but Rene calls Hunter "sweetheart" in her utterance because she wants to. Hunter knows that Rene loves her and that she is not alone in dealing with Sionis. The word "amazing" is used when Rene reveals her admiration to themselves when they are able to beat a dozen of men. Empty adjectives Super polite forms The use of empty adjectives in women’s language indicates that the speaker wants to show the relationship of her emotional expression with her addressee. In this movie, the female characters use five words of empty adjective. Women use the super polite form or speak indirectly so that people they are talking to do not feel offended and to sound more polite. Thus, women also use tag orders, tag questions, and other requests to state polite commands. HQ: “Hello nice baby.” HQ: “but still, that felt pretty great.” HQ: “Oh no no not that one, sentimental value.” HQ: “You heard the cop said, Sionis is gone.” H: “He’s gone,” RM: “Sionis is gone “ © 2022 by Al-Ta’lim. This work is licensed under (CC-BY-SA) 259 260 Volume 29, Number 3, November, 2022, Page 253-266 When they finally beat Sionis, Harley, Hunter, and Rene express their joy by saying "he's gone?" and "Sionis is gone". Instead of using the word ‘death’, they use "gone" because they do not want to sound harsh on Sionis deaths. H: “I need someone.” your help locating When Hunter meets Sir Kebab for the first time, she asks him for help to find someone. She says, "I need your help." because she is trying to look polite and does not want the addressee to feel like he is being given orders. BC: “Yeah, I do not know who you think I am lady, but I’m not her.” Black Canary and Harley both use the word "lady" in their speech. "Lady" is a term used to refer to a woman who has a high social status. They use the word "lady" to honour the woman they are talking to. Canary uses it because she respects Harley even though Harley has broken up with Joker. Harley uses it to honour every girl she met. She uses "lady" to make the addressee feel less offended. RM: “I’m sorry for your loss.” Rene uses super polite form when she talks to Black Canary to express her condolences even though it is too late. Rene still says it so that the addressee does not feel offended by what Rene says earlier about Black Canary's mother. Rene does not want Black Canary to think that she does not care about her mother's death. She expresses her condolence so that Black Canary would not feel offended and to reduce her embarrassment for not being able to help Black Canary's mother. Avoidance of Strong Swear Words HQ: “Oh god, stop.” HQ: “oh Jesus Christ.” Harley prefers to use the words "oh god" and "Jesus Christ" instead of swearing when talking to Sionis. Harley usually prefers to swear rather than avoid using swear words, but when dealing with Sionis under threat, she prefers to use the words "Oh God" and "Jesus Christ". According to Allan & Burridge (2006), the use of swear words is closely related to one's emotions because using swear words can diverge someone's feelings effectively. Even though Harley is threatened and upset, she chooses to avoid using swear words because she does not want Sionis to feel that he does not respect him. BC: “Oh Thank god, don’t worry I’m gonna get you outta here.” Black Canary avoids using swear words when she finds Cassandra tied up in the toilet of an abandoned building. She feels grateful that Cassandra is still alive. The difference between using swear words and avoiding using swear words can be seen from the speaker's feelings. When she find Cassandra, Black Canary feel more grateful than angry, and that is why she is saying "thank God" instead of swearing. RM: “Jesus who the fuck is this?” When Rene gets multiple phone calls, she is annoyed because the calls interfere with her meeting with her boss. She says "Jesus" after saying "who the fuck is this" to relieve her annoyance because she still wants to sound polite even though she cursed afterward. Emphatic Stress Women typically use a greater range of pitch than those used by men. HQ: “I could do whatever I want.” Harley uses empathic stress in her utterances to emphasize words he thinks are important. She adds the word "whatever" to emphasize that she really can do whatever she wants. In this context, the use elongation is obvious so that the listener focuses on the word she is emphasizing. H: “It’s not, a fucking, bow and arrow.” Hunter convinces the addressee that the weapon she is using was not a bow and arrow © 2022 by Al-Ta’lim. This work is licensed under (CC-BY-SA) Zulfadli Abdul Aziz, Masrizal Mahmud, Dista Nurhasanah: Women’s Languange… but a crossbow by emphasizing the words "it is not" and "a fucking". Hunter uses pitch when saying "it is not" and makes the vowels last longer when saying "a fucking". She does not want the addressee to take her words as a joke. H: “I don’t have, rage issue!” When she is talking with hunter about the crossbow, Black Canary teases hunter by saying that she has rage issues. Hunter does not accept that and says, "I don't have a range issue." She emphasizes the word "I don't" to make black canary stop teasing her and take her words seriously. RM: “This was their spot. She just publicly updated her relationship status. Harley Quinn just called open season on herself.” Just like Harley and Hunter, Rene emphasizes a few words in her speech so that the addressee does not take her words lightly. Rene emphasizes the word "spot.", "relationship status", and "herself" as she explains why Harley blow up a building and throw away the necklace he used to wear. Rene pauses between each word at the end of her sentence to further clarify her statement. Based on the findings, out of Lakoff’s (2004) ten linguistic features which characterize women's speech, the researchers only found six in the movie; lexical hedges/filler, tag question, empty adjective, super polite form, avoidance of swear words, and emphatic stress. Super polite form is a feature that is mostly used by the female characters. There are eight super polite forms used by all female characters except Cassandra. The female characters do not use rising intonation, precise colour terms, intensifiers, and hypercorrect grammar. In contrast to the current findings, Leaper & Robnett (2011) only found one utterance related to super polite form. The most used feature in her study is lexical hedges/fillers, which are found in 19 utterances. Similar to Amanda (2017), she found that lexical hedges/fillers were the most used form (41.54%) by Hillary Clinton in her Presidential Debates. The use of female language by female characters also varies. Harley uses the most women's speech features in the movie. She uses 15 words for each different feature. Her use of female language is as a form of lack of confidence and friendliness when talking to others. This can be seen from the many uses of question tags, super polite forms, lexical hedges/fillers, and empty adjectives in her speech. Despite being a female criminal, Harley is a character who uses the most female language in the movie. She often uses women's language due to her past as a psychiatrist where she was required to speak politely and gently to her patients. The inharmonious environment and her untrustworthy relationship with the surrounding community are one of the reasons she uses women's language a lot in her speeches. The use of women's language in Harley's speech is a form of femininity and also covers her lack of confidence. Hunter and Rene also use women's language features as a form of respect to the addressee and lack of confidence. They use emphatic stress in their speech so the addressee does not take their words lightly and to emphasise the serious tone of the situation. It is used to intensify the speaker’s utterances and emotional message (Bylund & Makoul, 2002; Palczewski et al., 2023). Black Canary tend to avoid swear words because she doesn't want to be considered rude by the addressee. She used this feature when talking to Cassandra who is much younger than her (Lakoff, 2004) notes that women tend to avoid using swear words because they will consider as unladylike. The linguistic devices that deeply distinguish women's language from that of men are lexical hedges or fillers. It is an utterance constructed with one or more words that expresses uncertainty from a lack of confidence (Purnata et al., 2021). Cassandra Cain used lexical hedge/filler and tag question to expresses her uncertainty and as a form of lack of confidence. The results suggest that the female characters used women's language features to keep their femininity. Women should talk like © 2022 by Al-Ta’lim. This work is licensed under (CC-BY-SA) 261 262 Volume 29, Number 3, November, 2022, Page 253-266 women; if they refuse, they are ridiculed and subjected to criticism as unfeminine (Lakoff, 2004). Men's social roles are designed to support the masculine concept, while women's social roles are to be feminine (Sun, 2013). The movie implies that the female characters use the women’s language features because they want to adhere to their social roles and didn’t want to be considered rude when talking to others. The Use of the Opposite of Women’s Language Unlike previous studies that are focusing on women’s language used only by women, the researcher found contrary evidences in this movie. The female characters freely use the opposite of women’s speech features more than the women’s speech features described by Lakoff (2004). The researcher found two uses of the word “cool”, which according to Lakoff (2004) is used more often by men than women. According to Aini (2016), women are free to choose between neutral words and feminine words in empty adjectives. Both genders can also use adverbs to their liking (Hanafiyeh & Afghari, 2014). However, it is also wrong to say that there is no risk for women to freely use neutral or women's words, because they will be considered as being unable to express their feelings by doing so. Chen (2021) claims that women use more adjectives than men do, although the qualitative differences are not significant. For this reason, we can conclude that the empty adjective feature is not gender specific. Both women and men use empty adjectives in their speech. The number of uses depends on the speaker’s social background or the topic being discussed. The researchers also found that the female characters use impolite forms of speech and offensive expressions to strengthen and emphasize their words so as not to be underestimated by others as well as to express their annoyance. This finding is not in line with Lakoff's (2004) theory suggesting that women use the super polite form more in their speech to avoid offending others. To explain this phenomenon, Cameron and Coates (1985) explain that the amount of discourse we produce is influenced by who we are with and what we are doing. Omen (2017) suggests that Linguistic politeness is the use of language in conversation to show consideration for the feelings and desires of one's interlocutors, to create and maintain interpersonal relationships (so-called political behaviour), and to follow the rules for what society or one's culture considers appropriate behaviour. Hobbs (2003) mentions that the frequency of negative politeness markers of male speakers is about the same as that of women, whereas men prefer to use more positive politeness techniques than women when they are in legal settings. It is suggested that women are often more polite than men is not true. According to Lakoff (2004), women tend to avoid the use of swear words, however the researchers found that all female characters use many swears words in their speech. There were nine ‘fucking’ words, eighteen ‘fuck’ words, four ‘motherfucking’ or ‘motherfucker’ words, fifteen ‘shit´ words, five ‘hell’ words, one ‘fuckbag’ word, two ‘damn’ words, two ‘asshole’ words, one ‘stupid-ass’ word, one ‘dick’ word, and one ‘crap’ word. The female characters used swear words as an affix. It was not used for swearing but to emphasize their subsequent words. In contrast to our findings, Walsh (2016) assert that male speakers use swear words more frequently than females. Interestingly, the female characters in the movie used more swear words than men, either when they were stress, in hurry, frustrated, habit, or in a precarious situation. Swearing can also be associated with their educational background. In the movie, the female characters grow up in a high crime rate environment. They live in a city where the average people are criminals and mafia. Furthermore, work-environments can also be the reason behind vulgar languages. The female characters, except Cassandra, work © 2022 by Al-Ta’lim. This work is licensed under (CC-BY-SA) Zulfadli Abdul Aziz, Masrizal Mahmud, Dista Nurhasanah: Women’s Languange… in a place that was closely related to criminals. Harley is unemployed, Hunter is an assassin, Rene is a low-ranking police officer, Black Canary is a club-singer, while Cassandra is just a child whose parents are always fighting. Based on social classes, the low-class people use swear words more often than those of the high class in their conversation. In other situation, people use swear words to create or show intimacy Wiranty (2015). Women use swear words more when they are communicating with their own gender (Varma & Tan, 2015). The words they use are mostly related to religion, bodily functions, and body parts, such as shit, fuck, hell, and God. It can be said that women use swear words in their speech because they want to express their feelings. Their low social status and living environment also motivate the use of swear words in their speech. Hay (2000) says that uncertainty and lack of confidence is not the only reason why women use women's speech features. She agrees that there are several factors aside from gender, topic and social status that influence the use of women's language. These factors include practice community, educational background, and social psychological perceptions (Arisandy, 2018). Nowadays, not only women, men also practice Lakoff's (2004) women’s language features in their daily conversations. Many linguists and researchers have also criticized and expressed opposing views on Lakoff's theory. Hall & Bucholtz (2012) point out that Lakoff's claims are not supported by empirical evidence. The majority of her findings were made by researchers in the empirically oriented social sciences (anthropology, psychology, sociology, and speech communication). Likewise, Holmes (2001) notes that Lakoff's assertions were based on her intuitions and observations, and that much of her initial research was methodologically unsatisfactory. Anderson et al. (2011) argued that Lakoff's description of women's linguistic characteristics is inaccurate. They demonstrate that the frequency of women's language features in speech is correlated with two other factors: the speaker's social status and the speaker's experiences. In her study, Aini (2016) found male characters in the movie used five types of women's language features. They were: raising intonation, super polite form, lexical hedge, intensifier, and tag questions. In the "I' Tonya" movie, three women's language features were used by the male characters, which are lexical hedges/fillers, intensifiers, and avoidance of swear words (Purnata et al., 2021). From the previous linguistic and studies above, we can conclude that women's language features can also be used by men. Furthermore, after analysing the use of women's language features used by female characters in the movie, the researchers concludes that the use of women's language in this movie does not always reflect selfconfidence, difficulty in making decisions, and uncertainty about their words, but there are several social factors and dimensions that can be the reason behind the use of women's language, such as educational background, social status, friendship, topics of conversation, formal settings, and work. Nowadays, many women have started using the male language as well as many men. Men have also started using some features of the female language. Women and men can interact with each other without having to think about their gender status (Pebrianti, 2013). We can observe that in social and work relationships, women and men can communicate without having to think about gender differences or language styles. CONCLUSION AND RECOMMENDATION The female characters use different women’s language features in their speech to express specific purposes. In the Birds of Prey and the Fantabulous Emancipation of One Harley Quinn movie, the researchers found six features used by the female characters, they are lexical hedges/fillers, empty adjectives, tag questions, super polite forms, avoiding strong words, and emphatic stress. The female characters use female language features to express their femininity. © 2022 by Al-Ta’lim. This work is licensed under (CC-BY-SA) 263 264 Volume 29, Number 3, November, 2022, Page 253-266 They do not seem to be perceived as impolite when conversing with people in higher positions. They also use female language features to express their emotions when they speak. It does not always reflect selfconfidence, difficulty in making decisions, and uncertainty about their words, but there are several social factors and dimensions which can be considered as a reflection of the use of women's language, such as educational background, social status, friendship, topics of conversation, formal settings, and work. Arisandy, A. (2018). Women’s Language Features Found In Nora Dominguez’s And Mary Dominguez’s Utterances In From Prada To Nada Movie [PhD Thesis]. Sanata Dharma University. The researchers also found the use of neutral words, swear words, offensive words, and impolite requests by the female characters in the movie. This provides evidence that women also began to use the opposite of women's language. When women make fast decisions, they often express their annoyance using harsh words like men do and become more confident in speaking. Blommaert, J. (2015). Chronotopes, scales, and complexity in the study of language in society. 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